Inhibitors of DNA-dependent protein kinase and compositions and uses thereof - Patents.com

Abstract

The present disclosure relates to inhibitors of DNA protein kinases, and compositions and methods of use thereof. In some embodiments, the inhibitor has Formula I: [Formula 1] or a salt thereof, wherein x1 is C-R3 or N, R1 is C1-C3 alkyl, R2 is cycloalkyl or heterocyclyl, where cycloalkyl and heterocyclyl are optionally substituted with one or more R6, R3 is H or C1-C3 alkyl, R4 is H or C1-C3 alkyl, R5 is C1-C3 alkyl, each R6 is independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R6 together with the atom(s) to which they are attached form a spiro or fused ring, and R7 is H or C1-C3 alkyl.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 176,225, filed April 17, 2021, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] The ability to modify the genome of any cell at precise locations has been improved with the recent discovery and introduction of CRISPR / Cas9 editing technology. However, the ability to introduce specific and directed changes at a given locus is hindered by the fact that the main cellular repair pathway following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homologous recombination (HDR) is less efficient than NHEJ, reducing the editing efficiency in eukaryotic cells.

[0004] DNA-dependent protein kinase (DNA-PK) is a nuclear serine / threonine kinase that has been shown to be essential in the DNA double-strand break repair mechanism. In mammals, the major pathway for repair of double-strand DNA breaks is the non-homologous end joining (NHEJ) pathway, which functions regardless of cell cycle phase and acts by removing non-ligatable ends and ligating the ends of double-strand breaks. DNA-PK inhibitors (DNA-PKIs) are a structurally diverse class of inhibitors of the DNA-PK and NHEJ pathways.

[0005] There is a great need for efficient systems and techniques for modifying genomes, as well as efficient methods for editing nucleic acid molecules using template nucleic acids. Summary of the Invention

[0006] The present disclosure relates to DNA-PKIs, as well as compositions and methods of use thereof.

[0007] In certain embodiments, the DNA-PKI has formula I: [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 Although it is an alkyl Provided that at least one of the following applies: (a)x 1 CR 3 That is, (b)R 1 C 2 -C 3 is alkyl, (c)R 4 C 1-C 3 is alkyl, (d)R 2 There is one R 6 is replaced by R 6 is the halo, (e) Two R groups that, together with the atom(s) to which they are attached, form a spirocyclic or fused ring. 6 R 2 is replaced, and (f)R 2 But there is one or more R 6 C optionally substituted with 3 -C 5 It is cycloalkyl.

[0008] In a preferred embodiment, the present disclosure provides [ka] [ka] or a salt thereof.

[0009] In certain embodiments, the present disclosure provides: a) DNA protein kinase inhibitors (DNA-PKIs), b) DNA cutting substances; c) optionally, cells, and d) optionally donor DNA; The present invention relates to a DNA-PKI composition comprising: [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl.

[0010] In certain embodiments, the present disclosure relates to a method for performing targeted genome editing in a cell, or a method for repairing a double-stranded DNA break in the genome of a cell, or a method for inhibiting or suppressing repair of a DNA break in a cell via the non-homologous end joining (NHEJ) pathway, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is represented by formula I [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl. [Brief description of the drawings]

[0011] [Figure 1A] 1 shows the effect of DNA-PKI compounds on GFP insertion into the TRAC locus. The percentage of CD3- cells after GFP insertion into the TRAC locus using compounds (Compound 1, Compound 2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound 8, and Compound 9) is shown. [Figure 1B] Figure 1 shows the effect of DNA-PKI compounds on GFP insertion into the TRAC locus. Insertion efficiency is shown as the percentage of CD3- cells that were GFP+. [Figure 2A] Editing at the TRAC locus with Compound 1, Compound 3, and Compound 4 is shown. Percent CD8+ cells are shown. [Figure 2B] 1 shows editing at the TRAC locus with Compound 1, Compound 3, and Compound 4. Remaining TCR+ cells after editing are shown. [Figure 2C] Shown is editing at the TRAC locus with Compound 1, Compound 3, and Compound 4. Shown is the percentage of WT1-TCR+ cells after editing. [Figure 3A]Figure 1 shows the cytotoxicity of engineered WT1-T cells with compound Compound 1 or Compound 3. Figure 2 shows the specific lysis of luciferase-expressing 697 ALL cells incubated with engineered WT1-T cells from donor 007HD. [Figure 3B] Figure 1 shows the cytotoxicity of engineered WT1-T cells with compound Compound 1 or Compound 3. Figure 2 shows the specific lysis of luciferase-expressing 697 ALL cells incubated with engineered WT1-T cells from donor 008HD. [Figure 3C] Figure 1 shows the cytotoxicity of engineered WT1-T cells with compound 1 or compound 3. Figure 2 shows the specific lysis of K562-luc2 cells transduced to express HLA-A*02:01 after incubation with engineered WT1-T cells from donor 007HD. [Figure 3D] Figure 1 shows the cytotoxicity of engineered WT1-T cells with compound 1 or compound 3. Figure 2 shows the specific lysis of K562-luc2 cells transduced to express HLA-A*02:01 after incubation with engineered WT1-T cells from donor 008HD. [Figure 4A] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows Granzyme B release after incubation with 697 ALL cells. [Figure 4B] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows granzyme B release after incubation with K562-luc2 cells transduced to express HLA-A*02:01. [Figure 4C] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows interferon-gamma (IFNg) release after incubation with 697 ALL cells. [Figure 4D]Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows interferon-gamma (IFNg) release after incubation with K562-luc2 cells transduced to express HLA-A*02:01. [Figure 4E] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows interleukin-2 (IL-2) release after incubation with 697 ALL cells. [Figure 4F] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows interleukin-2 (IL-2) release after incubation with K562-luc2 cells transduced to express HLA-A*02:01. [Figure 4G] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows TNF-α release after incubation with 697 ALL cells and . [Figure 4H] Figure 1 shows cytokine release by T cells engineered with Compound 1 or Compound 3 after incubation with target cells. Figure 2 shows TNF-α release after incubation with K562-luc2 cells transduced to express HLA-A*02:01. [Diagram 5] Shown is the percentage of B2M negative cells, which represents the population of B cells with effective gene disruption after editing with Compound 1 or Compound 4. [Figure 6A] Shown is the average percent editing in AAVS1 assessed by NGS after treatment with LNP compositions and various doses of Compound 1 or Compound 4. [Figure 6B] The percentage of NK cells with high GFP expression (GFP++) after editing the AAVS1 locus with Compound 1 or Compound 4 to insert GFP is shown. [Figure 7A]The percentage of CD3eta+, Vb8- cells, which represent the population of T cells without gene disruptions at the TRAC or TRBC1 / 2 loci, is shown. [Figure 7B] The percentage of CD3eta+, Vb8+ cells, which represent the population of T cells with a WT1 TCR insertion in TRAC, is shown. [Figure 7C] The percentage of HLA-A2- cells, which represents the population of T cells with effective gene disruptions at the HLA locus, is shown. [Figure 7D] The percentage of HLA-DRDPDQ- cells, which represent the population of T cells carrying an effective gene disruption at the CIITA locus, is shown. [Figure 7E] The percentage of GFP+ cells, representing the population of T cells harboring a GFP insertion at the AAVS1 locus, is shown. [Figure 7F] The percentage of Vb8+GFP+HLA-A-HLA-DRDPDQ- cells representing the population of T cells with five genome edits is shown. [Figure 8A] A and B show the percentage of GFP+ cells representing the population of T cells after editing with alternative media conditions for two LNP compositions. A shows cells treated with an LNP composition with a lipid molar ratio of 50% ionizable lipid / 38.5% cholesterol / 10% DSPC / 1.5% PEG lipid. B shows cells treated with an LNP composition with a lipid molar ratio of 35% ionizable lipid / 47.5% cholesterol / 15% DSPC / 2.5% PEG lipid. [Figure 8B] A and B show the percentage of GFP+ cells representing the population of T cells after editing with alternative media conditions for two LNP compositions. A shows cells treated with an LNP composition with a lipid molar ratio of 50% ionizable lipid / 38.5% cholesterol / 10% DSPC / 1.5% PEG lipid. B shows cells treated with an LNP composition with a lipid molar ratio of 35% ionizable lipid / 47.5% cholesterol / 15% DSPC / 2.5% PEG lipid. [Figure 9A] The percentage of unintended structural differences after editing with compound 3 and compound 4 is shown. [Figure 9B] The percentage of GFP-positive cells after editing with Compound 3 and Compound 4 is shown. [Figure 10A] A and B show the percentage of indels and the percentage of HD3 TCR insertions in the presence and absence of DNApki compound 4 at different doses of sgRNA. A shows the percentage of TRAC editing. B shows the percentage of CD3+Vβ7.2+T cells. [Figure 10B] A and B show the percentage of indels and the percentage of HD3 TCR insertions in the presence and absence of DNApki compound 4 at different doses of sgRNA. A shows the percentage of TRAC editing. B shows the percentage of CD3+Vβ7.2+T cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Described herein are small molecule inhibitors of DNA-dependent protein kinase (DNA-PKI) that are useful for reducing NHEJ-mediated mutagenesis events or increasing the rate or probability of HDR after double-strand break (DSB) generation caused by Cas9 cleavage.Exemplary DNA-PKIs are provided in, for example, WO2018 / 114999, WO2014 / 183850, WO03 / 024949, Fok, JHL, et al., Nat, Commun, 10, 5065 (2019), Griffin, RJ et al., J.Med.Chem. 2005, 48, 569-585, Goldberg, FW, et al., J.Med.Chem. 2020, 63, 3461-3471, and U.S. Patent No. 10,786,512.

[0013] In some embodiments, DNAPK inhibitors (DNA-PKIs) are used in compositions and methods for delivering biologically active agents, such as nucleic acids such as CRISPR / Cas component RNAs and / or gRNAs ("cargo"), to cells.

[0014] Methods of gene editing and methods of producing engineered cells using the DNA-PKIs described herein, as well as compositions comprising same, are also provided.

[0015] In some embodiments, the compositions and methods provided herein provide an editing efficiency of greater than about 80%, greater than about 90%, or greater than about 95%. In some embodiments, the compositions and methods provide an editing efficiency of about 80-95%, about 90-95%, about 80-99%, about 90-99%, or about 95-99%.

[0016] DNA PK inhibitors The present disclosure relates to DNA-PKIs, as well as compositions and methods of use thereof.

[0017] In certain embodiments, the present disclosure provides a compound of formula I: [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 Although it is an alkyl Provided that at least one of the following applies: (a)x 1 CR 3 That is, (b)R 1 C 2 -C 3 is alkyl, (c)R 4 C 1 -C 3 is alkyl, (d)R 2 There is one R 6 is replaced by R 6 is the halo, (e) Two R groups that, together with the atom(s) to which they are attached, form a spirocyclic or fused ring. 6 R 2 is replaced, and (f)R 2 But there is one or more R 6 C optionally substituted with 3 -C 5 It is cycloalkyl.

[0018] In certain embodiments, the present disclosure provides a method for the preparation of a compound comprising: 1 CR 3 For example, R 3 can be H or methyl. In other embodiments, the compound is 1 The present invention relates to any of the compounds described herein wherein

[0019] In certain embodiments, the present disclosure provides 1 C 2 -C 3alkyl, e.g., R 1 is selected from methyl and ethyl, preferably R 1 is methyl.

[0020] In some embodiments, the present disclosure provides 4 C 1 -C 3 alkyl, e.g., R 4 is H or methyl, preferably R 4 is H.

[0021] In certain embodiments, the present disclosure provides 2 is cycloalkyl, for example, R 2 is C 3 -C 7 Cycloalkyl, preferably R 2 is cyclohexyl or C 3 -C 5 This relates to any of the compounds described herein which is cycloalkyl.

[0022] In certain embodiments, the present disclosure provides 2 is heterocyclyl, for example, R 2 is 5-7 membered heterocyclyl, preferably R 2 In certain embodiments, the present disclosure relates to any of the compounds described herein, wherein R is tetrahydropyranyl or tetrahydrofuranyl. 2 one or more R are independently selected from hydroxy, halo, and cycloalkyl; 6 or two R which, taken together with the atom(s) to which they are attached, form a spirocyclic or fused ring; 6 Optionally substituted with, for example, R 2 is one or more R 6 Each R 6 is halo or hydroxyl, e.g., R 2 There is one R 6 is replaced by R6 In some embodiments, each R 6 In some embodiments, the present disclosure provides for two R s which, taken together with the atom(s) to which they are attached, form a spirocyclic or fused ring. 6 R 2 In certain embodiments, R 2 is one or more R independently selected from hydroxy, methoxy, and methyl; 6 is optionally replaced by

[0023] In certain embodiments, the present disclosure provides 5 is methyl.

[0024] In some embodiments, the present disclosure provides 7 is H or methyl.

[0025] In a preferred embodiment, the present disclosure provides [ka] [ka] or a salt thereof.

[0026] In a specific embodiment, the compound is [ka] or a salt thereof.

[0027] In a specific embodiment, the compound is [ka] or a salt thereof.

[0028] In a specific embodiment, the compound is [ka] or a salt thereof.

[0029] In a specific embodiment, the compound is [ka] or a salt thereof.

[0030] In a specific embodiment, the compound is [ka] or a salt thereof.

[0031] In a specific embodiment, the compound is [ka] or a salt thereof.

[0032] In a specific embodiment, the compound is [ka] or a salt thereof.

[0033] In certain embodiments, the disclosure relates to any of the compounds described herein, wherein the compound is a free base.

[0034] In certain embodiments, the disclosure relates to any of the compounds described herein, wherein the compound is a salt, e.g., a triflate salt.

[0035] DNA-PKI composition In this specification, a) DNA protein kinase inhibitors (DNA-PKIs), b) DNA cutting substances; c) optionally, cells, and d) optionally donor DNA; The present invention describes a DNA-PKI composition comprising the formula I [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl.

[0036] In certain embodiments, the present disclosure provides a method for the preparation of a compound comprising: 1 is N.

[0037] In certain embodiments, the present disclosure provides 1 is methyl.

[0038] In certain embodiments, the present disclosure provides 4 is H.

[0039] In certain embodiments, the present disclosure provides 2 In another embodiment, R is cyclohexyl. 2 is tetrahydropyranyl. In yet another embodiment, R 2 is tetrahydrofuranyl. In certain embodiments, the present disclosure provides 2 one or more R are independently selected from hydroxy, methoxy, and methyl; 6 The present invention relates to any of the compositions described herein, optionally substituted with

[0040] In certain embodiments, the present disclosure provides 5 is methyl.

[0041] In certain embodiments, the present disclosure provides 7 is H or methyl.

[0042] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the DNA-PKI is any of the compounds described herein.

[0043] In certain embodiments, the present disclosure provides: a) DNA protein kinase inhibitors (DNA-PKIs), b) DNA cutting substances; c) optionally, cells, and d) optionally donor DNA; The composition comprises: [ka] [ka] or a salt thereof.

[0044] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0045] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0046] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0047] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0048] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0049] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0050] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0051] In a specific embodiment, the DNA-PKI in the composition is [ka] or a salt thereof.

[0052] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the concentration of DNA-PKI in the composition is about 1 μM or less, such as about 0.25 μM or less, for example, about 0.1 to 1 μM, preferably about 0.1 to 0.5 μM.

[0053] In some embodiments, the disclosure relates to any of the compositions described herein comprising a cell, e.g., a eukaryotic cell, such as a hepatocyte or an immune cell. In some embodiments, the disclosure relates to any of the compositions described herein, wherein the cell is useful in adoptive cell therapy (ACT). Examples of ACT include autologous cell therapy and allogeneic cell therapy. In some embodiments, the disclosure relates to any of the compositions described herein, wherein the cell is a stem cell. In some embodiments, the disclosure relates to any of the compositions described herein, wherein the cell is a stem cell. In some embodiments, the disclosure relates to any of the compositions described herein, wherein the cell is a hematopoietic stem cell (HSC) or an induced pluripotent stem cell (iPSC). In certain embodiments, the disclosure relates to any of the compositions described herein, wherein the immune cell is a white blood cell or lymphocyte, e.g., the immune cell is a lymphocyte, such as a T cell, a B cell, or a NK cell, preferably, the lymphocyte is a T cell. In some embodiments, the disclosure relates to any of the compositions described herein, wherein the T cell is a primary T cell. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the T cells are regulatory T cells. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the lymphocytes are activated T cells or non-activated T cells.

[0054] In certain embodiments, the disclosure relates to any of the compositions described herein, wherein the cell is a human cell.

[0055] In some embodiments, the present disclosure relates to any of the compositions described herein, wherein the DNA cleaving agent comprises a CRISPR / Cas nuclease component and optionally a guide RNA component. In some embodiments, the present disclosure relates to any of the compositions described herein, wherein the DNA cleaving agent comprises a DNA cleaving agent or a nucleic acid encoding the DNA cleaving agent, e.g., an mRNA encoding the DNA cleaving agent, wherein the DNA cleaving agent is selected from a zinc finger nuclease, a TALE effector domain nuclease (TALEN), a CRISPR / Cas nuclease component, and combinations thereof, preferably, the DNA cleaving agent is a CRISPR / Cas nuclease component. In some embodiments, the DNA cleaving agent is a CRISPR / Cas nuclease component and a guide RNA component. In some embodiments, the present disclosure relates to any of the compositions described herein, wherein the CRISPR / Cas nuclease component comprises a Cas nuclease or an mRNA encoding a Cas nuclease, e.g., the CRISPR / Cas nuclease component comprises an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the Cas nuclease is a Cas9 nuclease, such as S. pyogenes Cas9 nuclease or N. meningitidis Cas9 nuclease. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the Cas nuclease is Nme2Cas9. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the Cas nuclease is a Cas12a nuclease.

[0056] In some embodiments, the disclosure relates to any of the compositions described herein comprising a modified RNA.

[0057] In certain embodiments, the disclosure relates to any of the compositions described herein comprising a guide RNA nucleic acid, such as a gRNA. In certain embodiments, the disclosure relates to any of the compositions described herein, in which the guide RNA nucleic acid is or encodes a dual-guide (dgRNA). In certain embodiments, the disclosure relates to any of the compositions described herein, in which the guide RNA nucleic acid is or encodes a single-guide (sgRNA). In some embodiments, the disclosure relates to any of the compositions described herein, in which the gRNA is a modified gRNA, e.g., the modified gRNA comprises a modification in one or more of the first five nucleotides of the 5' end, or the modified gRNA comprises a modification in one or more of the last five nucleotides of the 3' end. In some embodiments, the gRNA is complexed with a Cas nuclease, such as a Cas9 nuclease.

[0058] In certain embodiments, the present disclosure relates to any of the compositions described herein comprising a guide RNA nucleic acid and a class 2 Cas nuclease mRNA, wherein the weight ratio of mRNA to guide RNA nucleic acid is about 2:1 to 1:4.

[0059] In some embodiments, the disclosure relates to any of the compositions described herein that include a DNA cleaving agent, wherein the DNA cleaving agent is present in a lipid-nucleic acid assembly composition.

[0060] In some embodiments, the disclosure relates to any of the compositions described herein comprising donor DNA.

[0061] In certain embodiments, the disclosure relates to any of the compositions described herein, wherein the donor DNA (also referred to herein as a "template nucleic acid" or "exogenous nucleic acid") comprises a sequence encoding a protein, a regulatory sequence, or a sequence encoding structural RNA.

[0062] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the lipid-nucleic acid assembly composition is a lipid nanoparticle (LNP) composition. In some embodiments, the LNP composition is any of the LNP compositions described herein.

[0063] In certain embodiments, the disclosure relates to any of the compositions described herein, wherein the LNPs are 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 diameter. In certain embodiments, the disclosure relates to any of the compositions described herein, comprising 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. For example, the average diameter can be the Z-average diameter.

[0064] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the lipid-nucleic acid assembly composition is a lipoplex.

[0065] In some embodiments, the disclosure relates to any of the compositions described herein, wherein the lipid-nucleic acid assembly composition comprises an ionizable lipid, e.g., any of the ionizable lipids described herein. In certain embodiments, the disclosure relates to any of the compositions described herein, wherein the ionizable lipid has a pKa of about 5.1 to about 8.0, e.g., about 5.5 to about 7.6, or about 5.1 to 7.4, e.g., about 5.5 to 6.6, about 5.6 to 6.4, about 5.8 to 6.2, or about 5.8 to 6.5.

[0066] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the lipid-nucleic acid assembly composition comprises a helper lipid.

[0067] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the lipid-nucleic acid assembly composition comprises a neutral lipid.

[0068] In some embodiments, the present disclosure relates to any of the compositions described herein, wherein the lipid-nucleic acid assembly composition comprises a PEG-lipid.

[0069] In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the N / P ratio of the lipid-nucleic acid assembly composition is about 3-10, e.g., about 5-7, preferably about 6.

[0070] In some embodiments, the present disclosure relates to any of the compositions described herein, for example, further comprising a vector, wherein the vector encodes a DNA cleavage agent or a donor DNA. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the vector is a viral vector. In other embodiments, the present disclosure relates to any of the compositions described herein, wherein the vector is a non-viral vector. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the vector is a lentiviral vector. In some embodiments, the present disclosure relates to any of the compositions described herein, wherein the vector is a retroviral vector. In certain embodiments, the present disclosure relates to any of the compositions described herein, wherein the vector is an AAV.

[0071] In certain embodiments, the disclosure relates to any of the compositions described herein, eg, comprising a cell, wherein the cell is not a cancer cell.

[0072] DNA-PKI method In certain embodiments, the present disclosure relates to a method for performing targeted genome editing in a cell, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI has the formula I [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl.

[0073] In some embodiments, the present disclosure relates to a method of repairing a double-stranded DNA break in the genome of a cell, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI has the formula I [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl.

[0074] In some embodiments, the present disclosure relates to a method of inhibiting or suppressing the repair of a DNA break in a cell via the non-homologous end joining (NHEJ) pathway, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI has the structure of Formula I [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C 1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl.

[0075] In some embodiments, the present disclosure relates to a method for targeted insertion of donor DNA into the genome of a cell, comprising contacting the cell with a DNA cleaving agent, donor DNA, and a DNA-PKI, wherein the DNA-PKI is a polypeptide represented by formula I. [ka] or a salt thereof, During the ceremony, x 1 CR 3 or N, R 1 is C 1 -C 3 is alkyl, R 2 is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R 6 is optionally replaced by R 3 is H or C 1 -C 3 is alkyl, R 4 is H or C1 -C 3 is alkyl, R 5 is C 1 -C 3 is alkyl, Each R 6 are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R 6 together with the atom(s) to which they are attached form a spiro or fused ring; and R 7 is H or C 1 -C 3 It is an alkyl.

[0076] In some embodiments, the present description relates to methods for adoptive cell transfer (ACT) therapy, such as tumor immunology. For example, in certain embodiments, the methods described herein result in cells whose genomes are modified at one or more specific target sequences, including those modified by the introduction of a CRISPR system that includes a gRNA molecule that targets the target sequence. In certain embodiments, gRNA molecules, CRISPR systems, cells, and methods are provided that are useful for genome editing of immune cells, e.g., T cells engineered to lack endogenous expression of a TCR, 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 that are useful for genome editing of B cells, e.g., B cells engineered to lack endogenous expression of a B cell receptor (BCR), 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 that are useful for expression of antibodies and useful for ACT therapy.

[0077] In certain embodiments, the disclosure relates to any of the methods of gene editing described herein, comprising administering the LNP composition to an animal, e.g., a human. In certain embodiments, the method comprises administering the LNP composition to a cell, e.g., a eukaryotic cell, particularly a human cell. In some embodiments, the cell is a type of cell useful for therapy, e.g., adoptive cell therapy (ACT). Examples of ACT include autologous cell therapy and allogeneic cell therapy. In some embodiments, the cell is a stem cell, such as a hematopoietic stem cell, an induced pluripotent stem cell, or another multipotent or multipotent cell. In some embodiments, the cell is a stem cell, e.g., a mesenchymal stem cell that can develop into bone, cartilage, muscle, or fat cells. In some embodiments, the stem cell comprises an ocular stem cell. In certain embodiments, the cells are selected from mesenchymal stem cells, hematopoietic stem cells (HSCs), mononuclear cells, endothelial progenitor cells (EPCs), neural stem cells (NSCs), limbal stem cells (LSCs), tissue-specific primary cells or cells derived therefrom (TSCs), induced pluripotent stem cells (iPSCs), ocular stem cells, pluripotent stem cells (PSCs), embryonic stem cells (ESCs), and cells for organ or tissue transplantation.

[0078] In certain embodiments, the present disclosure relates to any of the methods described herein, comprising growing cells in a cell culture medium that does not contain DNA-PKI and adding DNA-PKI to the cell culture medium.

[0079] In certain embodiments, the present disclosure relates to any of the methods described herein, comprising contacting the cell with a DNA-PKI after contacting the cell with a DNA cleaving agent, for example within about 6 hours after contacting the cell with the DNA cleaving agent, preferably within about 3 hours after contacting the cell with the DNA cleaving agent.

[0080] In other embodiments, the disclosure relates to any of the methods described herein comprising contacting the cell with a DNA cleaving agent simultaneously with the DNA-PKI.

[0081] In yet other embodiments, the present disclosure relates to any of the methods described herein, comprising contacting the cell with a DNA-PKI, for example, within about 3 hours of contacting the cell with the DNA-PKI, followed by contacting the cell with a DNA cleaving agent.

[0082] In certain embodiments, the present disclosure relates to any of the methods described herein comprising growing cells in a cell culture medium comprising DNA-PKI.

[0083] In some embodiments, the present disclosure relates to any of the methods described herein, wherein the cells are contacted with the DNA cleaving agent and the DNA-PKI for at least about one day, for example, from about one day to one week, preferably for about five days.

[0084] In certain embodiments, the present disclosure provides a method for the preparation of a compound comprising: 1 is N.

[0085] In certain embodiments, the present disclosure provides 1 is methyl.

[0086] In certain embodiments, the present disclosure provides 4 is H.

[0087] In some embodiments, the present disclosure provides 2 is cyclohexyl, tetrahydropyranyl, or tetrahydrofuranyl. 2 one or more R are independently selected from hydroxy, methoxy, and methyl; 6 The present invention relates to any of the methods described herein, optionally substituted with

[0088] In certain embodiments, the present disclosure provides 5is methyl.

[0089] In certain embodiments, the present disclosure provides 7 is H or methyl.

[0090] In a preferred embodiment, the present disclosure relates to any of the methods described herein, wherein the DNA-PKI is any of the compounds described herein.

[0091] In certain embodiments, the present disclosure relates to a method for performing targeted genome editing in a cell, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI comprises: [ka] [ka] or a salt thereof.

[0092] In certain embodiments, the present disclosure relates to a method of repairing a double-stranded DNA break in the genome of a cell, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, the DNA-PKI comprising: [ka] [ka] or a salt thereof.

[0093] In certain embodiments, the present disclosure relates to a method for inhibiting or suppressing repair of a DNA break in a cell via the non-homologous end joining (NHEJ) pathway, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is [ka] [ka] or a salt thereof.

[0094] In certain embodiments, the present disclosure relates to a method for targeted insertion of donor DNA into the genome of a cell, comprising contacting the cell with a DNA cleaving agent, donor DNA, and a DNA-PKI, wherein the DNA-PKI: [ka] [ka] or a salt thereof.

[0095] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0096] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0097] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0098] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0099] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0100] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0101] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0102] In a specific embodiment, the DNA-PKI used in the method comprises: [ka] or a salt thereof.

[0103] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the cells are contacted with DNA-PKI in cell culture medium, and the concentration of DNA-PKI in the cell culture medium is about 1 μM or less, for example, about 0.25 μM or less, for example, about 0.1 to 1 μM, preferably about 0.1 to 0.5 μM.

[0104] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the cell is a eukaryotic cell.

[0105] In some embodiments, the disclosure relates to any of the methods described herein, wherein the composition comprises a cell, e.g., a eukaryotic cell, such as a hepatocyte or an immune cell. In certain embodiments, the cell is useful for adoptive cell therapy (ACT). Examples of ACT include autologous cell therapy and allogeneic cell therapy. In certain embodiments, the cell is a stem cell. In certain embodiments, the stem cell is a hematopoietic stem cell (HSC). In certain embodiments, the cell is an induced pluripotent stem cell (iPSC). In certain embodiments, the disclosure relates to any of the methods described herein, wherein the immune cell is a white blood cell or a lymphocyte, e.g., the immune cell is a lymphocyte, such as a T cell, a B cell, or a NK cell, preferably, the lymphocyte is a T cell. In some embodiments, the disclosure relates to any of the methods described herein, wherein the T cell is a primary T cell. In certain embodiments, the disclosure relates to any of the methods described herein, wherein the T cell is a regulatory T cell. In certain embodiments, the disclosure relates to any of the methods described herein, wherein the lymphocyte is an activated T cell or a non-activated T cell.

[0106] In certain embodiments, the disclosure relates to any of the methods described herein, wherein the cell is a human cell.

[0107] In some embodiments, the present disclosure relates to any of the methods described herein, e.g., comprising a DNA cleaving agent, wherein the DNA cleaving agent is selected from a zinc finger nuclease, a TALE effector domain nuclease (TALEN), a CRISPR / Cas nuclease component, and combinations thereof, preferably, the DNA cleaving agent is a CRISPR / Cas nuclease component.

[0108] In some embodiments, the disclosure relates to any of the methods described herein, where the CRISPR / Cas nuclease components comprise a Cas nuclease or an mRNA encoding a Cas nuclease, e.g., the CRISPR / Cas nuclease components comprise an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease. In certain embodiments, the disclosure relates to any of the methods described herein, where the Cas nuclease is a Cas9 nuclease, such as a S. pyogenes Cas9 nuclease or a N. meningitidis Cas9 nuclease. In certain embodiments, the disclosure relates to any of the methods described herein, where the Cas nuclease is an Nme2Cas9. In certain embodiments, the disclosure relates to any of the methods described herein, where the Cas nuclease is a Cas12a nuclease.

[0109] In some embodiments, the disclosure relates to any of the methods described herein involving modified RNA.

[0110] In certain embodiments, the disclosure relates to any of the methods described herein that include a guide RNA nucleic acid such as a gRNA. In certain embodiments, the disclosure relates to any of the methods described herein, where the guide RNA nucleic acid is or encodes a dual guide RNA (dgRNA). In certain embodiments, the disclosure relates to any of the methods described herein, where the guide RNA nucleic acid is or encodes a single guide (sgRNA). In some embodiments, the disclosure relates to any of the methods described herein, where the gRNA is a modified gRNA, for example, the modified gRNA includes a modification in one or more of the first five nucleotides at the 5' end, or the modified gRNA includes a modification in one or more of the last five nucleotides at the 3' end.

[0111] In certain embodiments, the disclosure relates to any of the methods described herein, wherein the composition comprises a guide RNA nucleic acid and a class 2 Cas nuclease mRNA, and the weight ratio of the mRNA to the guide RNA nucleic acid is about 2:1 to 1:4. In some embodiments, the composition comprises a complex of a class 2 Cas nuclease and a guide RNA.

[0112] In some embodiments, the disclosure relates to any of the methods described herein that include a DNA cleaving agent, wherein the DNA cleaving agent is present in a lipid nucleic acid assembly composition.

[0113] In some embodiments, the disclosure relates to any of the methods described herein involving donor DNA.

[0114] In certain embodiments, the disclosure relates to any of the methods described herein, wherein the donor DNA comprises a sequence encoding a protein, a regulatory sequence, or a sequence encoding structural RNA.

[0115] In certain embodiments, the disclosure relates to any of the methods described herein, wherein the template sequence is integrated into the genome of the cell via homology directed repair (HDR).

[0116] In certain embodiments, the disclosure relates to any of the methods described herein comprising contacting a cell with a lipid nucleic acid assembly composition that includes a DNA cleaving agent.

[0117] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the lipid-nucleic acid assembly composition is a lipid nanoparticle (LNP) composition. In some embodiments, the LNP composition is any of the LNP compositions described herein.

[0118] In certain embodiments, the disclosure relates to any of the compositions and methods described herein, wherein the LNPs are 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 diameter. In certain embodiments, the disclosure relates to any of the methods described herein, wherein the composition comprises 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. For example, the average diameter can be the Z-average diameter.

[0119] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the lipid-nucleic acid assembly composition is a lipoplex.

[0120] In some embodiments, the disclosure relates to any of the methods described herein, wherein the lipid-nucleic acid assembly composition comprises an ionizable lipid, e.g., any of the ionizable lipids described herein. In certain embodiments, the disclosure relates to any of the methods described herein, wherein the ionizable lipid has a pKa of about 5.1-7.4, e.g., about 5.5-6.6, about 5.6-6.4, about 5.8-6.2, or about 5.8-6.5.

[0121] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the lipid-nucleic acid assembly composition comprises a helper lipid.

[0122] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the lipid-nucleic acid assembly composition comprises neutral lipids.

[0123] In some embodiments, the disclosure relates to any of the methods described herein, wherein the lipid-nucleic acid assembly composition comprises a PEG-lipid.

[0124] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the N / P ratio of the lipid-nucleic acid assembly composition is about 3-10, such as about 5-7, and preferably about 6.

[0125] In some embodiments, the present disclosure relates to any of the methods described herein, for example, further comprising a vector, wherein the vector encodes a DNA cleavage agent or a donor DNA. In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the vector is a viral vector. In other embodiments, the present disclosure relates to any of the methods described herein, wherein the vector is a non-viral vector. In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the vector is a lentiviral vector. In some embodiments, the present disclosure relates to any of the methods described herein, wherein the vector is a retroviral vector. In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the vector is an AAV.

[0126] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the cell is not a cancer cell.

[0127] In certain embodiments, the disclosure relates to any of the methods described herein, wherein a DNA cleaving agent interacts with a target sequence in the genome of a cell to generate a double-stranded DNA break (DSB).

[0128] In some preferred embodiments, the disclosure relates to any of the methods described herein that result in a gene knockout.

[0129] In some preferred embodiments, the present disclosure relates to any of the methods described herein that result in gene correction.

[0130] In certain embodiments, the disclosure relates to any of the methods of gene editing described herein that result in an insertion, hi some embodiments, the insertion is a gene insertion.

[0131] In certain embodiments, the present disclosure relates to any of the methods described herein, wherein the donor DNA comprises a template comprising an exogenous nucleic acid encoding a protein. In certain embodiments, the protein is selected from a cytokine, an immunosuppressant, an antibody, a receptor, and an enzyme. In certain embodiments, the protein is a receptor. In certain embodiments, the receptor is selected from an immune receptor, a T cell receptor (TCR), and a chimeric antigen receptor. In certain embodiments, the receptor is an immune receptor. In certain embodiments, the receptor is a TCR. In certain embodiments, the exogenous nucleic acid encodes the TCR alpha and / or beta chains of a TCR. In certain embodiments, the receptor is a chimeric antigen receptor.

[0132] In certain embodiments, the present disclosure relates to any of the methods of gene editing described herein, in which a DNA cleavage agent interacts with a target sequence in the genome of a cell to generate a double-stranded DNA break (DSB). In certain embodiments, the DNA cleavage agent interacts with a target sequence in the TRAC gene of a T cell. In certain embodiments, a template is integrated into the TRAC gene of a T cell. In certain embodiments, the template comprises a first homology arm and a second homology arm that are complementary to the sequence located upstream and downstream of the cleavage site, respectively.

[0133] DNA-cutting agents, such as proteins, RNA, or the nucleic acids encoding them, can be delivered to cells by electroporation, lipid-based delivery, e.g., via lipid-nucleic acid assemblies such as lipid nanoparticles, or other delivery techniques known in the art.

[0134] Ionizable lipids In some embodiments, methods and compositions are provided in which the nucleic acid assembly comprises a DNA cleavage agent and serves to deliver the DNA cleavage agent to cells.The ionizable lipid and other "biodegradable lipids" suitable for use in the lipid nucleic acid assembly described herein are biodegradable in vivo or ex vivo.The ionizable lipid has low toxicity (e.g., is tolerable in animal models and does not have adverse effects at doses of 10mg / kg or more). Biodegradable lipids suitable for use in the lipid-nucleic acid assemblies described herein include, for example, the biodegradable lipids of WO / 2020 / 219876, WO / 2020 / 118041, WO / 2020 / 072605, WO / 2019 / 067992, WO / 2017 / 173054, WO2015 / 095340, and WO2014 / 136086, each of which is incorporated by reference in its entirety, and specifically the ionizable lipids and compositions described in each are incorporated by reference herein.

[0135] In some embodiments, the lipid-nucleic acid assembly composition comprises an ionizable lipid, such as lipid A or its equivalent, for example, an acetal analog of lipid A.

[0136] In some embodiments, the ionizable 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 referred to 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]

[0137] Lipid A can be synthesized according to WO2015 / 095340 (e.g., pp. 84-86).

[0138] In some embodiments, the ionizable lipid is lipid D, which is nonyl 8-((7,7-bis(octyloxy)heptyl)(2-hydroxyethyl)amino)octanoate. Lipid D can be depicted as follows: [ka]

[0139] Lipid D can be synthesized according to WO2020 / 072605.

[0140] The ionizable lipids of the present disclosure can form salts depending on the pH of the medium in which they are placed.For example, in a slightly acidic medium, the ionizable lipids can be protonated and therefore have a positive charge. Conversely, in a slightly basic medium, such as blood, which has a pH of approximately 7.35, the ionizable lipids can be unprotonated and therefore have no charge.In some embodiments, the ionizable lipids of the present disclosure can be mostly protonated at a pH of at least about 9.In some embodiments, the ionizable lipids of the present disclosure can be mostly protonated at a pH of at least about 10.

[0141] The pH at which an ionizable lipid is largely protonated is related to its inherent pKa. In some embodiments, the ionizable lipid salts of the present disclosure have a pKa in the range of about 5.1 to about 8.0, even more preferably about 5.5 to about 7.6. In some embodiments, the ionizable lipid salts of the present disclosure have a pKa in the range of about 5.7 to about 8, about 5.7 to about 7.6, about 6 to about 8, about 6 to about 7.5, about 6 to about 7, or about 6 to about 6.5. In some embodiments, the ionizable lipid salts of the present disclosure have a pKa of about 6.0, about 6.1, about 6.1, about 6.2, about 6.3, about 6.4, about 6.6, or about 6.6. Alternatively, the ionizable lipid salts of the present disclosure have a pKa in the range of about 6 to about 8. pKa can be an important consideration when formulating LNPs, as LNPs formulated with certain lipids having a pKa in the range of about 5.5 to about 7.0 have been found to be effective for in vivo cargo delivery, e.g., to the liver. Additionally, LNPs formulated with certain lipids having a pKa in the range of about 5.3 to about 6.4 have been found to be effective for in vivo delivery, e.g., to tumors. See, e.g., WO2014 / 136086. In some embodiments, the ionizable lipid is positively charged at acidic pH but neutral in blood.

[0142] Additional lipids Suitable "neutral lipids" for use in the lipid compositions of the present disclosure include, for example, a variety of neutral lipids, uncharged lipids, or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (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-stearoylphosphatidylcholine (PMP ... phosphatidylcholine (PSPC), 1,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1-stearoyl-2-palmitoylphosphatidylcholine (SPPC), 1,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoylphosphatidylcholine (POPC), lysophosphatidylcholine, dioleoylphosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine, distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), dipalmitoylphosphatidylethanolamine (DPPE), palmitoyloleoylphosphatidylethanolamine (POPE), lysophosphatidylethanolamine and combinations thereof. In certain embodiments, the neutral phospholipid may be selected from distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylethanolamine (DMPE), preferably distearoylphosphatidylcholine (DSPC).

[0143] "Helper lipid" includes steroids, sterols, and alkylresorcinols. Suitable helper lipids for use in the present disclosure include, but are not limited to, cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In certain embodiments, the helper lipid may be cholesterol or a derivative thereof, such as cholesterol hemisuccinate.

[0144] In some embodiments, the LNP composition includes a polymeric lipid, such as a PEG lipid, which can affect the length of time that the nanoparticles can reside in vivo or ex vivo (e.g., in blood or medium). The PEG lipid can aid in the formulation process, for example, by inhibiting particle aggregation and controlling particle size. The PEG lipid used herein can modulate the pharmacokinetic properties of the LNP composition. Typically, the PEG lipid includes a lipid portion and a polymer portion (PEG portion) based on PEG (sometimes referred to as poly(ethylene oxide)). Information regarding PEG lipids suitable for use in the lipid compositions of the present disclosure and the biochemistry of such lipids can be found in Romberg et al., Pharmaceutical Research 25(1), 2008, pp. 55-71 and Hoekstra et al., Biochimica et Biophysica Acta 1660(2004) 41-52. Additional suitable PEG lipids are disclosed, for example, in WO2015 / 095340 (p. 31, line 14 to p. 37, line 6), WO2006 / 007712, and WO2011 / 076807 ("stealth lipids"), which are incorporated by reference.

[0145] In some embodiments, the lipid moiety may be derived from a diacylglycerol or diacylglycamide, e.g., comprising, independently, a dialkylglycerol or dialkylglycamide group having an alkyl chain length of about C4 to about C40 saturated or unsaturated carbon atoms, the chain may include one or more functional groups, e.g., amide or ester. In some embodiments, the alkyl chain length includes about C10 to C20. The dialkylglycerol or dialkylglycamide group may further include one or more substituted alkyl groups. The chain length may be symmetric or asymmetric.

[0146] Unless otherwise indicated, the term "PEG" as used herein refers to any polyethylene glycol or other polyalkylene ether polymer, e.g., an optionally substituted linear or branched polymer of ethylene glycol or ethylene oxide. In certain embodiments, the PEG moiety is unsubstituted. Alternatively, the PEG moiety may be substituted, e.g., by one or more alkyl, alkoxy, acyl, hydroxy, or aryl groups. For example, the PEG moiety may comprise a PEG copolymer, such as a PEG-polyurethane or a PEG-polypropylene (see, e.g., J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992)), or alternatively, the PEG moiety may be a PEG homopolymer. In certain embodiments, the PEG moiety has a molecular weight of about 130 to about 50,000, e.g., about 150 to about 30,000, or about 150 to about 20,000. Similarly, the PEG moiety can have a molecular weight of about 150 to about 15,000, about 150 to about 10,000, about 150 to about 6,000, or about 150 to about 5,000. In certain preferred embodiments, the PEG moiety has a molecular weight of about 150 to about 4,000, about 150 to about 3,000, about 300 to about 3,000, about 1,000 to about 3,000, or about 1,500 to about 2,500.

[0147] In certain preferred embodiments, the PEG moiety is "PEG-2K," also known as "PEG 2000," and has an average molecular weight of about 2,000 daltons. PEG-2K is represented herein by the following formula (III): [ka] where n is about 45, meaning that the PEG contains subunits with a number average degree of polymerization of about 45. However, other embodiments of PEG known in the art may be used, including, for example, those containing subunits with a number average degree of polymerization of about 23 (n=23) and / or 68 subunits (n=68). In some embodiments, n may range from about 30 to about 60. In some embodiments, n may range from about 35 to about 55. In some embodiments, n may range from about 40 to about 50. In some embodiments, n may range from about 42 to about 48. In some embodiments, n may be 45. In some embodiments, R may be selected from H, substituted alkyl, and unsubstituted alkyl. In some embodiments, R may be unsubstituted alkyl, such as methyl.

[0148] In any of the embodiments described herein, the PEG lipid may be PEG-dilaurylglycerol, PEG-dimyristoylglycerol (PEG-DMG) (catalog number GM-020, manufactured by NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE) (catalog number DSPE-020CN, manufactured by NOF, Tokyo, Japan), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxazone-1-yl)-1,1-dimethylethyl ether (DMG), PEG-dimyristoylglycerol (DMG), PEG-DMG (catalog number GM-020, manufactured by NOF, Tokyo, Japan), PEG-dipalmitoylglycerol, PEG-distearoylglycamide (DSPE), PEG-DSPE (DSPE-020CN), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1-[8'-(cholest-5-ene-3[beta]-oxy)carboxamido-3',6'-dioxazone-1-yl)-1,1-dimethylethyl ether (DMG), PEG-DMG ... [butanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol) ether), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMPE), or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE) (catalog number 880120C, Avanti The PEG lipid may be selected from PEG2k-DMG, PEG2k-DSG, 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol (PEG2k-DSG; GS-020, NOF Tokyo, Japan), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA). In certain such embodiments, the PEG lipid may be PEG2k-DMG. In some embodiments, the PEG lipid may be PEG2k-DSG. In other embodiments, the PEG lipid may be PEG2k-DSPE. In some embodiments, the PEG lipid may be PEG2k-DMA.In yet another embodiment, the PEG lipid may be PEG2k-C-DMA. In certain embodiments, the PEG lipid may be compound S027, as disclosed in WO2016 / 010840 (paragraphs

[0240] to

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

[0149] In a preferred embodiment, the PEG lipid comprises a glycerol group. In a preferred embodiment, the PEG lipid comprises a dimyristoyl glycerol (DMG) group. In a preferred embodiment, the PEG lipid comprises PEG-2k. In a preferred embodiment, the PEG lipid is PEG-DMG. In a preferred embodiment, the PEG lipid is PEG-2k-DMG. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In a preferred embodiment, the PEG-2k-DMG is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0150] In some embodiments, methods and compositions are provided in which the nucleic acid assembly comprises a cationic lipid composition and a DNA cleavage agent and serves to deliver the DNA cleavage agent to cells. Suitable cationic lipids for use in the lipid compositions described herein include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N-(1-(2,3- Dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dioleoylcarbamyl-3-dimethylammonium-propane (DOCDAP), 1,2-dilineoyl-3-dimethylammonium-propane (DLINDAP), dilauryl (C12:0) trimethylammonium propane (DLTAP), dioctadecylamidoglycylspermine (DOGS), DC-Choi, dioleoyloxy-N-[2-(s Perminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-ene-3-beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), N,N-dimethyl-2,3-dioleyloxy Cationic lipids include, but are not limited to, N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), and 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP). In some embodiments, the cationic lipid is DOTAP or DLTAP.

[0151] In further embodiments, methods and compositions are provided, wherein nucleic acid assembly comprises anionic lipid composition and DNA cutting agent, and serves to deliver DNA cutting agent to cells.The anionic lipid suitable for use in the compositions described herein includes, but is not limited to, phosphatidylglycerol, cardiolipin, diacyl phosphatidylserine, diacyl phosphatidic acid, N-dodecanoyl phosphatidylethanolamine, N-succinyl phosphatidylethanolamine, N-glutaryl phosphatidylethanolamine, cholesterol hemisuccinate (CHEMS), and lysyl phosphatidylglycerol.

[0152] lipid composition Described herein is a lipid composition comprising at least one ionizable lipid, cationic lipid, or anionic lipid, such as an ionizable lipid, or a salt thereof (e.g., a pharma- ceutically acceptable salt thereof), and optionally comprising at least one helper lipid, at least one neutral lipid, and at least one polymeric lipid. In some embodiments, the lipid composition comprises an ionizable lipid, or a salt thereof, a neutral lipid, a helper lipid, and a PEG lipid. In some embodiments, the neutral lipid is DSPC or DPME. In some embodiments, the helper lipid is cholesterol, 5-heptadecylresorcinol, or cholesterol hemisuccinate.

[0153] In a preferred embodiment, the ionizable lipid is [ka] In a preferred embodiment, the neutral lipid is DSPC. In a preferred embodiment, the helper lipid is cholesterol. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In a particularly preferred embodiment, the ionizable lipid is [ka] where the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0154] In a preferred embodiment, the ionizable lipid is [ka] In a preferred embodiment, the neutral lipid is DSPC. In a preferred embodiment, the helper lipid is cholesterol. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0155] In a particularly preferred embodiment, the ionizable lipid is [ka] where the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0156] In some embodiments, the lipid composition further comprises one or more additional lipid components. In some embodiments, the lipid composition further comprises at least one cationic lipid and / or at least one anionic lipid. In further embodiments, the lipid composition further comprises a cationic lipid, optionally with one or more additional lipid components. In further embodiments, the lipid composition further comprises an anionic lipid, optionally with one or more additional lipid components.

[0157] In some embodiments, the lipid composition is in liposome form. In a preferred embodiment, the lipid composition is in lipid nanoparticle (LNP) composition form. "Lipid nanoparticle" or "LNP" refers, without limitation, to a particle that includes multiple (i.e., more than one) LNP components that are physically associated with each other by intermolecular forces. In certain embodiments, the lipid composition is suitable for in vivo delivery. In certain embodiments, the lipid composition is suitable for delivery to an organ, such as the liver. In certain embodiments, the lipid composition is suitable for delivery to tissues ex vivo. In certain embodiments, the lipid composition is suitable for delivery to cells in vitro.

[0158] The lipid composition may be in various forms, including but not limited to particle-forming delivery agents such as microparticles, nanoparticles and transfection agents, which are useful for delivering various molecules to cells. Certain compositions are effective for transfecting or delivering bioactive agents. Preferred bioactive agents are RNA and DNA. In further embodiments, the bioactive agent is selected from mRNA, gRNA, and DNA. The gRNA can be dgRNA or sgRNA. In certain embodiments, the cargo includes an mRNA encoding an RNA-guided DNA cleavage agent (e.g., Cas nuclease, class 2 Cas nuclease, or Cas9), a gRNA or a nucleic acid encoding a gRNA, or a combination of mRNA and gRNA.

[0159] A compound or composition generally, but not necessarily, includes one or more pharma- ceutically acceptable excipients. The term "excipient" includes any component other than the compound(s) of the present disclosure, other lipid component(s) and biologically active substances. An excipient can provide both functional (e.g., drug release rate control) and / or non-functional (e.g., processing aid or diluent) properties to the composition. The choice of excipient is largely dependent on factors such as the specific mode of administration, the effect of the excipient on solubility and stability, and the nature of the dosage form.

[0160] Parenteral formulations are typically aqueous or oily solutions or suspensions. When the formulations are aqueous, the excipients are sugars (including but not limited to glucose, mannitol, sorbitol, etc.), salts, carbohydrates, and buffers (preferably pH 3-9), etc., although in some applications they may be more suitably formulated using sterile non-aqueous solutions or as a dry form in combination with a suitable vehicle such as sterile pyrogen-free water (WFI).

[0161] LNP composition The lipid composition may be provided as an LNP composition, and the LNP compositions described herein may be provided as a lipid composition. The lipid nanoparticle may be, for example, a microsphere (which includes unilamellar and multilamellar vesicles, e.g., lamellar phase lipid bilayers referred to as "liposomes," which in some embodiments are substantially spherical, and which in certain embodiments may include an aqueous core that contains, for example, a substantial portion of the RNA molecules), the dispersed phase of an emulsion, a micelle, or the internal phase of a suspension.

[0162] Described herein are LNP compositions comprising at least one ionizable lipid, or a salt thereof (e.g., a pharma- ceutically acceptable salt thereof), at least one helper lipid, at least one neutral lipid, and at least one polymeric lipid. In some embodiments, the LNP composition comprises at least one ionizable lipid, or a pharma- ceutically acceptable salt thereof, at least one neutral lipid, at least one helper lipid, and at least one PEG lipid. In some embodiments, the neutral lipid is DSPC or DPME. In some embodiments, the helper lipid is cholesterol, 5-heptadecylresorcinol, or cholesterol hemisuccinate.

[0163] In a preferred embodiment, the ionizable lipid is [ka] In a preferred embodiment, the neutral lipid is DSPC. In a preferred embodiment, the helper lipid is cholesterol. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000. In a particularly preferred embodiment, the ionizable lipid is [ka] where the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0164] In a preferred embodiment, the ionizable lipid is [ka] In a preferred embodiment, the neutral lipid is DSPC. In a preferred embodiment, the helper lipid is cholesterol. In a preferred embodiment, the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0165] In a particularly preferred embodiment, the ionizable lipid is [ka] where the neutral lipid is DSPC, the helper lipid is cholesterol, and the PEG lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000.

[0166] In an embodiment of the present disclosure, lipid compositions are provided that are expressed according to the respective molar ratios of the component lipids in the composition. In certain embodiments, the amount of ionizable lipid is about 25 mol% to about 60 mol%, the amount of neutral lipid is about 5 mol% to about 30 mol%, the amount of helper lipid is about 20 mol% to about 65 mol%, and the amount of PEG lipid is about 0.5 mol% to about 10 mol%. All mol% figures are given as a percentage of the lipid component of the lipid composition, or more specifically, the LNP composition. In some embodiments, the lipid mol% for a lipid component of a lipid is ±30%, ±25%, ±20%, ±15%, ±10%, ±5%, or ±2.5% of the specified mol%, nominal mol%, or actual mol%. In some embodiments, the lipid mol% for a lipid component of a lipid is ±4 mol%, ±3 mol%, ±2 mol%, ±1.5 mol%, ±1 mol%, ±0.5 mol%, ±0.25 mol%, or ±0.05 mol% of the specified mol%, nominal mol%, or actual mol% of the lipid component. In certain embodiments, the lipid mol% varies by less than 15%, less than 10%, less than 5%, less than 1%, or less than 0.5% from the specified mol%, nominal mol%, or actual mol% of the lipid. In some embodiments, the mol% number is based on the nominal concentration. As used herein, "nominal concentration" refers to a concentration based on the input amount of a substance that is combined to form a resulting composition. For example, if 100 mg of solute is added to 1 L of water, the nominal concentration is 100 mg / L. In some embodiments, the mol% number is based on the actual concentration, e.g., the concentration determined by analytical methods. In some embodiments, the actual concentration of lipids in the lipid component may be determined from a detection method, such as, for example, chromatography, such as liquid chromatography, followed by charged particle detection. In some embodiments, the actual concentration of lipids in the lipid component may be characterized by lipid analysis, AF4-MALS, NTA, and / or cryo-EM. All numbers in mol% are given as the percentage of lipids in the lipid component of the LNP composition.

[0167] In some embodiments, the aqueous component comprises a DNA cleaving agent. In some embodiments, the aqueous component comprises a polypeptide DNA cleaving agent, optionally in combination with a nucleic acid. In some embodiments, the aqueous component comprises a nucleic acid DNA cleaving agent, such as an RNA encoding a nuclease or nickase. In some embodiments, the aqueous component is a nucleic acid component. In some embodiments, the nucleic acid component comprises DNA and may be referred to as a DNA component. In some embodiments, the nucleic acid component comprises RNA. In some embodiments, the aqueous component, such as an RNA component, may comprise an mRNA, such as an mRNA encoding an RNA-guided DNA cleaving agent. In some embodiments, the RNA-guided DNA cleaving agent is a Cas nuclease. In certain embodiments, the aqueous component may comprise an mRNA encoding a Cas nuclease, such as Cas9. In certain embodiments, the DNA cleaving agent is a Cas nuclease mRNA. In certain embodiments, the DNA cleaving agent is a class 2 Cas nuclease mRNA. In certain embodiments, the DNA cleaving agent is a Cas9 nuclease mRNA. In certain embodiments, the aqueous component may comprise a modified RNA. In some embodiments, the aqueous component may comprise a guide RNA nucleic acid. In certain embodiments, the aqueous component may comprise a gRNA. In certain embodiments, the aqueous component may comprise a dgRNA. In certain embodiments, the aqueous component may comprise a modified gRNA. In some compositions that include an mRNA encoding an RNA-guided DNA cleaving agent, the composition further comprises a gRNA nucleic acid, such as a gRNA. In some embodiments, the aqueous component comprises an RNA-guided DNA cleaving agent and a gRNA. In some embodiments, the aqueous component comprises a Cas nuclease mRNA and a gRNA. In some embodiments, the aqueous component comprises a class 2 Cas nuclease mRNA and a gRNA.

[0168] In certain embodiments, lipid compositions, such as LNP compositions, may include an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, an ionizable lipid or a pharma- ceutically acceptable salt thereof, a helper lipid, optionally a neutral lipid, and a PEG lipid. In certain compositions comprising an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the helper lipid is cholesterol. In other compositions comprising an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, the neutral lipid is DSPC. In additional embodiments comprising an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease (e.g., Cas9), the PEG lipid is PEG2k-DMG. In certain compositions, the composition further comprises an mRNA encoding a Cas nuclease, such as a class 2 Cas nuclease, and an ionizable lipid or a pharma- ceutically acceptable salt thereof. In certain compositions, the composition further comprises a gRNA, such as a dgRNA or sgRNA.

[0169] In some embodiments, a lipid composition, such as an LNP composition, may include a gRNA. In certain embodiments, the composition may include an ionizable lipid or a pharma- ceutically acceptable salt thereof, a gRNA, a helper lipid, optionally a neutral lipid, and a PEG lipid. In certain LNP compositions that include a gRNA, the helper lipid is cholesterol. In some compositions that include a gRNA, the neutral lipid is DSPC. In additional embodiments that include a gRNA, the PEG lipid is PEG2k-DMG. In certain compositions, the gRNA is selected from a dgRNA and an sgRNA.

[0170] In certain embodiments, lipid compositions, such as LNP compositions, include mRNA encoding an RNA-guided DNA cleavage agent and gRNA, which may be sgRNA, in the aqueous component, and ionizable lipids in the lipid component. For example, LNP compositions may include ionizable lipids or pharma- ceutically acceptable salts thereof, mRNA encoding a Cas nuclease, gRNA, helper lipids, neutral lipids, and PEG lipids. In certain compositions that include mRNA and gRNA encoding a Cas nuclease, the helper lipid is cholesterol. In some compositions that include mRNA and gRNA encoding a Cas nuclease, the neutral lipid is DSPC. In additional embodiments that include mRNA and gRNA encoding a Cas nuclease, the PEG lipid is PEG2k-DMG.

[0171] In certain embodiments, a lipid composition, such as an LNP composition, includes an RNA-guided DNA cleaving agent, such as a class 2 Cas mRNA and at least one gRNA. In some embodiments, the gRNA is an sgRNA. In some embodiments, the RNA-guided DNA cleaving agent is a Cas9 mRNA. In certain embodiments, the LNP composition includes a gRNA and an RNA-guided DNA cleaving agent mRNA, such as a class 2 Cas nuclease mRNA, in a ratio of about 1:1 or about 1:2. In some embodiments, the weight ratio is about 25:1 to about 1:25, about 10:1 to about 1:10, about 8:1 to about 1:8, about 4:1 to about 1:4, about 2:1 to about 1:2, about 2:1 to 1:4, or about 1:1 to about 1:2, by weight.

[0172] The compositions and methods disclosed herein may include template nucleic acid, e.g., DNA template. Template nucleic acid may be delivered simultaneously with or separately from lipid composition (including as LNP composition) that includes ionizable lipid or its pharma-ceutically acceptable salt. In some embodiments, template nucleic acid may be single-stranded or double-stranded depending on the desired repair mechanism. Template may have homology region with target DNA and / or adjacent sequence of target DNA, e.g., within target DNA sequence.

[0173] In some embodiments, the LNP composition is formed by mixing an aqueous RNA solution with an organic solvent-based lipid solution. Suitable solutions or solvents may include or contain water, PBS, Tris buffer, NaCl, citrate buffer, acetate buffer, ethanol, chloroform, diethyl ether, cyclohexane, tetrahydrofuran, methanol, isopropanol. For example, the organic solvent may be 100% ethanol. A pharma- ceutically acceptable buffer, for example for in vivo administration of the LNP composition, may be used. In certain embodiments, a buffer is used to maintain the pH of the LNP-containing composition at pH 6.5 or higher. In certain embodiments, a buffer is used to maintain the pH of the LNP-containing composition at pH 7.0 or higher. In certain embodiments, the composition has a pH in the range of about 7.2 to about 7.7. In additional embodiments, the composition has a pH in the range of about 7.3 to about 7.7 or about 7.4 to about 7.6. The pH of the composition may be measured with a micro pH probe. In certain embodiments, a cryoprotectant is included in the composition. Non-limiting examples of cryoprotectants include sucrose, trehalose, glycerol, DMSO, and ethylene glycol. Exemplary compositions may include up to 10% cryoprotectant, such as sucrose. In certain embodiments, the composition may include tris saline sucrose (TSS). In certain embodiments, the composition is an LNP composition and may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% cryoprotectant. In certain embodiments, the composition is an LNP composition and may include about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% sucrose. In some embodiments, the composition includes a buffer. In some embodiments, the buffer may include phosphate buffer (PBS), Tris buffer, citrate buffer, and mixtures thereof. In certain exemplary embodiments, the buffer includes NaCl. In certain embodiments, the buffer lacks NaCl. Exemplary amounts of NaCl can range from about 20 mM to about 45 mM. Exemplary amounts of NaCl can range from about 40 mM to about 50 mM. In some embodiments, the amount of NaCl is about 45 mM.In some embodiments, the buffer is a Tris buffer. Exemplary amounts of Tris can range from about 20 mM to about 60 mM. Exemplary amounts of Tris can range from about 40 mM to about 60 mM. In some embodiments, the amount of Tris is about 50 mM. In some embodiments, the buffer comprises NaCl and Tris. Certain exemplary embodiments of the composition contain 5% sucrose and a Tris buffer containing 45 mM NaCl. In other exemplary embodiments, the composition contains sucrose in an amount of about 5 w / v%, about 45 mM NaCl, and about 50 mM Tris at pH 7.5. The salt, buffer, and cryoprotectant amounts can be modified to maintain the osmolality of the entire composition. For example, the final osmolality can be maintained below 450 mOsm / L. In further embodiments, the osmolality is between 350 and 250 mOsm / L. In certain embodiments, the final osmolality is 300+ / -20 mOsm / L or 310+ / -40 mOsm / L.

[0174] In some embodiments, microfluidic mixing, T-mixing, or cross mixing of an aqueous RNA solution and an organic solvent containing a lipid solution is used to generate the LNP composition. In certain aspects, the flow rate, junction size, junction geometry, junction shape, tube diameter, solution, and / or RNA and lipid concentration can vary. The LNP or LNP composition can be concentrated or purified, for example, by dialysis, centrifugal filters, tangential flow filtration, or chromatography. The LNP composition can be stored, for example, as a suspension, emulsion, or lyophilized powder. In some embodiments, the LNP composition is stored at 2-8°C, and in certain aspects, the LNP composition is stored at room temperature. In additional embodiments, the LNP composition is stored frozen, for example, at -20°C or -80°C. In other embodiments, the LNP composition is stored at a temperature ranging from about 0°C to about -80°C. The frozen LNP composition can be thawed, for example, on ice, at room temperature, or at 25°C, prior to use.

[0175] Preferred lipid compositions, for example LNP compositions, are biodegradable in that they do not accumulate to cytotoxic levels in vivo at therapeutically effective doses.In some embodiments, the composition does not induce a natural immune response that causes substantial adverse effects at therapeutic doses.In some embodiments, the composition provided herein does not induce toxicity at therapeutic doses.

[0176] In some embodiments, the concentration of LNP in the LNP composition is about 1-10 μg / mL, about 2-10 μg / mL, about 2.5-10 μg / mL, about 1-5 μg / mL, about 2-5 μg / mL, about 2.5-5 μg / mL, about 0.04 μg / mL, about 0.08 μg / mL, about 0.16 μg / mL, about 0.25 μg / mL, about 0.63 μg / mL, about 1.25 μg / mL, about 2.5 μg / mL, or about 5 μg / mL.

[0177] In some embodiments, dynamic light scattering ("DLS") can be used to characterize the polydispersity index (PDI) and size of the LNPs of the present disclosure. DLS measures the scattering of light resulting from exposing a sample to a light source. The PDI determined from a DLS measurement represents the particle size (approximate mean particle size) distribution in a population; a completely homogenous population would have a PDI of zero.

[0178] In some embodiments, the LNPs disclosed herein have a PDI of about 0.005 to about 0.75. In some embodiments, the LNPs disclosed herein have a PDI of about 0.005 to about 0.1. In some embodiments, the LNPs disclosed herein have a PDI of about 0.005 to about 0.09, about 0.005 to about 0.08, about 0.005 to about 0.07, or about 0.006 to about 0.05. In some embodiments, the LNPs have a PDI of about 0.01 to about 0.5. In some embodiments, the LNPs have a PDI of about zero to about 0.4. In some embodiments, the LNPs have a PDI of about zero to about 0.35. In some embodiments, the LNPs may have a PDI in the range of about zero to about 0.3. In some embodiments, the LNPs may have a PDI in the range of about zero to about 0.25. In some embodiments, the LNPs may have a PDI in the range of about zero to about 0.2. In some embodiments, the LNPs have a PDI of about zero to about 0.05. In some embodiments, the LNPs have a PDI of about zero to about 0.01. In some embodiments, the LNPs have a PDI of less than about 0.01, about 0.02, about 0.05, about 0.08, about 0.1, about 0.15, about 0.2, or about 0.4.

[0179] The size of LNPs can be measured by various analytical methods known in the art. In some embodiments, the size of LNPs can be measured using asymmetric flow field separation-multi-angle light scattering (AF4-MALS). In certain embodiments, the size of LNPs can be measured by separating particles in a composition by hydrodynamic radius, and then measuring the molecular weight, hydrodynamic radius, and root mean square radius of the fractionated particles. In some embodiments, the size and particle concentration of LNPs can be measured by Nanoparticle Tracking Analysis (NTA, Malvern Nanosight). In certain embodiments, LNP samples are appropriately diluted and injected onto a microscope slide. As the particles are slowly injected through the field of view, a camera records the scattered light. After a video is taken, Nanoparticle Tracking Analysis processes the video by tracking pixels and calculates the diffusion coefficient. This diffusion coefficient can be converted to the hydrodynamic radius of the particle. Such methods can also count the number of particles to obtain the particle concentration. In some embodiments, the size, morphology, and structural characteristics of LNPs may be determined by cryo-electron microscopy ("cryo-EM").

[0180] The LNPs of the LNP compositions disclosed herein are, for example, about 1 to about 250 nm in size (e.g., Z-average diameter). In some embodiments, the LNPs are about 10 to about 200 nm in size. In further embodiments, the LNPs are about 20 to about 150 nm in size. In some embodiments, the LNPs are about 50 to about 150 nm in size or about 70 to 130 nm in size. In some embodiments, the LNPs are about 50 to about 100 nm in size. In some embodiments, the LNPs are about 50 to about 120 nm in size. In some embodiments, the LNPs are about 60 to about 100 nm in size. In some embodiments, the LNPs are about 75 to about 150 nm in size. In some embodiments, the LNPs are about 75 to about 120 nm in size. In some embodiments, the LNPs are about 75 to about 100 nm in size. In some embodiments, the LNPs are about 50 to about 145 nm, about 50 to about 120 nm, about 50 to about 120 nm, about 50 to about 115 nm, about 50 to about 100 nm, about 60 to about 145 nm, about 60 to about 120 nm, about 60 to about 115 nm, or about 60 to about 100 nm in size. In some embodiments, the LNPs are less than about 145 nm, less than about 120 nm, less than about 115 nm, or less than about 100 nm in size. In some embodiments, the LNPs are greater than about 50 nm or greater than about 60 nm in size. In some embodiments, the particle size is the Z-average particle size. In some embodiments, the particle size is the number-average particle size. In some embodiments, the particle size is the size of an individual LNP. Unless otherwise indicated, all sizes referred to herein are the average size (diameter) of fully formed nanoparticles as measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. Nanoparticle samples are diluted in phosphate buffered saline (PBS) to give a count rate of approximately 200-400 kcps.

[0181] The LNPs can be about 1 to about 250 nm in size (e.g., Z-average diameter). In some embodiments, the LNPs are about 10 to about 200 nm in size. In further embodiments, the LNPs are about 20 to about 150 nm in size. In some embodiments, the LNPs are about 50 to about 150 nm in size or about 70 to 130 nm in size. In some embodiments, the LNPs are about 50 to about 100 nm in size. In some embodiments, the LNPs are about 50 to about 120 nm in size. In some embodiments, the LNPs are about 60 to about 100 nm in size. In some embodiments, the LNPs are about 75 to about 150 nm in size. In some embodiments, the LNPs are about 75 to about 120 nm in size. In some embodiments, the LNPs are about 75 to about 100 nm in size. In some embodiments, the LNPs are about 40 to about 125 nm, about 40 to about 110 nm, about 40 to about 100 nm, about 40 to about 90 nm in size. In some embodiments, the particle size is the Z-average particle size. In some embodiments, the particle size is the number-average particle size. In some embodiments, the particle size is the size of an individual LNP. Unless otherwise indicated, all sizes referred to herein are the average size (diameter) of fully formed nanoparticles as measured by dynamic light scattering on a Malvern Zetasizer or Wyatt NanoStar. Nanoparticle samples are diluted in phosphate buffered saline (PBS) to obtain a count rate of approximately 200-400 kcps.

[0182] In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 50% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 50% to about 95%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 70% to about 90%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 90% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 75% to about 95%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 90% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 95% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 98% to about 100%. In some embodiments, the LNP compositions are formed with an average encapsulation rate ranging from about 99% to about 100%.

[0183] cargo The cargo delivered via the LNP composition can be a DNA cleavage agent, such as an RNA-guided DNA cleavage agent. In certain embodiments, the cargo is or comprises one or more DNA cleavage agents, such as an mRNA, a gRNA, an expression vector, an RNA-guided DNA cleavage agent, such as a CRISPR Cas nuclease or an mRNA encoding a nuclease, optionally combined with a guide RNA. The DNA cleavage agents listed above are merely exemplary and are not intended to be limiting. Such compounds may be purified or partially purified, may be naturally occurring or synthetic, and may be chemically modified.

[0184] The cargo delivered via the LNP composition can be RNA, such as an mRNA molecule encoding a DNA cleavage agent. Examples include mRNA for expressing an RNA-guided DNA cleavage agent or a protein such as a Cas nuclease. LNP compositions are provided that include a Cas nuclease mRNA, for example, a class 2 Cas nuclease mRNA that allows expression of a class 2 Cas nuclease, such as a Cas9 or Cpf1 (also called Cas12a) protein, in cells. Additionally, the cargo can contain one or more gRNAs or a nucleic acid encoding a gRNA. Template nucleic acids, for example for repair or recombination, can also be used in the methods described herein. In a subembodiment, the cargo includes an mRNA encoding Streptococcus pyogenes Cas9 and, optionally, an S. pyogenes gRNA. In a subembodiment, the cargo includes an mRNA encoding Neisseria meningitidis Cas9 and, optionally, an Nme (Neisseria meningitidis) gRNA.

[0185] "mRNA" refers to a polynucleotide and 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 contain a phosphate-sugar backbone, such as ribose residues or analogs thereof, e.g., 2'-methoxyribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2'-methoxyribose residues, or combinations thereof. In general, an mRNA does not contain a substantial amount of thymidine residues (e.g., 0 residues or fewer than 30, 20, 10, 5, 4, 3, or 2 thymidine residues, or a thymidine content of less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%). An mRNA can contain modified uridines at some or all of its uridine positions.

[0186] DNA cutting substance In some embodiments, the composition or method includes a DNA cleaving agent, such as a protein or RNA component or a nucleic acid encoding the same. As used herein, the term DNA cleaving agent is any component of a genome editing system (or gene editing system) that is necessary or useful for generating an edit in the genome of a cell. In some embodiments, the present disclosure provides a method for delivering a DNA cleaving agent of a genome editing system (e.g., a zinc finger nuclease system, a TALEN system, a meganuclease system, or a CRISPR / Cas system) to a cell (or a population of cells). DNA cleaving agents 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, and nucleic acids, such as RNAs, that encode them. DNA cleaving agents, e.g., nucleases, can optionally modify the genome of a cell without cleavage or nickases of nucleic acids. DNA cleaving nuclease or nickase agents can be encoded by mRNA. Such nucleases and nickases include, for example, RNA-guided DNA cleaving agents, and CRISPR / Cas components. DNA cleavage agents include fusion proteins, such as, for example, a nickase fused to an effector domain, such as an editor domain. DNA cleavage agents include any component necessary or useful for achieving genome editing to introduce DNA cleavage, such as, for example, a guide RNA, sgRNA, dgRNA, etc.

[0187] A variety of suitable gene editing systems, including DNA cleavage agents for use with the DNA-PKI compounds described herein, include, but are not limited to, CRISPR / Cas systems, zinc finger nuclease (ZFN) systems, and transcription activator-like effector nuclease (TALEN) systems.Generally, DNA cleavage agents 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, TALENs, or through the use of CRISPR / Cas systems with engineered guide RNAs to induce specific cleavage or nicking of target DNA sequences. Additionally, targeted nucleases based on the Argonaute system (e.g., known as "TtAgo," derived from T. thermophilus, see Swarts et al (2014) Nature 507(7491):258-261) are being developed and may also have the potential to be used in genome editing and gene therapy.

[0188] In certain embodiments, the disclosed compositions include one or more DNA modifying agents, such as DNA cleavage agents. A variety of DNA modifying agents can be included in the LNP compositions described herein. For example, DNA modifying agents include nucleases (both sequence specific and non-specific), topoisomerases, methylases, acetylases, chemicals, pharmaceuticals, and other agents. In some embodiments, proteins that bind to a given DNA sequence or set of sequences can be used to induce DNA modifications, such as strand breaks. Proteins can be used to induce DNA modifications, such as strand breaks, such as those that result from radioactive decay. 125Protein-DNA crosslinks can be modified by a number of means, such as the incorporation of I or by modifying crosslinking reagents such as 4-azidophenacyl bromide, which forms crosslinks with DNA upon exposure to UV light. Such protein-DNA crosslinks can then be converted to double-stranded DNA breaks by treatment with piperidine. Yet another approach to DNA modification involves antibodies raised against specific proteins bound at one or more DNA sites, such as transcription factors or chromatin structural proteins, and used to isolate DNA from nucleoprotein complexes.

[0189] In certain embodiments, the disclosed compositions include one or more DNA cleavage agents. DNA cleavage agents include technologies such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), mito-TALEN, and meganuclease systems. TALEN and ZFN technologies use a strategy of tethering an endonuclease catalytic domain to a modular DNA binding protein to induce targeted DNA double-strand breaks (DSBs) in specific genomic loci. Additional DNA cleavage agents include small interfering RNA, microRNA, anti-microRNA, antagonists, short hairpin RNA, and aptamers (based on RNA, DNA, or peptides (including affimers)).

[0190] In some embodiments, the gene editing system is a TALEN system. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cleave specific sequences of DNA. They are created by fusing the DNA binding domain of a TAL effector 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 to promote DNA cleavage at specific locations (see, e.g., Boch, 2011, Nature Biotech). Restriction enzymes can be introduced into cells for use in in situ gene editing or genome editing, a technique known as genome editing with engineered nucleases. Such methods and compositions for use therein are known in the art. See, e.g., WO2019147805, WO2014040370, WO2018073393, the contents of which are hereby incorporated in their entirety.

[0191] In some embodiments, the gene editing system is a zinc finger system. Zinc finger nucleases (ZFNs) are artificial restriction enzymes that are generated by fusing the DNA binding domain of zinc fingers to a DNA cleavage domain. The zinc finger domain targets a specific desired DNA sequence, and can be engineered to allow zinc finger nucleases to target unique sequences within complex genomes. ZFNs typically use a non-specific cleavage domain from type II restriction endonuclease FokI as the cleavage domain. The cleavage is repaired by endogenous DNA repair mechanisms, allowing ZFNs to precisely alter the genome of higher organisms. Such methods and compositions for use therein are known in the art. See, for example, WO2011091324, the contents of which are hereby incorporated in their entirety.

[0192] In preferred embodiments, the disclosed compositions comprise an mRNA encoding a DNA cleaving agent, such as a Cas nuclease. In certain embodiments, the disclosed compositions comprise an mRNA encoding a class 2 Cas nuclease, such as S. pyogenes Cas9.

[0193] As used herein, "RNA-guided DNA cleavage agent" refers to a polypeptide or polypeptide complex having DNA binding and cleavage activity, or a DNA-binding subunit of such a complex, where the DNA-binding activity is sequence-specific and dependent on the RNA sequence capable of inducing ssDNA or dsDNA cleavage. Exemplary RNA-guided DNA cleavage agents include Cas cleavase / nickases and their inactivated forms ("dCas DNA cleavage agents"). As used herein, "Cas nuclease" encompasses Cas cleavase, Cas nickase, and dCas DNA cleavage agents. Cas cleavase / nickases and dCas DNA cleavage agents include the Csm complex or Cmr complex of type III CRISPR system, their subunits Cas10, Csm1, or Cmr2, the Cascade complex of type I CRISPR system, its Cas3 subunit, and class 2 Cas nuclease. As used herein, "class 2 Cas nuclease" is a single-chain polypeptide with RNA-guided DNA cleavage activity. Class 2 Cas nucleases include Class 2 Cas cleavase / nickases that further have RNA-guided DNA cleavase or nickase activity (e.g., H840A, D10A, or N863A variants), and Class 2 dCas DNA cleavage agents in which the cleavase / nickase activity has been inactivated. Class 2 Cas nucleases that may be used with the LNP compositions described herein include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modified versions thereof. The Cpf1 protein (Zetsche et al., Cell, 163:1-13 (2015)) is homologous to Cas9 and contains a RuvC-like nuclease domain.The Cpf1 sequence of Zetsche is incorporated by reference in its entirety. See, e.g., Tables 2 and 4 of Zetsche. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11):722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0194] Non-limiting exemplary species from which Cas nucleases may be derived include Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Listeria innocua, Lactobacillus gasseri, Francisella novicida, Wolinella succinogenes, Sutterella wadsworthensis, Gammaproteobacterium, Neisseria meningitidis, Campylobacter jejuni, Pasteurella multocida, Fibrobacter succinogene, Rhodospirillum rubrum, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, Alicyclobacillus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus buchneri, Treponema denticola, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp.、Crocosphaera watsonii、Cyanothece sp.、Microcystis aeruginosa、Synechococcus sp., Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor becscii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Ktedonobacter racemifer, Methanohlobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, Streptococcus pasteurianus, Neisseria cinerea, Campylobacter lari, Parvibaculum lavamentivorans, Corynebacterium diphtheria, Acidaminococcus sp.、Lachnospiraceae bacterium ND2006、、、Acaryochloris marina is included。.

[0195] In some embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus pyogenes. In other embodiments, the Cas nuclease is a Cas9 nuclease from Streptococcus thermophilus. In still other embodiments, the Cas nuclease is a Cas9 nuclease from Neisseria meningitidis. In some embodiments, the Cas nuclease is a Cas9 nuclease from Staphylococcus aureus. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella novicida. In other embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus sp. In still other embodiments, the Cas nuclease is a Cpf1 nuclease from Lachnospiraceae bacterium ND2006. In further embodiments, the Cas nuclease is a Cpf1 nuclease from Francisella tularensis, Lachnospiraceae bacterium, Butyrivibrio proteoclasticus, Peregrinibacteria bacterium, Parcubacteria bacterium, Smithella, Acidaminococcus, Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi, Leptospira inadai, Porphyromonas crevioricanis, Prevotella disiens, or Porphyromonas macacae. In some embodiments, the Cas nuclease is a Cpf1 nuclease from Acidaminococcus or Lachnospiraceae.

[0196] 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 multiple RuvC domains and / or multiple HNH domains. In some embodiments, the Cas9 nuclease is wild-type Cas9. In some embodiments, Cas9 can induce double-stranded breaks in the target DNA. In other embodiments, the Cas nuclease can cleave dsDNA or a single strand of dsDNA, and may not have DNA cleavage or nickase activity. In some embodiments, two nickases are combined to generate dsDNA cleavage.

[0197] In some embodiments, a Cas nuclease, such as a chimeric Cas nuclease, is used in which one domain or region of a protein is fused to a portion of a different protein, e.g., a heterologous polypeptide, and optionally includes a linker polypeptide between the Cas nuclease portion and the heterologous functional domain portion of the chimeric Cas9. In some embodiments, the Cas nuclease domain can be fused, e.g., via a linker, to a domain from a different nuclease, such as Fok1. In some embodiments, the Cas nuclease can be a nickase or a modified nuclease, such as dCas9.

[0198] 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.

[0199] In some embodiments, the RNA-guided DNA cleaving agent has single-stranded nickase activity, i.e., it can cleave one DNA strand to produce a single-stranded break, also known as a "nick." In some embodiments, the RNA-guided DNA cleaving agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cleaves one strand of a DNA double helix but not the other. In some embodiments, the Cas nickase is a variant of a Cas nuclease (e.g., the Cas nucleases described above) in which the endonucleolytic activity site has been inactivated, for example, by one or more alterations (e.g., point mutations) in the catalytic domain. For a discussion of Cas nickases and exemplary catalytic domain modifications, see, for example, U.S. Patent No. 8,889,356. In some embodiments, the Cas nickase, such as the Cas9 nickase, has an inactivated RuvC domain or HNH domain.

[0200] In some embodiments, the RNA-guided DNA cleavage agent is modified to contain only one functional nuclease domain. For example, the cleavage agent protein can be modified to reduce its nucleic acid cleavage activity by mutating or completely or partially deleting one of the nuclease domains. In some embodiments, a nickase with a RuvC domain with 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 with reduced activity is used. In some embodiments, a nickase with an inactive HNH domain is used.

[0201] In some embodiments, conserved amino acids in the Cas protein nuclease domain are substituted to reduce or alter nuclease activity. In some embodiments, the Cas nuclease may include an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3):759-771. In some embodiments, the Cas nuclease may include an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015). Further exemplary amino acid substitutions include D917A, E1006A, and D1255A (based on the Francisella novicida U112 Cpf1 (FnCpf1) sequence (UniProtKB-A0Q7Q2(CPF1_FRATN)).

[0202] In some embodiments, the mRNA encoding the nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNA guides the nickase to the target sequence, where it introduces a DSB by nicking the opposite strand of the target sequence (i.e., double nicking). In some embodiments, the use of double nicking can improve specificity and reduce off-target effects. In some embodiments, the nickase is used with two separate guide RNAs that target opposite strands of DNA to generate a double nick in the target DNA. In some embodiments, the nickase is used with two separate guide RNAs that are selected to be adjacent to generate a double nick in the target DNA. In some embodiments, the nickase, such as the Cas9 nickase, is fused to a heterologous functional domain, such as a deaminase polypeptide.

[0203] In some embodiments, the RNA-guided DNA cleavage agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA cleavage agent comprises a dCas DNA-binding polypeptide. The dCas polypeptide has DNA-binding activity but essentially lacks catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA cleavage agent or dCas DNA-binding polypeptide lacking cleavase and nickase activity is a variant of a Cas nuclease (e.g., the Cas nuclease described above), whose endonuclease decomposition active site is inactivated, for example, by one or more changes (e.g., point mutations) in its catalytic domain. See, for example, US2014 / 0186958A1, US2015 / 0166980A1.

[0204] In some embodiments, the RNA-guided DNA cleaving agent comprises one or more heterologous functional domains (eg, is or comprises a fusion polypeptide).

[0205] In some embodiments, the heterologous functional domain can facilitate the transport of the RNA-guided DNA cleavage agent into the nucleus of the cell. For example, the heterologous functional domain can be a nuclear localization signal (NLS).

[0206] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA cleavage agent. In some embodiments, the half-life of the RNA-guided DNA cleavage agent may be extended. In some embodiments, the half-life of the RNA-guided DNA cleavage agent may be shortened. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA cleavage agent. In some embodiments, the heterologous functional domain may be capable of decreasing the stability of the RNA-guided DNA cleavage agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may include a PEST sequence. In some embodiments, the RNA-guided DNA cleavage agent may be modified by the addition of ubiquitin or polyubiquitin chains. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include small ubiquitin-like modifiers (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon stimulated gene-15 (ISG15)), ubiquitin-related modifier 1 (URM1), neuronal-precursor-cell expressed developmentally downregulated protein-8 (NEDD8, also known as Rub1 in S. cerevisiae), human leukocyte antigen F-related (FAT10), autophagy-8 (ATG8) and autophagy-12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0207] In some embodiments, the heterologous functional domain can 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 can 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 protein (mOrange, mKO, Kusabira-Orange, monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag and / or an epitope tag.Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin-binding protein (CBP), maltose-binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0208] In additional embodiments, the heterologous functional domain may direct the RNA-guided DNA cleaving agent to a particular organelle, cell type, tissue, or organ. In some embodiments, the heterologous functional domain may direct the RNA-guided DNA cleaving agent to mitochondria.

[0209] In further embodiments, the heterologous functional domain can be an effector domain, such as an editor domain. When an RNA-guided DNA cleavage 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 transcription activation domain, or a transcription repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, for example, U.S. Patent No. 9,023,649. In some embodiments, the heterologous functional domain is a transcription activator or a transcription repressor. See, e.g., 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, in essence, the RNA-guided DNA cleavage agent becomes a transcription factor that can be oriented to bind to a desired target sequence using a guide RNA.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 a specific modification, such as a deaminase domain. See, for example, WO2015 / 089406, US2016 / 0304846. The nucleic acid editing domains, deaminase domains, and Cas9 variants described in WO2015 / 089406 and US2016 / 0304846 are incorporated herein by reference.

[0210] In some embodiments, the RNA-guided DNA cleavage agent or Cas nickase, such as Cas9 nickase, comprises an APOBEC deaminase. In some embodiments, the APOBEC deaminase is an APOBEC3 deaminase, such as APOBEC3A (A3A). In some embodiments, the A3A is human A3A. In some embodiments, the A3A is wild-type A3A.

[0211] In some embodiments, the RNA-guided DNA cleavage 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. In some embodiments, the editor is provided with a uracil glycosylase inhibitor ("UGI"). In some embodiments, the editor is fused to a UGI. In some embodiments, the mRNA encoding the editor and the mRNA encoding the UGI are formulated together in the LNP composition. In other embodiments, the editor and the UGI are provided in separate LNP compositions.

[0212] The RNA-guided DNA cleavage agent may include at least one domain that interacts with a guide RNA ("gRNA"). In addition, it may be directed to a target sequence by the gRNA. In a Class 2 Cas nuclease system, the gRNA interacts with the nuclease and the target sequence to guide binding to the target sequence. In some embodiments, the gRNA provides specificity for target cleavage, and the nuclease may be versatile and pair with different gRNAs to cleave different target sequences. Class 2 Cas nucleases may pair with gRNA scaffold structures of the types, orthologs, and exemplary species listed above.

[0213] As used herein, "ribonucleoprotein" (RNP) or "RNP complex" refers to a gRNA together with an RNA-guided DNA cleavage agent (e.g., Cas9), such as a Cas nuclease, e.g., a Cas cleavase, a Cas nickase, or a dCas DNA cleavage agent, such as a dCas9 fusion protein. In some embodiments, the gRNA guides an RNA-guided DNA cleavage agent, such as Cas9, to a target sequence to which the gRNA hybridizes and the cleavage agent binds, and if the cleavage agent is a cleavase or nickase, cleavage or nicking may occur after binding.

[0214] In some embodiments of the present disclosure, the cargo for the LNP composition includes at least one gRNA that includes a guide sequence that directs an RNA-guided DNA cleavage agent, which may be a nuclease (e.g., a Cas nuclease, such as Cas9), to a target DNA. 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 to provide specificity for cleavage by such a nuclease. In some embodiments, the gRNA and the Cas nuclease may form a ribonucleoprotein (RNP), e.g., a CRISPR / Cas complex, such as a CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a type II CRISPR / Cas9 complex. In some embodiments, the CRISPR / Cas complex may be a type V CRISPR / Cas complex, such as a Cpf1 / gRNA complex. The Cas nuclease and the corresponding gRNA may be paired. The gRNA scaffold structure that pairs with each Class 2 Cas nuclease varies depending on the individual CRISPR / Cas system.

[0215] "Guide RNA", "gRNA" and simply "guide" are used interchangeably herein and refer to the corresponding guide nucleic acid for an RNA-guided DNA cleavage agent. Guide RNA may include modified RNA as described herein. gRNA may be either crRNA (also known as CRISPR RNA) or a combination of crRNA and trRNA (also known as tracrRNA). crRNA and trRNA may be associated as a single RNA molecule (single guide RNA, sgRNA) or may be two or more separate RNA molecules, optionally covalently linked (dual guide RNA, dgRNA). "Guide RNA" or "gRNA" refer to each type. trRNA may be a naturally occurring sequence or a trRNA sequence with a modification or variation compared to the naturally occurring sequence.

[0216] In some embodiments, the mRNA encoding the RNA-guided DNA cleavage agent is formulated in a first LNP composition and the gRNA nucleic acid is formulated in a second LNP composition. In some embodiments, the first and second 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 preincubation step. In other embodiments, the first and second lipid nucleic acid assembly compositions are preincubated separately.

[0217] In certain embodiments, the compositions and methods described herein involve modified RNA. In some embodiments, the compositions and methods described herein involve guide RNA nucleic acids. In certain embodiments, the compositions and methods described herein involve gRNA, such as dgRNA or modified gRNA. In some compositions that include an mRNA encoding an RNA-guided DNA cleaving agent, the composition further includes a gRNA nucleic acid, such as a gRNA. In some embodiments, the compositions and methods described herein involve an RNA-guided DNA cleaving agent and a gRNA. In some embodiments, the compositions and methods described herein involve a Cas nuclease mRNA and a gRNA, such as a class 2 Cas nuclease mRNA and a gRNA.

[0218] In some embodiments, the cargo may comprise a DNA molecule. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a crRNA. In some embodiments, the nucleotide sequence encoding the crRNA comprises a targeting sequence flanked by all or part of a repeat sequence from a naturally occurring CRISPR / Cas system. In some embodiments, the nucleic acid may comprise a nucleotide sequence encoding a tracrRNA. In certain 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 a sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cas9 nuclease sgRNA. In some embodiments, the gRNA nucleic acid encodes a Cpf1 nuclease sgRNA.

[0219] The nucleotide sequence encoding the guide RNA may be operably linked to at least one transcriptional or regulatory control sequence, such as a promoter, a 3'UTR, or a 5'UTR. In one example, the promoter may be a tRNA promoter, such as a tRNALys3, or a tRNA chimera. See Mefferd et al., RNA. 2015 21:1683-9, Scherer et al., Nucleic Acids Res. 2007 35:2620-2628. In some embodiments, the promoter may be recognized by RNA polymerase III (Pol III). Non-limiting examples of Pol III promoters also include the U6 promoter and the H1 promoter. In some embodiments, the nucleotide sequence encoding the guide RNA may be operably linked to a mouse or human U6 promoter. In some embodiments, the gRNA nucleic acid is a modified nucleic acid. In some embodiments, the gRNA nucleic acid includes modified nucleosides or modified 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 of the nucleic acid. In other embodiments, the gRNA nucleic acid includes a 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, e.g., FAM, ROX, TAMRA, and AlexaFluor.

[0220] As used herein, "guide sequence" refers to a sequence in a gRNA that is complementary to a target sequence and functions to guide the gRNA to the target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA cleavage agent. A "guide sequence" may also be referred to as a "targeting sequence" or a "spacer sequence." A guide sequence may be, for example, 20 base pairs in length for Streptococcus pyogenes (i.e., Spy Cas9) and related Cas9 homologs / orthologues. Shorter or longer sequences, for example, 15, 16, 17, 18, 19, 21, 22, 23, 24, or 25 nucleotides in length, may 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 or at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the guide sequence and the target region may be 100% complementary or identical over a region of at least 15, 16, 17, 18, 19, or 20 contiguous nucleotides. In other embodiments, the guide sequence and the target region may contain at least one mismatch. For example, the guide sequence and the target sequence may contain one, two, three, or four mismatches, in which case the total length of the target sequence is at least 17, 18, 19, 20, or more base pairs. In some embodiments, the guide sequence and the target region may contain one to four mismatches, in which case the guide sequence includes at least 17, 18, 19, 20, or more nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches, in which case the guide sequence comprises 20 nucleotides.

[0221] In certain embodiments, multiple LNP compositions may be used cooperatively and / or for separate purposes. In some embodiments, cells may be contacted with a first and a second LNP composition described herein. In some embodiments, the first and second LNP compositions each independently comprise, for example, one or more of mRNA, gRNA, and gRNA nucleic acid. In some embodiments, the first and second LNP compositions are administered simultaneously. In some embodiments, the first and second LNP compositions are administered sequentially.

[0222] In some embodiments, methods are provided for generating multiple genome edits in a cell, sometimes referred to herein and elsewhere as "multiplexing" or "multiplex gene editing" or "multiplex genome editing." The ability to engineer multiple attributes in a single cell depends on the ability to efficiently perform edits in multiple target genes, such as knockouts and in locus insertions, while retaining viability and the desired cellular phenotype. In some embodiments, the method includes culturing a cell in vitro, contacting the cell with two or more lipid nucleic acid assembly compositions, each lipid nucleic acid assembly composition comprising a nucleic acid genome editing tool capable of editing a target site, and propagating the cell in vitro. The method results in a cell with multiple genome edits, where the genome edits are different. In certain embodiments, the first LNP composition comprises a first gRNA and the second LNP composition comprises a second gRNA, where the first gRNA and the second gRNA comprise different guide sequences complementary to different targets. In such embodiments, the LNP compositions may enable multiplex gene editing.

[0223] The target sequence of an RNA-guided DNA cleavage protein, such as a Cas protein, includes both the positive and negative strands of genomic DNA (i.e., a given sequence and its reverse complementary strand), since the nucleic acid substrate of the Cas protein is a double-stranded nucleic acid. Thus, when a guide sequence is said to be "complementary to a target sequence," it should be understood that the guide sequence can guide the gRNA to bind to the reverse complementary strand of the target sequence. Thus, in some embodiments, when the guide sequence binds to the reverse complementary strand of the target sequence, the guide sequence is identical to a particular nucleotide of the target sequence (e.g., the target sequence without the PAM), except for the substitution of T for U in the guide sequence.

[0224] The length of the targeting sequence may vary depending on the CRISPR / Cas system and components used. For example, different class 2 Cas nucleases from different 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 targeting sequence length is 0, 1, 2, 3, 4, or 5 nucleotides longer or shorter than the guide sequence of a naturally occurring CRISPR / Cas system. In certain embodiments, the Cas nuclease and the gRNA scaffold are derived from the same CRISPR / Cas system. In some embodiments, the targeting sequence may comprise or consist of 18-24 nucleotides. In some embodiments, the targeting sequence may comprise or consist of 19-21 nucleotides, hi some embodiments, the targeting sequence may comprise or consist of 20 nucleotides.

[0225] 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 certain embodiments, the gRNA comprises a crRNA and a tracrRNA sufficient to form an active complex with the Cas9 protein and mediate RNA-guided DNA cleavage. In certain embodiments, the gRNA comprises a crRNA sufficient to form an active complex with the Cpf1 protein and mediate RNA-guided DNA cleavage. See Zetsche 2015.

[0226] Certain embodiments also provide nucleic acids, e.g., expression cassettes, encoding the gRNAs described herein. "Guide RNA nucleic acid" is used herein to refer to gRNAs (e.g., sgRNAs or dgRNAs) and gRNA expression cassettes, which are nucleic acids that encode one or more gRNAs.

[0227] modified RNA In certain embodiments, a lipid composition, such as an LNP composition, comprises a modified nucleic acid, such as a modified RNA.

[0228] Modified nucleosides or nucleotides may be present in an RNA, e.g., a gRNA or an mRNA. A gRNA or an mRNA that includes one or more modified nucleosides or nucleotides is referred to, for example, as a "modified" RNA, denoting the presence of one or more non-naturally occurring and / or naturally occurring components or structures used in place of or in addition to the standard residues A, G, C, and U. In some embodiments, the modified RNA is synthesized with non-standard nucleosides or nucleotides, and is referred to herein as "modified."

[0229] Modified nucleosides and nucleotides can include one or more of the following: (i) an alteration, e.g., replacement, of one or both of the non-linked oxygens of the phosphate and / or one or more of the linked oxygens of the phosphate in the phosphodiester backbone linkages (exemplary backbone modifications); (ii) an alteration, e.g., replacement, of a component of the ribose sugar, e.g., the hydroxyl at the 2' position of the ribose sugar (exemplary sugar modifications); (iii) a substantial replacement of the phosphate moiety with a "dephospho" linker (exemplary backbone modifications); (iv) a modification or replacement of a naturally occurring nucleobase, such as 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 a polynucleotide, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, cap or linker (such 3' or 5' cap modifications can include sugar and / or backbone modifications); and (vii) a modification or replacement of the sugar (exemplary sugar modifications). Certain embodiments include a 5'-end modification to the mRNA, gRNA, or nucleic acid. Certain embodiments include a modification to the mRNA, gRNA, or nucleic acid. Certain embodiments include a 3'-end modification to the mRNA, gRNA, or nucleic acid. The modified RNA can contain modifications at the 5'-end and the 3'-end. The modified RNA can contain one or more modified residues at a position other than the end. In certain embodiments, the gRNA includes at least one modified residue. In certain embodiments, the mRNA includes at least one modified residue. In certain embodiments, the modified gRNA includes a modification in one or more of the first five nucleotides at the 5'-end. In certain embodiments, the modified gRNA includes a modification in one or more of the first five nucleotides at the 5'-end. The LNP composition of claim 52 or 53, wherein the modified gRNA includes a modification in one or more of the last five nucleotides at the 3'-end.

[0230] Unmodified nucleic acids may be susceptible to degradation, for example, by intracellular nucleases or nucleases found in serum. For example, nucleases can hydrolyze phosphodiester bonds of nucleic acids. Thus, in one aspect, the RNAs (e.g., mRNA, gRNA) described herein can contain one or more modified nucleosides or nucleotides to introduce stability against, for example, intracellular or serum nucleases. In some embodiments, the modified RNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term "innate immune response" includes the cellular response to exogenous nucleic acids, such as single-stranded nucleic acids, involving the expression and release of cytokines, particularly interferons, and the induction of cell death.

[0231] Thus, in some embodiments, the RNA or nucleic acid comprises at least one modification that confers improved or enhanced stability to the nucleic acid, such as improved resistance to digestion by nucleases in vivo. As used herein, the terms "modification" and "modified" as they relate to the nucleic acids provided herein, preferably include at least one change that enhances stability and renders the RNA or nucleic acid more stable (e.g., resistant to digestion by nucleases) than a wild-type or naturally occurring form of the RNA or nucleic acid. As used herein, the terms "stable" and "stability," and such terms, as they relate to the nucleic acids described herein, particularly RNA, refer to increased or enhanced resistance to degradation, such as by nucleases (i.e., endonucleases or exonucleases) that are typically capable of degrading such RNA. Improved stability can include, for example, less susceptibility to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within a target cell or tissue, thereby prolonging or enhancing the residence of such RNA or nucleic acid in a target cell, tissue, subject, and / or cytoplasm. The stabilized RNA or nucleic acid molecules provided herein exhibit a longer half-life compared to naturally occurring unmodified RNA or nucleic acid molecules (e.g., wild-type versions of the molecules). As the terms "modification" and "modification" relate to the mRNA of the LNP compositions disclosed herein, such terms also contemplate changes that improve or enhance translation of the mRNA nucleic acid, such as, for example, the inclusion of a sequence that functions in the initiation of protein translation (e.g., a Kozak consensus sequence). (Kozak, M., Nucleic Acids Res 15(20):8125-48(1987)).

[0232] In some embodiments, RNA or nucleic acid is chemically or biologically modified to make it more stable.Exemplary modifications to RNA or nucleic acid include removing bases (e.g., by deleting a nucleotide or replacing it with another nucleotide) or modifying bases, e.g., chemically modifying bases.As used herein, the term "chemical modification" includes modification that introduces chemistry different from that found in naturally occurring RNA or nucleic acid, e.g., covalent modification such as introducing modified nucleotides (e.g., including nucleotide analogs or pendant groups that are not naturally found in such RNA (e.g., deoxynucleosides) or nucleic acid molecules).

[0233] In some embodiments of backbone modification, the phosphate group of the modified residue can be modified by replacing one or more of the oxygens with different substituents.Furthermore, modified residues, such as modified residues present in modified nucleic acids, can include largely replacing unmodified phosphate moieties with modified phosphate groups as described herein.In some embodiments, backbone modification of phosphate backbone can include changes that result in either uncharged linkers or charged linkers with asymmetric charge distribution.

[0234] Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphates, hydrogen phosphonates, phosphoramidates, alkyl or aryl phosphonates, and phosphotriesters. The phosphorus atom of an unmodified phosphate group is achiral. However, the phosphorus atom can be made chiral by replacing one of the non-bridging oxygens with the atoms or groups listed above. The asymmetric phosphorus atom can have either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). The backbone can also be modified by replacing the bridging oxygens (i.e., the oxygens that link the phosphate group to the nucleoside) with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates). Replacement can occur at either or both of the linking oxygens. In certain backbone modifications, the phosphate group can be replaced with a phosphorus-free connector. In some embodiments, the charged phosphate group can be replaced with a neutral moiety. Examples of moieties that can replace the phosphate group include, without limitation, for example, methylphosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo, and methyleneoxymethylimino.

[0235] In some embodiments, a composition or formulation disclosed herein comprises an mRNA that includes an open reading frame (ORF), such as an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or a class 2 Cas nuclease as described herein. In some embodiments, an mRNA is provided, used, or administered that includes an ORF encoding an RNA-guided DNA-binding agent, such as a Cas nuclease or a class 2 Cas nuclease. In some embodiments, the ORF is codon-optimized. In some embodiments, an ORF encoding an RNA-guided DNA-binding agent is a "modified RNA-guided DNA-binding agent ORF" or simply a "modified ORF," which is used as shorthand 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 is comprised of a set of codons, at least 75% of which are the minimal uridine codon(s) for a given amino acid, e.g., the codon(s) with the fewest uridines (usually 0 or 1, with the exception that the codon for phenylalanine has 2 uridines in its minimal uridine codon); or (4) 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 above methods. 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 (3) above.

[0236] "Modified uridine" is used herein to refer to a nucleoside other than thymidine in which the hydrogen bond acceptor is the same as in uridine and which 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 non-proton substituents (e.g., alkoxy, such as methoxy) replace a proton. In some embodiments, the modified uridine is a pseudouridine. In some embodiments, the modified uridine is a substituted pseudouridine, i.e., a pseudouridine in which one or more non-proton substituents (e.g., alkyl, such as methyl) replace a proton. In some embodiments, the modified uridine is any of a substituted uridine, pseudouridine, or substituted pseudouridine.

[0237] As used herein, a "uridine position" refers to a position in a polynucleotide that is occupied by a uridine or modified uridine. Thus, for example, a polynucleotide with "100% of the uridine positions being modified uridines" contains a modified uridine at every position that would be a uridine in a conventional RNA of the same sequence (wherein all bases are the standard bases A, U, C, or G). Unless otherwise indicated, U in the polynucleotide sequences set forth in this disclosure or in the sequence tables or sequence listings attached hereto may be a uridine or modified uridine. [Table 1]

[0238] In any of the foregoing embodiments, the modified ORF may consist of a codon set in which at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the codons are codons set forth in the minimal uridine codon table above.

[0239] In any of the foregoing embodiments, the modified ORF may have a uridine content ranging 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.

[0240] In any of the foregoing embodiments, the modified ORF may have a uridine dinucleotide content ranging 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.

[0241] In any of the foregoing embodiments, the modified ORF may include modified uridines at at least one, more than one, or all of the 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 may 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.

[0242] 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 of an mRNA according to the disclosure are modified uridines. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions of an mRNA according to the disclosure are modified uridines, such as 5-methoxyuridine, 5-iodouridine, N1-methylpseudouridine, pseudouridine, or combinations thereof. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of an mRNA according to the present disclosure are 5-methoxyuridine. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of an mRNA according to the present disclosure are pseudouridine. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of an mRNA according to the disclosure are N1-methylpseudouridine. In some embodiments, 10%-25%, 15%-25%, 25%-35%, 35%-45%, 45%-55%, 55%-65%, 65%-75%, 75%-85%, 85%-95%, or 90%-100% of the uridine positions of an mRNA according to the disclosure are 5-iodouridine. In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA according to the present disclosure are 5-methoxyuridine, with the remainder being N1-methylpseudouridine.In some embodiments, 10%-25%, 15-25%, 25-35%, 35-45%, 45-55%, 55-65%, 65-75%, 75-85%, 85-95%, or 90-100% of the uridine positions in an mRNA of the present disclosure are 5-iodouridine and the remainder are N1-methylpseudouridine.

[0243] In any of the foregoing embodiments, the modified ORF may include a low uridine dinucleotide content, such as the lowest possible uridine dinucleotide (UU) content, e.g., the ORF (a) uses a minimum of uridine codons (described above) at every position, and (b) encodes the same amino acid sequence as the given ORF. The uridine dinucleotide (UU) content may be expressed in absolute terms as a count of UU dinucleotides in the ORF, or expressed as a ratio as a percentage of positions occupied by uridine dinucleotides (e.g., in the case of AUUAU, 2 out of 5 positions are occupied by uridine dinucleotides, resulting in a uridine dinucleotide content of 40%). For purposes of assessing the minimum uridine dinucleotide content, modified uridine residues are considered equivalent to uridine.

[0244] In some embodiments, the mRNA comprises at least one UTR from an expressed mammalian mRNA, such as a constitutively expressed mRNA. An mRNA is considered to be constitutively expressed in a mammal if it 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'UTRs from an expressed mammalian RNA, such as a constitutively expressed mammalian mRNA. Actin mRNA is an example of a constitutively expressed mRNA.

[0245] In some embodiments, the mRNA comprises at least one UTR from 17-beta hydroxysteroid 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 a 5'UTR, a 3'UTR, or 5' and 3'UTRs from a globin mRNA, such as HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 5'UTR from bovine growth hormone, cytomegalovirus (CMV), mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises a 3' UTR from bovine growth hormone, cytomegalovirus, mouse Hba-a1, HSD, albumin gene, HBA, HBB, or XBG. In some embodiments, the mRNA comprises 5' and 3' UTRs 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).

[0246] In some embodiments, the mRNA comprises 5' and 3' UTRs from the same source, for example, a constitutively expressed mRNA such as actin, albumin, or a globin such as HBA, HBB, or XBG.

[0247] 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.

[0248] In some embodiments, the mRNA comprises a Kozak sequence. The Kozak sequence can affect translation initiation and the overall yield of polypeptides translated from the mRNA. The Kozak sequence includes a methionine codon that can function as a start codon. The 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 mismatches or with a maximum of one or two mismatches to the lowercase positions. In some embodiments, the Kozak sequence is rccAUGg, with zero mismatches or with a maximum of one or two mismatches to the lowercase positions. In some embodiments, the Kozak sequence is gccRccAUGG with zero mismatches or with a maximum of one, two, or three mismatches at the lowercase positions. In some embodiments, the Kozak sequence is gccAccAUG with zero mismatches or with a maximum of one, two, three, or four mismatches at the lowercase positions. In some embodiments, the Kozak sequence is GCCACCAUG. In some embodiments, the Kozak sequence is gccgccRccAUGG with zero mismatches or with a maximum of one, two, three, or four mismatches at the lowercase positions.

[0249] In some embodiments, the mRNAs disclosed herein comprise a 5' cap, such as Cap0, Cap1, or Cap2. The 5' cap is generally a 7-methylguanine ribonucleotide (which may be further modified, e.g., with respect to ARCA, as discussed below) linked via a 5'-triphosphate to the first nucleotide of the 5' to 3' strand of the mRNA, i.e., the 5' position of the first cap-proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2'-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA each comprise a 2'-methoxy and a 2'-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2'-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous mRNAs in higher eukaryotes, including mammalian mRNAs such as human mRNAs, contain Cap1 or Cap2. Cap0, as well as other cap structures distinct from Cap1 and Cap2, can be immunogenic in mammals, such as humans, because they are recognized as "non-self" by components of the innate immune system, such as IFIT-1 and IFIT-5, which can lead to increased levels of cytokines, such as 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, which can inhibit translation of the mRNA.

[0250] A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific catalog no. AM8045) is a cap analog containing 7-methylguanine 3'-methoxy-5'-triphosphate linked to the 5' position of a guanine ribonucleotide that can be incorporated into transcripts during transcription initiation in vitro. ARCA results in a cap, Cap0, in which the 2' position of the first cap-proximal nucleotide is a hydroxyl. See, e.g., 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 ARCA structure is shown below. [ka]

[0251] To obtain the Cap1 structure by co-transcription, 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. 3'-O-methylated versions of CleanCap™ AG and CleanCap™ GG are also available from TriLink Biotechnologies under catalog numbers N-7413 and N-7433, respectively. The CleanCap™ AG structure is shown below. [ka]

[0252] Alternatively, a cap can be added to RNA post-transcriptionally. For example, vaccinia capping enzyme is commercially available (New England Biolabs Catalog No. M2080S), which has RNA triphosphatase and guanylyltransferase activities provided by its D1 subunit, and guanine methyltransferase provided by its D12 subunit. Thus, in the presence of S-adenosylmethionine and GTP, this enzyme can add 7-methylguanine 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.

[0253] In some embodiments, the mRNA further comprises a polyadenylation (polyA) tail. In some embodiments, the polyA tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the polyA tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some cases, the polyA tail is "interrupted" with one or more non-adenine nucleotide "anchors" at one or more positions within the polyA tail. The polyA tail may comprise at least eight consecutive adenine nucleotides, but may also comprise one or more non-adenine nucleotides. As used herein, "non-adenine nucleotides" refers to any natural or non-natural nucleotide that does not contain adenine. Guanine nucleotides, thymine nucleotides, and cytosine nucleotides are exemplary non-adenine nucleotides. Thus, the poly A tail of 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 of the mRNA includes 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.

[0254] 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 of the mRNA described herein may contain consecutive adenine nucleotides located 3' of the nucleotides encoding the RNA-guided DNA binding agent or sequence of interest. In some cases, the poly-A tail of the mRNA contains non-consecutive adenine nucleotides located 3' of 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.

[0255] In some embodiments, the mRNA is purified. In some embodiments, the mRNA is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., as described herein). In some embodiments, the mRNA is purified using a chromatographic method, such as a HPLC method, or equivalent method (e.g., as described herein). In some embodiments, the mRNA is purified using both a precipitation method (e.g., LiCl precipitation) and a HPLC method.

[0256] 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 separate molecule 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.

[0257] mRNA In some embodiments, the compositions or formulations disclosed herein include an mRNA that includes an open reading frame (ORF) that encodes an RNA-guided DNA cleaving agent, such as a Cas nuclease, or a DNA cleaving agent, such as a class 2 Cas nuclease as described herein. In some embodiments, an mRNA that includes an ORF that encodes an RNA-guided DNA cleaving agent, such as a Cas nuclease or a class 2 Cas nuclease, is provided, used, or administered. The mRNA may include one or more of a 5' cap, a 5' untranslated region (UTR), a 3' UTR, and a polyadenine tail. The mRNA may include a modified open reading frame, for example, to encode a nuclear localization sequence or to use alternative codons to encode a protein.

[0258] The mRNA of the LNP compositions of the present disclosure may, for example, encode a secreted hormone, enzyme, receptor, polypeptide, peptide, or other protein of interest that is normally secreted. In some embodiments, the mRNA may optionally have chemical or biological modifications that, for example, improve the stability and / or half-life of such mRNA, or improve or otherwise facilitate protein production.

[0259] In addition, suitable modifications include changing one or more nucleotides of a codon to code for the same amino acid as the codon found in the wild-type version of the mRNA, but to be more stable than the codon. For example, an inverse correlation has been shown between RNA stability and a high number of cytidine (C) and / or uridine (U) residues, and RNA lacking C and U residues has been found to be stable to most RNases (Heidenreich, et al. J Biol Chem 269, 2131-8 (1994)). In some embodiments, the number of C and / or U residues in the mRNA sequence is reduced. In another embodiment, the number of C and / or U residues is reduced by substituting one codon that codes for a particular amino acid with another codon that codes for the same or a related amino acid. Contemplated modifications to mRNA nucleic acids also include the incorporation of pseudouridine. The incorporation of pseudouridine into mRNA nucleic acids can increase stability and translational capacity, and reduce immunogenicity in vivo. See, e.g., Kariko, K., et al., Molecular Therapy 16(11):1833-1840 (2008). Substitutions and modifications to mRNA can be carried out by methods readily known to those of skill in the art.

[0260] The constraints on reducing the number of C and U residues in a sequence may be greater in the coding regions of an mRNA compared to untranslated regions (i.e., it may not be possible to remove all of the C and U residues present in the message while still retaining the capacity of the message to code for the desired amino acid sequence), but the degeneracy of the genetic code provides the opportunity to reduce the number of C and / or U residues present in a sequence while still maintaining the same coding capacity (i.e., depending on which amino acids are coded for by the codons, several different possibilities for modification of the RNA sequence may be possible).

[0261] The term modification also includes, for example, the incorporation of non-nucleotide bonds or modified nucleotides into an mRNA sequence (e.g., modifications to one or both of the 3' and 5' ends of an mRNA molecule encoding a functional secreted protein or enzyme). Such modifications include the addition of bases to the mRNA sequence (e.g., inclusion of a polyA tail or a long polyA tail), alteration of the 3' or 5' UTR, complexing the mRNA with a substance (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements that alter the structure of the mRNA molecule (e.g., forming a secondary structure).

[0262] PolyA tails are believed to stabilize natural messengers. Thus, long polyA tails may be added to mRNA molecules, thereby making the mRNA more stable. PolyA tails can be added using a variety of art-recognized techniques. For example, long polyA tails can be added to synthetic or in vitro transcribed mRNAs using polyA polymerase (Yokoe, et al. Nature Biotechnology. 1996; 14: 1252-1256). Transcription vectors can also encode long polyA tails. Additionally, polyA tails can be added by direct transcription from PCR products. In some embodiments, the length of the polyA tail is at least about 90, 200, 300, 400 or at least 500 nucleotides. In certain embodiments, the length of the polyA tail can be adjusted to control the stability of the modified mRNA molecule, and thus the transcription of the protein. For example, the length of the polyA tail can affect the half-life of an mRNA molecule, and therefore the length of the polyA tail can be adjusted to alter the level of resistance of the mRNA to nucleases, thereby controlling the time course of protein expression in a cell. In some embodiments, the stabilized mRNA molecules are sufficiently resistant to in vivo degradation (e.g., by nucleases) so that they can be delivered to a target cell without the use of a delivery vehicle.

[0263] In certain embodiments, the mRNA may be modified by the incorporation of 3' and / or 5' untranslated (UTR) sequences not naturally found in the wild-type mRNA. In some embodiments, 3' and / or 5' flanking sequences that naturally flank the mRNA and encode a second, unrelated protein may be incorporated into the nucleotide sequence of the mRNA molecule encoding a therapeutic or functional protein to modify it. For example, 3' or 5' sequences from a stable mRNA molecule (e.g., globin, actin, GAPDH, tubulin, histones, or citric acid cycle enzymes) may be incorporated into the 3' and / or 5' regions of the sense mRNA nucleic acid molecule to enhance the stability of the sense mRNA molecule. See, e.g., US2003 / 0083272.

[0264] A more detailed description of mRNA modifications can be found in US2017 / 0210698A1, pages 57-68, the contents of which are incorporated herein.

[0265] Template nucleic acid The methods disclosed herein may include using template nucleic acid. The template may be used to change or insert a nucleic acid sequence at or near a target site for an RNA-guided DNA cleavage protein, such as a Cas nuclease, e.g., class 2 Cas nuclease. In some embodiments, the method includes introducing a 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 occurs 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.

[0266] In some embodiments, the template can be used in homologous recombination. In some embodiments, homologous recombination can result in the incorporation of the template sequence or a portion of the template sequence into the target nucleic acid molecule. In other embodiments, the template can be used in homologous recombination repair, which involves the invasion of a DNA strand at the site of the nucleic acid break. In some embodiments, homologous recombination repair results in the inclusion of the template sequence in the edited target nucleic acid molecule. In yet another embodiment, the template can be used in gene editing mediated by non-homologous end joining. In some embodiments, the template sequence has no similarity to the nucleic acid sequence near the break site. In some embodiments, the template or a portion of the template sequence is incorporated. In some embodiments, the template includes adjacent inverted terminal repeat (ITR) sequences.

[0267] In some embodiments, the template may include a first homology arm and a second homology arm (also referred to as a first nucleotide sequence and a second nucleotide sequence), which are complementary to the sequence located upstream and downstream of the cleavage site, respectively. When the template contains two homology arms, each arm may be the same length or different length, and the sequence flanked by the homology arms may be substantially similar or identical to the target sequence flanked by the homology arms, or may be a completely unrelated sequence. 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 the degree of complementarity or percent identity between the second nucleotide sequence on the template and the sequence downstream of the cleavage site may allow homologous recombination, such as high fidelity homologous recombination, between the template and the target nucleic acid molecule. In some embodiments, the degree of complementarity may 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%.

[0268] In some embodiments, the template sequence may correspond to, comprise, or consist of an endogenous sequence of the target cell. Additionally or alternatively, the template sequence may correspond to, comprise, or consist of an exogenous sequence of the target cell. As used herein, the term "endogenous sequence" refers to a sequence that is native to the cell. The term "exogenous sequence" refers to a sequence that is not native to the cell or that is at a different location than its natural 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 sequence or an 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 of the cell at or near the cleavage site, but may include at least one nucleotide change. In some embodiments, template editing of the cleaved target nucleic acid molecule may result in a mutation that includes an insertion, deletion, or substitution of one or more nucleotides of 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 that includes the target sequence.

[0269] 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 change 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, the templated editing of the cleaved target nucleic acid molecule may result in changes in exon sequences, intron sequences, control sequences, transcriptional regulatory sequences, translational regulatory sequences, splicing sites, or non-coding sequences of the target nucleic acid molecule, such as DNA.

[0270] 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., an ORF) operably linked to an exogenous promoter sequence such that upon integration of the exogenous sequence into the 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 the 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 that codes for a protein or a portion of the protein. In yet another embodiment, the exogenous sequence may include or consist of an exon sequence, an intron sequence, a control sequence, a transcriptional regulatory sequence, a translational regulatory sequence, a splicing site, or a non-coding sequence. 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.

[0271] The template may be of any suitable length. In some embodiments, the template may be comprised of 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, or more nucleotides in length. The template may be a single-stranded nucleic acid. The template may be a double-stranded or partially double-stranded nucleic acid. In some embodiments, the single-stranded template is 20, 30, 40, 50, 75, 100, 125, 150, 175, or 200 nucleotides in length. In some embodiments, the template may comprise a nucleotide sequence 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 may contain homology arms that are complementary to sequences located upstream or downstream of the cleavage site on the target nucleic acid molecule.

[0272] In some embodiments, the template contains ssDNA or dsDNA flanked by 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.

[0273] In some embodiments, the nucleic acid is purified. In some embodiments, the nucleic acid is purified using a precipitation method (e.g., LiCl precipitation, alcohol precipitation, or equivalent method, e.g., as described herein). In some embodiments, the nucleic acid is purified using a chromatographic method, such as a HPLC method, or equivalent method (e.g., as described herein). In some embodiments, the nucleic acid is purified using both a precipitation method (e.g., LiCl precipitation) and a HPLC method. In some embodiments, the nucleic acid is purified by tangential flow filtration (TFF).

[0274] cell type In some embodiments, the cell is a eukaryotic cell, such as a human cell in a subject. In some embodiments, the cell is an in vivo cell, e.g., a cell of a tissue, organ, or organism. In some embodiments, the cell is an in vitro cell. 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 may be selected from CD3+, CD4+, and CD8+ T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DCs). In some embodiments, the immune cell is allogeneic. In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a NK cell.

[0275] As used herein, a T cell can be defined as a cell that expresses a T cell receptor ("TCR" or "αβ TCR" or "γδ TCR"), although in some embodiments, the TCR of a T cell may be genetically modified to reduce its expression (e.g., by genetic modification of the TRAC or TRBC genes), such that expression of the protein CD3 can be used as a marker to identify T cells by standard flow cytometry methods. CD3 is a multi-subunit signaling complex that associates with the TCR. Thus, T cells may be referred to as CD3+. In some embodiments, a T cell is a cell that expresses the CD3+ marker and either the CD4+ or CD8+ marker.

[0276] In some embodiments, the T cells are CD8+ by standard flow cytometry because they express the glycoprotein CD8, which may be referred to as "cytotoxic" T cells. In some embodiments, the T cells are CD4+ by standard flow cytometry because they express the glycoprotein CD4, which may be referred to as "helper" T cells. CD4+ T cells can differentiate into subsets, which may be referred to as Th1 cells, Th2 cells, Th9 cells, Th17 cells, Th22 cells, regulatory T ("Treg") cells, or follicular helper T 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.

[0277] In some embodiments, the T cells are memory T cells. In the body, memory T cells have encountered antigen. Memory T cells may be located in secondary lymphoid organs (central memory T cells) or in recently infected tissues (effector memory T cells). Memory T cells may be CD8+ T cells. Memory T cells may be CD4+ T cells. As used herein, "central memory T cells" may be defined as T cells that have experienced antigen and may express, for example, CD62L and CD45RO. Central memory T cells may be detected as CD62L+ and CD45RO+ by central memory T cells that also express CCR7, and therefore may be detected as CCR7+ by standard flow cytometry methods.

[0278] As used herein, "early stem cell memory T cells" (or "Tscm") can be defined as T cells that express CD27 and CD45RA, and thus are CD27+ and CD45RA+ by standard flow cytometry. Tscm do not express CD45RO, an isoform of CD45, and therefore when stained for this isoform, Tscm are also CD45RO- by standard flow cytometry. Thus, CD45RO-CD27+ cells are also early stem cell memory T cells. Tscm cells further express CD62L and CCR7, and thus can be detected as CD62L+ and CCR7+ by standard flow cytometry. Early stem cell memory T cells have been shown to correlate with increased persistence and therapeutic efficacy of cell therapy products.

[0279] In some embodiments, the cell is a B cell. As used herein, a "B cell" can be defined as a cell that expresses CD19 and / or CD20, and / or B cell maturation antigen ("BCMA"), and thus the B cell is CD19+, and / or CD20+, and / or BCMA+ by standard flow cytometry. The B cell is further CD3 negative and CD56 negative by standard flow cytometry. The B cell can be a plasma cell. The B cell can be a memory B cell. The B cell can be a naive B cell. The B cell can be IgM+ or has a class-switched B cell receptor (e.g., IgG+, or IgA+).

[0280] Cells used in ACT therapy include mesenchymal stem cells (e.g., isolated from bone marrow (BM), peripheral blood (PB), placenta, umbilical cord (UC) or fat), hematopoietic stem cells (HSC, e.g., isolated from BM), mononuclear cells (e.g., isolated from BM or PB), endothelial progenitor cells (EPC; isolated from BM, PB, and UC), neural stem cells (NSC), limbal stem cells (LSC), or tissue-specific primary or derived cells (TSC), etc. Cells used in ACT therapy further include induced pluripotent stem cells (iPSC), which can be induced to differentiate into other cell types, such as pancreatic islet cells, neurons, and blood cells; ocular stem cells; pluripotent stem cells (PSC); embryonic stem cells (ESC); cells for organ or tissue transplantation, such as pancreatic islet cells, cardiomyocytes, thyroid cells, thymus cells, nerve cells, skin cells, retinal cells, chondrocytes, muscle cells, and keratinocytes.

[0281] In some embodiments, the cells are human cells, such as cells from a subject. In some embodiments, the cells are isolated from a human subject, such as a human donor. In some embodiments, the cells are isolated from PBMCs or leukopaks of a human donor. 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").

[0282] 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").

[0283] In some embodiments, the method is carried out ex vivo. As used herein, "ex vivo" refers to an in vitro method in which cells can be transferred to a subject, for example, as 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.

[0284] In some embodiments, the cells are maintained in culture. In some embodiments, the cells are transplanted into a patient. In some embodiments, the cells are removed from a subject, genetically modified ex vivo, and then administered back to the same patient. In some embodiments, the cells are removed from a subject, genetically modified ex vivo, and then administered to a subject other than the one from which the cells were removed.

[0285] 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.

[0286] In some embodiments, the cells are from a cell bank. In some embodiments, the cells are genetically modified and then transferred to a cell bank. In some embodiments, the cells are removed from a subject, genetically modified ex vivo, and transferred to a cell bank. In some embodiments, 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 a first and a second subpopulation, the first and second subpopulations having at least one common genetic modification and at least one different genetic modification, is transferred to a cell bank.

[0287] In some embodiments, T cells are activated by polyclonal activation (or "polyclonal stimulation") (not antigen-specific stimulation). In some embodiments, T cells are activated by CD3 stimulation (e.g., 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 a ready-to-use reagent for activating T cells (e.g., via CD3 / CD28 stimulation). In some embodiments, T cells are activated by via CD3 / CD28 stimulation provided by beads. In some embodiments, T cells are activated by 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, e.g., concanavalin A ("ConA"), or PHA).

[0288] In some embodiments, one or more cytokines are used for T cell activation. IL-2 is provided for T cell activation and / or to promote T cell survival. In some embodiments, the cytokine(s) for T cell activation are cytokines that bind to the common gamma chain (γc) receptor. In some embodiments, IL-2 is provided for T cell activation. In some embodiments, IL-7 is provided for T cell activation. In some embodiments, IL-15 is provided for T cell activation. In some embodiments, IL-21 is provided for T cell activation. In some embodiments, a combination of cytokines is provided for T cell activation, e.g., IL-2, IL-7, IL-15, and / or IL-21.

[0289] 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 on a major histocompatibility complex ("MHC") molecule (peptide-MHC complex). Cognate antigens can be presented to T cells by co-culturing T cells with antigen-presenting cells (feeder cells) and antigen. In some embodiments, T cells are activated by co-culture with antigen-pulsed antigen-presenting cells. In some embodiments, the antigen-presenting cells are pulsed with a peptide of the antigen.

[0290] In some embodiments, the T cells may be activated for 12 to 72 hours. In some embodiments, the T cells may be activated for 12 to 48 hours. In some embodiments, the T cells may be activated for 12 to 24 hours. In some embodiments, the T cells may be activated for 24 to 48 hours. In some embodiments, the T cells may be activated for 24 to 72 hours. In some embodiments, the T cells may be activated for 12 hours. In some embodiments, the T cells may be activated for 48 hours. In some embodiments, the T cells may be activated for 72 hours.

[0291] definition It should be noted that, as used in this application, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a composition" includes a plurality of compositions, reference to "a cell" includes a plurality of cells, and so forth. The use of "or" is inclusive and means "and / or" unless specifically stated otherwise.

[0292] Unless specifically stated in the specification above, embodiments herein that describe "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the described components, embodiments herein that describe "consisting of" various components are also contemplated as "comprising" or "consisting essentially of" the described components, embodiments herein that describe "about" various components are also contemplated as "at" the described components, and embodiments herein that describe "consisting essentially of" various components are also contemplated as "consisting of" or "comprising" the described components (this interchangeability does not apply to the use of these terms in the claims).

[0293] Numeric ranges include the numbers that define the range. Measurements and measurable values ​​are understood to be approximate, taking into account significant digits and errors associated with measurements. As used in this application, the terms "about" and "approximately" have their art-understood meanings and do not necessarily imply different ranges when used in one form than when used in the other form. Unless otherwise indicated, numbers used in this application, with or without modifiers such as "about" or "approximately", should be understood to encompass normal differences and / or variations as would be understood by one of ordinary skill in the relevant art. In certain embodiments, the term "approximately" or "about" may refer to a range of values ​​that is 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1% or less in either direction (above or below) from a stated reference value, unless otherwise stated or otherwise clear from the context (except when such number would exceed 100% of the possible values).

[0294] As used herein, the term "contacting" refers to establishing a physical connection between two or more entities. For example, contacting a mammalian cell with a nanoparticle composition means that the mammalian cell and the nanoparticle share a physical connection. Methods for contacting cells with external entities, both in vivo and ex vivo, are well known in the field of biology. For example, contacting a mammalian cell with a nanoparticle composition disposed within a mammal can be performed by various routes of administration (e.g., intravenous, intramuscular, intradermal, and subcutaneous) and can involve various amounts of the nanoparticle composition. Additionally, multiple mammalian cells can be contacted by the nanoparticle composition.

[0295] As used herein, the term "delivering" means providing an entity to a target. For example, delivery of a therapeutic and / or prophylactic agent to a subject can involve administering to the subject a nanoparticle composition comprising the therapeutic and / or prophylactic agent (e.g., by intravenous, intramuscular, intradermal, or subcutaneous routes). Administration of a nanoparticle composition to a mammal or mammalian cells can involve contacting one or more cells with the nanoparticle composition.

[0296] As used herein, "encapsulation rate" refers to the amount of therapeutic and / or prophylactic agent that becomes part of the nanoparticle composition relative to the initial total amount of therapeutic and / or prophylactic agent used in preparing the nanoparticle composition. For example, if 97 mg of therapeutic and / or prophylactic agent are encapsulated in the nanoparticle composition out of a total amount of 100 mg of therapeutic and / or prophylactic agent initially provided in the composition, the encapsulation rate may be 97%. As used herein, "encapsulation" may refer to complete, substantial, or partial surrounding, enclosing, surrounding, or enclosing.

[0297] As used herein, the terms "editing efficiency", "editing percentage", "indel efficiency" and "indel percent" refer to the total number of sequence reads with insertions or deletions relative to the total number of sequence reads. For example, editing efficiency at a target position in a genome can be measured by isolating and sequencing genomic DNA to identify the presence of insertions and deletions introduced by gene editing. In some embodiments, editing efficiency is measured as the percentage of cells that no longer contain a gene (e.g., CD3) after treatment relative to the number of cells that originally contained the gene (e.g., CD3+ cells).

[0298] 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 amount of protein in cells 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 for the protein. Methods for measuring mRNA knockdown are known, including sequencing the mRNA isolated from the sample of interest. In some embodiments, "knockdown" can refer to some loss of expression of a particular gene product, such as a reduction in the amount of transcribed mRNA or a reduction in the amount of protein expressed by a population of cells, including those found in a population in vivo, e.g., a tissue.

[0299] As used herein, "knockout" refers to the loss of expression from a particular gene or the loss of a particular protein in a cell. Knockout can be measured, for example, by detecting the total amount of intracellular protein in a cell, tissue, or population of cells. Knockout can also be detected, for example, at the genome or mRNA level.

[0300] As used herein, the term "biodegradable" is used to refer to a material that, when introduced into a cell, is degraded by cellular machinery (e.g., enzymatic degradation) or hydrolytically degraded into components that the cell can reuse or discard without significant toxic effect(s) to the cell. In certain embodiments, the components generated by degradation of the biodegradable material do not induce inflammation and / or other deleterious effects in vivo. In some embodiments, the biodegradable material is enzymatically degraded. Alternatively or additionally, in some embodiments, the biodegradable material is hydrolytically degraded.

[0301] As used herein, "N / P ratio" refers to the molar ratio of ionizable nitrogen atom-containing lipid (e.g., a compound of Formula I) to phosphate groups in RNA in a nanoparticle composition that includes, for example, a lipid component and RNA.

[0302] The composition may also include one or more salts of the compounds. The salt may be a pharmaceutically acceptable salt. As used herein, "pharmaceutically acceptable salt" refers to a derivative of the compound of the present disclosure, in which the parent compound is modified by converting an existing acid or base moiety into its salt form (e.g., by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfate, and the like. Representative salts of alkali metals include phonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, etc. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, etc., as well as non-toxic ammonium, quaternary ammonium, and amine cations, such as, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, etc. The pharma- ceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharma- ceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or an organic solvent, or a mixture of both, generally with non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile being preferred. A listing of suitable salts can be found in Remington's Pharmaceutical Sciences, 17. th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety.

[0303] As used herein, "polydispersity index" is a ratio that represents the uniformity of the particle size distribution of a system. A small value, for example less than 0.3, indicates a narrow particle size distribution. In some embodiments, the polydispersity index may be less than 0.1.

[0304] As used herein, "transfection" refers to the introduction of a seed (e.g., RNA) into a cell. Transfection can be performed, for example, in vitro, ex vivo, or in vivo.

[0305] The term "alkyl" as used herein refers to a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, s-butyl, t-butyl, n-pentyl, isopentyl, s-pentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. An alkyl group can be cyclic or acyclic. An alkyl group can be branched or unbranched (i.e., linear). An alkyl group can also be substituted or unsubstituted. For example, an alkyl group can be substituted with one or more groups, including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonic acid, carboxylic acid, or thiol, as described herein. A "lower alkyl" group is an alkyl group containing 1 to 6 (eg, 1 to 4) carbon atoms.

[0306] The term "alkenyl" as used herein refers to an aliphatic group containing at least one carbon-carbon double bond, and is intended to include both "unsubstituted alkenyl" and "substituted alkenyl," the latter of which refers to an alkenyl moiety having a substituent replacing a hydrogen on one or more carbons of the alkenyl group. Such substituents may occur on one or more carbons that are included or not included in one or more double bonds. Moreover, such substituents include all of those contemplated for alkyl groups, as discussed below, except where stability is critical. For example, an alkenyl group may be substituted with one or more alkyl groups, and carbocyclyl, aryl, heterocyclyl, or heteroaryl groups are contemplated. Exemplary alkenyl groups include vinyl (-CH=CH 2 ), allyl (-CH 2 CH=CH 2 ), cyclopentenyl (-C 5 H 7 ), and 5-hexenyl (-CH 2 CH 2 CH2 CH 2 CH=CH 2 ), but are not limited to these.

[0307] An "alkylene" group refers to a divalent alkyl radical, which can be branched or unbranched (i.e., linear). Any of the monovalent alkyl groups described above can be converted to an alkylene by abstracting a second hydrogen atom from the alkyl. Representative alkylenes include, but are not limited to, C 2-4 Alkylene and C 2-3 Exemplary alkylene groups include -CH(CH 3 )-, -C(CH 3 ) 2 -, -CH 2 CH 2 -, -CH 2 CH(CH 3 )-, -CH 2 C(CH 3 ) 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, etc. The alkylene group can be substituted or unsubstituted. For example, the alkylene group can be substituted with one or more groups, including, but not limited to, alkyl, aryl, heteroaryl, cycloalkyl, alkoxy, amino, ether, halide, hydroxy, nitro, silyl, sulfoxo, sulfonic acid, carboxylic acid, or thiol, as described herein.

[0308] The term "alkenylene" includes divalent linear or branched unsaturated acyclic hydrocarbyl groups having at least one carbon-carbon double bond and, in one embodiment, no carbon-carbon triple bonds. Any of the monovalent alkenyl groups described above can be converted to alkenylene by abstracting a second hydrogen atom from the alkenyl. Representative alkenylenes include C 2-6 Alkenylene is exemplified.

[0309] "C x-y The term "alkyl" when used in conjunction with a chemical moiety such as alkyl or alkylene is intended to include groups containing x to y carbons in the chain. For example, "C x-y The term "alkyl" refers to substituted or unsubstituted saturated hydrocarbon groups, including straight-chain and branched-chain alkyl and alkylene groups containing x to y carbons in the chain.

[0310] The term "alkoxy" refers to an alkyl group, preferably a lower alkyl group, having an oxygen attached thereto. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, and the like.

[0311] While the invention has been described in conjunction with exemplary embodiments, it will be understood that they are not intended to limit the invention to those embodiments. Rather, the present disclosure is intended to encompass all alternatives, modifications, and equivalents, such as equivalents of specific features, which may be included within the invention as defined by the appended claims.

[0312] Both the general description and detailed description above, as well as the following examples, are illustrative and explanatory only and are not intended to limit the teachings. The section headings used herein are for organizational purposes only and should not be construed as limiting the desired subject matter in any manner. In the event that a document incorporated by reference conflicts with a term defined herein, the present specification shall control. All ranges described in this application include endpoints unless otherwise specified.

[0313] Incorporation by Reference The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited in this specification are incorporated herein by reference in their entireties to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents. EXAMPLES

[0314] Example 1 - Materials and Methods 1.1. Preparation of T cell culture medium. The composition of the T cell culture medium used below is described here. "X-VIVO Basic Medium" consists of X-VIVO™ 15 medium, 1% Penstrep, 50 μM beta-mercaptoethanol, 10 mM NAC. In addition to the above components, other variable medium components used were: 1. serum (fetal bovine serum (FBS)), and 2. cytokines (IL-2, IL-7, IL-15).

[0315] 1.2. Preparation of T cells Leukapheresis of healthy human donors was obtained commercially (Hemacare). T cells were isolated on a MultiMACS™ Cell24 Separator Plus instrument by negative selection using the EasySep Human T cell Isolation Kit (Stem Cell Technology, Cat. No. 17951) or by positive CD4 / CD8 selection using StraightFrom® Leukopak® CD4 / CD8 MicroBeads (Milteny, Cat. No. 130-122-352) according to the manufacturer's instructions. T cells were cryopreserved in Cryostor CS10 cryopreservation medium (Cat. No. 07930) for future use.

[0316] Upon thawing, T cells were cultured in T cell complete growth medium consisting of CTS OpTmizer Base Media (CTS OpTmizer Media (Gibco, A3705001) supplemented with 1x GlutaMAX, 10 mM HEPES buffer (10 mM), and 1% pen-strep (Gibco, 15140-122), and further supplemented with 200 IU / mL IL-2 (Peprotech, 200-02), 5 ng / mL IL-7 (Peprotech, 200-07), 5 ng / mL IL-15 (Peprotech, 200-15), and 2.5% human serum (Gemini, 100-512). After overnight incubation, T cells were cultured in T cell complete growth medium consisting of CTS OpTmizer Base Media (CTS OpTmizer Media (Gibco, A3705001) supplemented with 1x GlutaMAX, 10 mM HEPES buffer (10 mM), and 1% pen-strep (Gibco, 15140-122). 6 T cells at a density of 0.5 × 10 / mL were activated with T cell TransAct reagent (1:100 dilution, Miltenyi) and incubated for 24 or 48 h at 37°C. After incubation, 0.5 × 10 6 Cells at a density of 100 / mL were used for editing purposes.

[0317] Unless otherwise indicated, the same process was used for non-activated T cells with the following exceptions: Upon thawing, non-activated T cells were cultured in CTS complete growth medium consisting of CTS OpTmizer Base Media (Thermofisher, A10485-01), 1% pen-strep (Corning, 30-002-CI), 1x GlutaMAX (Thermofisher, 35050061), 10 mM HEPES (Thermofisher, 15630080)) supplemented with 200U / mL IL-2 (Peprotech, 200-02), 5ng / mL IL-7 (Peprotech, 200-07), 5ng / mL IL-15 (Peprotech, 200-15) with 5% human AB serum (Gemini, 100-512) for 24 hours without activation. T cells were cultured at 10 in 100 uL of the above CTS OpTmizer basal medium containing 2.5% human serum and cytokines for editing. 6 Cells were seeded at a cell density of 100 / mL.

[0318] 1.3. Preparation of lipid nanoparticles. Unless otherwise stated, lipid components were dissolved in various molar ratios in 100% ethanol. RNA cargo (e.g., Cas9 mRNA and sgRNA) was dissolved in 25 mM citrate, 100 mM NaCl, pH 5.0 to a concentration of RNA cargo of approximately 0.45 mg / mL.

[0319] Unless otherwise stated, LNP refers to ionizable lipid A ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl(9Z,12Z)-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. The mixture contained 50:38:9:3 molar ratios of methyl)propyl octadeca-9,12-dienoate, cholesterol, DSPC, and PEG2k-DMG. The lipid-nucleic acid assemblies were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6 and a gRNA to mRNA weight ratio of 1:1 unless otherwise noted. In Examples 13-16, a gRNA to mRNA weight ratio of 1:2 was used unless otherwise noted.

[0320] Lipid nanoparticles (LNPs) were prepared using a cross-flow technique that utilizes impinging jet mixing of lipid-containing ethanol with two volumes of RNA solution and one volume of water. The lipid-containing ethanol was mixed with two volumes of RNA solution via a mixing cross. A fourth stream of water was mixed with the outlet stream from the cross via an in-line T-junction (see FIG. 2 of WO2016010840). The LNPs were kept at room temperature (RT) for 1 hour and further diluted with water (approximately 1:1 v / v). The LNPs were concentrated using tangential flow filtration, e.g., with a flat sheet cartridge (Sartorius, 100 kD MWCO), and buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) using a PD-10 desalting column (GE). Alternatively, the LNPs were optionally concentrated using a 100 kDa Amicon spin filter and buffer exchanged into TSS using a PD-10 desalting column (GE). The resulting mixture was then filtered using a 0.2 μm sterile filter. The final LNPs were stored at 4° C. or −80° C. until further use.

[0321] 1.4. Next Generation Sequencing ("NGS") and Analysis of On-Target Cleavage Efficiency Genomic DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Cat. No. QE09050) according to the manufacturer's instructions.

[0322] To quantitatively determine the editing efficiency at the target location of the genome, next generation sequencing was used to identify the presence of insertions and deletions introduced by gene editing. PCR primers were designed around the target site in the gene of interest (e.g., TRAC) to amplify the genomic region of interest. Primer sequence design was performed as standard in the art.

[0323] An additional PCR was performed according to the manufacturer's (Illumina) operating procedure, and sequencing chemistry was added. Amplicons were sequenced on an Illumina MiSeq instrument. After removing reads with low quality scores, the reads were aligned to the human (e.g., hg38) reference genome. The resulting files containing the reads were mapped to the reference genome (BAM files), reads overlapping the target region of interest were selected, and the number of reads containing insertions or deletions ("indels") relative to the number of wild-type reads was calculated.

[0324] Editing percentage (e.g., "editing efficiency" or "percent editing") is defined as the total number of sequence reads with insertions or deletions ("indels") relative to the total number of sequence reads, including the wild type.

[0325] 1.5. In Vitro Transcription ("IVT") of Nuclease mRNA Capped and polyadenylated mRNA containing N1-methylpseudo-U was generated by in vitro transcription using linearized plasmid DNA template and T7 RNA polymerase. Plasmid DNA containing the T7 promoter, transcription sequence, and polyadenylation region was linearized by incubation with XbaI for 2 hours at 37°C using the following conditions: 200 ng / μL plasmid, 2 U / μL XbaI (NEB), and 1× reaction buffer. XbaI was inactivated by heating the reaction at 65°C for 20 minutes. Linearized plasmid was purified from enzymes and buffer salts. IVT reactions to generate modified mRNA were performed by incubating for 1.5–4 h at 37°C with the following conditions: 50 ng / μL linearized plasmid, 2–5 mM each of GTP, ATP, CTP, and N1-methylpseudo-UTP (Trilink), 10–25 mM ARCA (Trilink), 5 U / μL T7 RNA polymerase (NEB), 1 U / μL mouse RNase inhibitor (NEB), 0.004 U / μL inorganic E. coli pyrophosphatase (NEB), and 1× reaction buffer. TURBO DNase (ThermoFisher) was added to a final concentration of 0.01 U / μL, and the reaction was incubated for an additional 30 min to remove the DNA template. mRNA was purified using the MegaClear Transcription Clean-up kit (ThermoFisher) or RNeasy Maxi kit (Qiagen) according to the manufacturer's operating instructions.

[0326] Alternatively, mRNA was purified by a precipitation procedure, followed in some cases by purification using HPLC. Briefly, after DNase digestion, mRNA was purified using LiCl, ammonium acetate and sodium acetate precipitation. For HPLC-purified mRNA, mRNA was purified by RP-IP HPLC after LiCl precipitation and reconstitution (see, for example, Kariko, et al. Nucleic Acids Research, 2011, Vol. 39, No. 21 e142). The fractions selected for pooling were combined and desalted by sodium acetate / ethanol precipitation as described above. In a further alternative, mRNA was purified by LiCl precipitation, followed by further purification by tangential flow filtration. RNA concentration was determined by measuring absorbance at 260 nm (Nanodrop) and transcripts were analyzed by capillary electrophoresis with a Bioanlayzer (Agilent).

[0327] Streptococcus pyogenes ("Spy") Cas9 mRNA was generated from plasmid DNA encoding the open reading frame set forth in SEQ ID NOs: 9 and 10 (see sequences in the Supplementary Sequence Listing). When the sequences cited in this paragraph are referred to below in relation to RNA, it is understood that T should be replaced with U (which may be a modified nucleoside as described above). The messenger RNA used in the examples includes a 5' cap and a 3' polyadenylation sequence (e.g., up to 100 nt), which are identified in the Supplementary Sequence Listing.

[0328] Guide RNAs are chemically synthesized by methods known in the art.

[0329] compound synthesis General information All reagents and solvents were purchased and used as received from commercial vendors or synthesized according to the cited procedures. All intermediates and final compounds were purified using flash silica gel column chromatography. NMR spectra were recorded on a Bruker or Varian 400 MHz spectrometer, and NMR data were collected at ambient temperature in CDCl3. Chemical shifts are reported in parts per million (ppm) relative to CDCl3 (7.26). 1H NMR data are reported as: chemical shift, multiplicity (br = broad line, s = singlet, d = doublet, t = triplet, q = quartet, dd = double doublet, dt = double triplet, m = multiplet), coupling constants, and integrals. MS data were recorded on a Waters SQD2 mass spectrometer using an electrospray ionization (ESI) source. The purity of the final compounds was determined by UPLC-MS-ELS using a Waters Acquity H-Class liquid chromatography system equipped with an SQD2 mass spectrometer with a photodiode array (PDA) detector and an evaporative light scattering (ELS) detector. [Table 2-1] [Table 2-2]

[0330] Example 2 - Compound 1 Intermediate 1a: (E)-N,N-dimethyl-N'-(4-methyl-5-nitropyridin-2-yl)formimidamide [ka] To a solution of 4-methyl-5-nitro-pyridin-2-amine (5 g, 1.0 equiv.) in toluene (0.3 M) was added DMF-DMA (3.0 equiv.). The mixture was stirred at 110° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue that was purified by column chromatography to give the product as a yellow solid (59%). 1H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.82 (s, 1H), 8.63 (s, 1H), 6.74 (s, 1H), 3.21 (m, 6H).

[0331] Intermediate 1b: (E)-N-hydroxy-N'-(4-methyl-5-nitropyridin-2-yl)formimidamide [ka] To a solution of intermediate 1a (4 g, 1.0 equiv.) in MeOH (0.2 M), NH 2 OH·HCl (2.0 equiv.) was added. The reaction mixture was stirred at 80° C. for 1 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by H 2 The mixture was partitioned between O and EtOAc and extracted twice with EtOAc. The organic phase was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a white solid (66%). 1H NMR (400 MHz, (CD 3 ) 2 SO) δ 10.52 (d, J = 3.8 Hz, 1H), 10.08 (dd, J = 9.9, 3.7 Hz, 1H), 8.84 (d, J = 3.8 Hz, 1H), 7.85 (dd, J = 9.7, 3.8 Hz, 1H), 7.01 (d, J = 3.9 Hz, 1H), 3.36 (s, 3H).

[0332] Intermediate 1c: 7-Methyl-6-nitro-[1,2,4]triazolo[1,5-a]pyridine [ka] To a solution of intermediate 1b (2.5 g, 1.0 equiv.) in THF (0.4 M) was added trifluoroacetic anhydride (1.0 equiv.) at 0° C. The mixture was stirred at 25° C. for 18 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a white solid (44%). 1H NMR (400 MHz, CDCl 3 ) δ 9.53 (s, 1H), 8.49 (s, 1H), 7.69 (s, 1H), 2.78 (d, J = 1.0 Hz, 3H).

[0333] Intermediate 1d: 7-Methyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine [ka] To a mixture of Pd / C (10 w / w%, 0.2 equiv.) in EtOH (0.1 M) was added intermediate 1c (1.0 equiv.) and ammonium formate (5.0 equiv.). The mixture was heated at 105° C. for 2 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a light brown solid. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.41 (s, 2H), 8.07 (d, J = 9.0 Hz, 2H), 7.43 (s, 1H), 2.22 (s, 3H).

[0334] Intermediate 1e: ethyl 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylate [ka] A solution of tetrahydropyran-4-amine (5 g, 1.0 equiv.) and ethyl 2,4-dichloropyrimidine-5-carboxylate (1.0 equiv.) in MeCN (0.25-2.0 M) was added to K 2 CO 3 (1.0-3.0 equiv.) was added. The mixture was stirred at 20-25°C for at least 12 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue. The residue was purified by column chromatography to give the product as a pale yellow solid (21%). 1H NMR (400 MHz, (CD 3 ) 2SO) δ 8.60 (s, 1H), 8.29 (d, J = 7.7 Hz, 1H), 4.28 (q, J = 7.1 Hz, 2H), 4.14 (dtt, J = 11.3, 8.3, 4.0 Hz, 1H), 3.82 (dt, J = 12.1, 3.6 Hz, 2H), 3.57 (s, 1H), 1.87 - 1.78 (m, 2H), 1.76 - 1.67 (m, 1H), 1.54 (qd, J = 10.9, 4.3 Hz, 2H), 1.28 (t, J = 7.1 Hz, 3H).

[0335] Intermediate 1f: 2-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)pyrimidine-5-carboxylic acid [ka] LiOH (2.5 equiv.) in THF / H 2 To a 1:1 solution of HO (0.25-1.0 M) was added intermediate 1e (3.0 g, 1.0 equiv.). The mixture was stirred at 25 °C for 12 h. The mixture was concentrated under reduced pressure to remove THF. The residue was adjusted to pH 2 with 2 M HCl and the resulting precipitate was collected by filtration, washed with water, and dried under vacuum to give a residue. The residue was purified by column chromatography to give the product as a white solid (74%) or used directly as crude.

[0336] Intermediate 1g: 2-chloro-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka] To a solution of intermediate 1f (2 g, 1.0 equiv.) in MeCN (0.2-0.5 M), 3N (1.0 equiv.) was added. The mixture was stirred at 25° C. for 30 min. Then DPPA (1.0 equiv.) was added to the mixture. The mixture was stirred at 100° C. for at least 7 h. The reaction mixture was poured into water and the resulting precipitate was collected by filtration, washed with water, and dried under vacuum to give a residue. The residue was purified by column chromatography to give the product as a white solid (56%). 1 H NMR (400 MHz, CDCl 3 ) δ 9.50 (s, 1H), 8.09 (s, 1H), 4.53 (tt, J = 12.4, 4.2 Hz, 1H), 4.07 (dt, J = 9.5, 4.8 Hz, 2H), 3.48 (td, J = 12.1, 1.9 Hz, 2H), 2.69 (qd, J = 12.5, 4.7 Hz, 2H), 1.67 (dd, J = 12.1, 3.9 Hz, 2H).

[0337] Intermediate 1h: 2-chloro-7-methyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka] A mixture of intermediate 1g (300 mg, 1.0 equiv.) and NaOH (5.0 equiv.) in THF / H 2 To a 1:1 solution of 2H2O (0.25-1.0 M) was added iodomethane (2.0 equiv.). The reaction mixture was stirred at 25° C. for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a white solid (47%). 1 H NMR (400 MHz, (CD 3 ) 2SO) δ 8.34 (s, 1H), 4.43 (ddt, J = 12.2, 8.5, 4.2 Hz, 1H), 3.95 (dd, J = 11.5, 4.6 Hz, 2H), 3.43 (td, J = 12.1, 1.9 Hz, 2H), 2.45 (s, 3H), 2.40 (td, J = 12.5, 4.7 Hz, 2H), 1.66 (ddd, J = 12.2, 4.4, 1.9 Hz, 2H).

[0338] Compound 1: 7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka] Intermediate 1h (1.3g, 1.0 equivalent), Intermediate 1d (1.0 equivalent), Pd(dppf)Cl 2 (0.1-0.2 equivalents), XantPhos (0.1-0.2 equivalents) and Cs 2 CO 3 (2.0 equiv.) in DMF (0.05–0.3 M) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 100-130°C under atmospheric conditions for at least 12 hours. The reaction mixture was then poured into water and extracted three times with DCM. The combined organic phase was washed with brine and diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the filtrate was concentrated in vacuo The residue was purified by column chromatography to give the product as a pale yellow solid. 1 H NMR (400 MHz, (CD 3 ) 2SO) δ 9.13 (s, 1H), 8.69 (s, 1H), 8.39 (s, 1H), 8.10 (s, 1H), 7.72 (s, 1H), 4.50 - 4.36 (m, 1H), 3.98 (dd, J = 11.6, 4.4 Hz, 2H), 3.44 (d, J = 11.9 Hz, 2H), 3.32 (s, 3H), 2.44 - 2.38 (m, 3H), 1.69 (d, J = 11.6 Hz, 2H). MS: 381.3 m / z [M+H].

[0339] Example 3 - Compound 2 Intermediate 2a: 2-chloro-7-ethyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka] A mixture of intermediate 1h (800 mg, 1.0 equiv.) and NaOH (5.0 equiv.) was dissolved in THF (0.4 M) and H 2 To the mixture with O (0.8 M) was added EtI (3.0 equiv.). The reaction mixture was stirred at 20° C. for 12 h. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography to give the product as a white solid (45%). 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.50 (s, 1H), 4.52 (tt, J = 12.2, 4.2 Hz, 1H), 4.03 (dd, J = 11.5, 4.6 Hz, 2H), 3.95 (q, J = 7.2 Hz, 2H), 3.51 (td, J = 12.1, 1.9 Hz, 2H), 2.48 (td, J = 12.5, 4.7 Hz, 2H), 1.79 - 1.71 (m, 2H), 1.31 (t, J = 7.2 Hz, 3H).

[0340] Compound 2: 7-Ethyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka] Compound 2 was synthesized as a TFA salt from intermediate 1d and intermediate 2a using the method used for compound 1, followed by purification by reverse phase HPLC. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.11 (s, 1H), 8.69 (s, 1H), 8.38 (s, 1H), 8.15 (s, 1H), 7.71 (t, J = 1.0 Hz, 1H), 4.42 (ddd, J = 12.1, 7.9, 4.1 Hz, 1H), 3.96 (dd, J = 11.7, 4.4 Hz, 2H), 3.83 (q, J = 7.2 Hz, 2H), 3.41 (t, J = 11.9 Hz, 2H), 2.40 (d, J = 1.0 Hz, 3H), 1.68 (d, J = 11.0 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H). MS: 395.3 m / z [M+H].

[0341] Example 4 - Compound 3 Intermediate 3a: Ethyl 2-chloro-4-((4,4-difluorocyclohexyl)amino)pyrimidine-5-carboxylate [ka] Intermediate 3a was synthesized from ethyl 2,4-dichloropyrimidine-5-carboxylate and 4,4-difluorocyclohexanamine hydrochloride using the method used for intermediate 1e. 1 H NMR (400 MHz, (CD 3 ) 2SO) δ 8.61 (s, 1H), 8.30 (d, J = 7.7 Hz, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.19 - 4.09 (m, 1H), 2.09 - 1.90 (m, 6H), 1.69 - 1.58 (m, 2H), 1.29 (t, J = 7.1 Hz, 3H).

[0342] Intermediate 3b: 2-chloro-4-((4,4-difluorocyclohexyl)amino)pyrimidine-5-carboxylic acid [ka] Intermediate 3b was synthesized from intermediate 3a (78%) using the method used for intermediate 1f. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 13.77 (s, 1H), 8.57 (s, 1H), 8.53 (d, J = 7.8 Hz, 1H), 4.12 (d, J = 10.2 Hz, 1H), 2.14 - 1.89 (m, 6H), 1.62 (ddt, J = 17.0, 10.3, 6.0 Hz, 2H).

[0343] Intermediate 3c: 2-chloro-9-(4,4-difluorocyclohexyl)-7,9-dihydro-8H-purin-8-one [ka] Intermediate 3c was synthesized from intermediate 3b (56%) using the method used for intermediate 1g. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 11.76 - 11.65 (m, 1H), 8.20 (s, 1H), 4.47 (dq, J = 12.6, 6.2, 4.3 Hz, 1H), 2.34 - 1.97 (m, 6H), 1.90 (d, J = 12.9 Hz, 2H).

[0344] Intermediate 3d: 2-chloro-9-(4,4-difluorocyclohexyl)-7-methyl-7,9-dihydro-8H-purin-8-one [ka] A mixture of intermediate 3c (1.4 g, 1.0 equiv.), NaOH (5.0 equiv.) and THF / H 2 To a 0.3 M solution of MeI in 5:1 N2O, MeI (2.0 equiv.) was added. The mixture was cooled to 5:1 with 1000 rpm for 2 h. 2 The mixture was stirred under atmosphere for 12 hours at 20° C. The reaction mixture was concentrated under reduced pressure to give a residue, which was purified by column chromatography to give the product as a yellow solid (47%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.01 (s, 1H), 4.53 - 4.39 (m, 1H), 3.43 (s, 3H), 2.73 (qd, J = 12.7, 12.1, 3.8 Hz, 2H), 2.32 - 2.20 (m, 2H), 2.03 - 1.82 (m, 4H).

[0345] Compound 3: 9-(4,4-difluorocyclohexyl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purin-8-one [ka] Compound 3 was synthesized from intermediate 1d and intermediate 3d using the method used for compound 1, followed by purification by reverse phase HPLC. 1 H NMR (400 MHz, (CD 3 ) 2SO) δ 9.03 (s, 1H), 8.66 (s, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.71 (d, J = 1.4 Hz, 1H), 4.36 (d, J = 12.3 Hz, 1H), 3.31 (s, 3H), 2.38 (d, J = 1.0 Hz, 3H), 2.11 - 1.96 (m, 4H), 1.81 (d, J = 12.6 Hz, 2H). MS: 415.5 m / z [M+H].

[0346] Example 5 - Compound 4 Intermediate 4a: 8-methylene-1,4-dioxaspiro[4.5]decane [ka] To a solution of methyl(triphenyl)phosphonium bromide (1.15 equiv.) in THF (0.6 M) was added n-BuLi (1.1 equiv.) dropwise at -78°C, and the mixture was stirred at 0°C for 1 h. Then, 1,4-dioxaspiro[4.5]decan-8-one (50 g, 1.0 equiv.) was added to the reaction mixture. The mixture was stirred at 25°C for 12 h. The reaction mixture was diluted with NH 4 Pour into aqueous Cl solution at 0°C and add H 2 The mixture was diluted with O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give a residue that was purified by column chromatography to give the product as a colorless oil (51%). 1 H NMR (400 MHz, CDCl 3 ) δ 4.67 (s, 1H), 3.96 (s, 4 H), 2.82 (t, J = 6.4 Hz, 4 H), 1.70 (t, J = 6.4 Hz, 4 H).

[0347] Intermediate 4b: 7,10-dioxadispiro[2.2.4 6 .2 3 ]Dodecane [ka] To a solution of intermediate 4a (5 g, 1.0 equiv.) in toluene (3 M), ZnEt 2(2.57 equiv.) was added dropwise at -40°C and the mixture was stirred at -40°C for 1 h. The mixture was then treated with diiodomethane (6.0 equiv.) 2 The mixture was then cooled to -40°C and cooled to 100°C. 2 The mixture was stirred under atmospheric conditions at 20° C. for 17 hours. 4 The mixture was poured into aqueous Cl at 0° C. and extracted twice with EtOAc. The combined organic phase was washed with brine (20 mL) and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the filtrate was concentrated in vacuo The residue was purified by column chromatography to give the product as a pale yellow oil (73%).

[0348] Intermediate 4c: spiro[2.5]octan-6-one [ka] Intermediate 4b (4g, 1.0 equiv.) in THF / H 2 To a 1.0 M solution of 2,4-dichlorophenyl ether (1:1) in N2O (1:1), TFA (3.0 equiv.) was added. The mixture was diluted with N 2 The mixture was stirred under atmospheric pressure at 20° C. for 2 h. The reaction mixture was concentrated under reduced pressure to remove THF, and the residue was adjusted to pH 7 with 2M NaOH (aq.). The mixture was poured into water and extracted three times with EtOAc. The combined organic phase was washed with brine and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the filtrate was concentrated in vacuo The residue was purified by column chromatography to give the product as a pale yellow oil (68%). 1 H NMR (400 MHz, CDCl 3 ) δ 2.35 (t, J = 6.6 Hz, 4H), 1.62 (t, J = 6.6 Hz, 4H), 0.42 (s, 4H).

[0349] Intermediate 4d: N-(4-methoxybenzyl)spiro[2.5]octan-6-amine [ka] To a mixture of intermediate 4c (2 g, 1.0 equiv.) and (4-methoxyphenyl)methanamine (1.1 equiv.) in DCM (0.3 M) was added AcOH (1.3 equiv.). The mixture was diluted with N 2 The mixture was stirred at 20° C. for 1 hour under atmospheric conditions. Then, NaBH(OAc) 3 (3.3 equiv.) was added at 0° C. and the mixture was cooled to 5° C. with N 2 The mixture was stirred at 20° C. for 17 hours under atmospheric pressure. The reaction mixture was concentrated under reduced pressure to remove DCM, and the resulting residue was diluted with H 2 The mixture was diluted with O and extracted three times with EtOAc. The combined organic layers were washed with brine and 2 SO 4 The mixture was dried at 40° C., filtered, and the filtrate was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a grey solid (51%). 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 7.15 - 7.07 (m, 2H), 6.77 - 6.68 (m, 2H), 3.58 (s, 3H), 3.54 (s, 2H), 2.30 (ddt, J = 10.1, 7.3, 3.7 Hz, 1H), 1.69 - 1.62 (m, 2H), 1.37 (td, J = 12.6, 3.5 Hz, 2H), 1.12 - 1.02 (m, 2H), 0.87 - 0.78 (m, 2H), 0.13 - 0.04 (m, 2H).

[0350] Intermediate 4e: spiro[2.5]octan-6-amine [ka] Intermediate 4d (2 g, 1.0 equiv.) was added to a suspension of Pd / C (10 w / w%, 1.0 equiv.) in MeOH (0.25 M) and the mixture was diluted with H 2 The mixture was stirred under atmosphere at 80° C. and 50 Psi for 24 h. The reaction mixture was filtered and the filtrate was concentrated under reduced pressure to give a residue which was purified by column chromatography to give the product as a white solid. 1 H NMR (400 MHz, (CD 3 )2 SO) δ 2.61 (tt, J = 10.8, 3.9 Hz, 1H), 1.63 (ddd, J = 9.6, 5.1, 2.2 Hz, 2H), 1.47 (td, J = 12.8, 3.5 Hz, 2H), 1.21 - 1.06 (m, 2H), 0.82 - 0.72 (m, 2H), 0.14 - 0.05 (m, 2H).

[0351] Intermediate 4f: ethyl 2-chloro-4-(spiro[2.5]octan-6-ylamino)pyrimidine-5-carboxylate [ka] Intermediate 4f was synthesized from intermediate 4e (54%) using the method used for intermediate 1e. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.64 (s, 1H), 8.41 (d, J = 7.9 Hz, 1H), 4.33 (q, J = 7.1 Hz, 2H), 4.08 (d, J = 9.8 Hz, 1H), 1.90 (dd, J = 12.7, 4.8 Hz, 2H), 1.64 (t, J = 12.3 Hz, 2H), 1.52 (q, J = 10.7, 9.1 Hz, 2H), 1.33 (t, J = 7.1 Hz, 3H), 1.12 (d, J = 13.0 Hz, 2H), 0.40 - 0.21 (m, 4H).

[0352] Intermediate 4g: 2-chloro-4-(spiro[2.5]octan-6-ylamino)pyrimidine-5-carboxylic acid [ka] Intermediate 4g was synthesized from intermediate 4f (82%) using the method used for intermediate 1f. 1 H NMR (400 MHz, (CD 3 ) 2SO) δ 13.54 (s, 1H), 8.38 (d, J = 8.0 Hz, 1H), 8.35 (s, 1H), 3.82 (qt, J = 8.2, 3.7 Hz, 1H), 1.66 (dq, J = 12.8, 4.1 Hz, 2H), 1.47 - 1.34 (m, 2H), 1.33 - 1.20 (m, 2H), 0.86 (dt, J = 13.6, 4.2 Hz, 2H), 0.08 (dd, J = 8.3, 4.8 Hz, 4H).

[0353] Intermediate 4h: 2-chloro-9-(spiro[2.5]octan-6-yl)-7,9-dihydro-8H-purin-8-one [ka] Intermediate 4h was synthesized from intermediate 4g (67%) using the method used for intermediate 1g. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 11.68 (s, 1H), 8.18 (s, 1H), 4.26 (ddt, J = 12.3, 7.5, 3.7 Hz, 1H), 2.42 (qd, J = 12.6, 3.7 Hz, 2H), 1.95 (td, J = 13.3, 3.5 Hz, 2H), 1.82 - 1.69 (m, 2H), 1.08 - 0.95 (m, 2H), 0.39 (tdq, J = 11.6, 8.7, 4.2, 3.5 Hz, 4H).

[0354] Intermediate 4i: 2-chloro-7-methyl-9-(spiro[2.5]octan-6-yl)-7,9-dihydro-8H-purin-8-one [ka] Intermediate 4i was synthesized from intermediate 4h (67%) using the method used for intermediate 1h. 1 H NMR (400 MHz, CDCl 3) δ 7.57 (s, 1H), 4.03 (tt, J = 12.5, 3.9 Hz, 1H), 3.03 (s, 3H), 2.17 (qd, J = 12.6, 3.8 Hz, 2H), 1.60 (td, J = 13.4, 3.6 Hz, 2H), 1.47 - 1.34 (m, 2H), 1.07 (s, 1H), 0.63 (dp, J = 14.0, 2.5 Hz, 2H), -0.05 (s, 4H).

[0355] Compound 4: 7-Methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-9-(spiro[2.5]octan-6-yl)-7,9-dihydro-8H-purin-8-one [ka] Compound 4 was synthesized from intermediate 4i and intermediate 1d using the method used for compound 1. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.09 (s, 1H), 8.73 (s, 1H), 8.44 (s, 1H), 8.16 (s, 1H), 7.78 (s, 1H), 4.21 (t, J = 12.5 Hz, 1H), 3.36 (s, 3H), 2.43 (s, 3H), 2.34 (dt, J = 13.0, 6.5 Hz, 2H), 1.93 - 1.77 (m, 2H), 1.77 - 1.62 (m, 2H), 0.91 (d, J = 13.2 Hz, 2H), 0.31 (t, J = 7.1 Hz, 2H). MS: 405.5 m / z [M+H].

[0356] Example 6 - Compound 5 Intermediate 5a: Ethyl 2-chloro-4-((3-hydroxycyclobutyl)amino)pyrimidine-5-carboxylate [ka] Intermediate 5a was synthesized from 3-aminocyclobutanol (49%) using the method used for intermediate 1e. 1 H NMR (400 MHz, (CD 3 ) 2 SO, mixture of rotamers) δ 8.62 (s, 1H), 8.45 (dd, J = 25.7, 7.1 Hz, 1H), 5.17 (dd, J = 6.0, 2.7 Hz, 1H), 4.32 (q, J = 7.1 Hz, 2H), 3.96 (dp, J = 50.4, 7.2 Hz, 2H), 2.67 (ddd, J = 11.6, 5.8, 2.8 Hz, 1H), 2.25 (td, J = 8.1, 7.0, 4.0 Hz, 1H), 1.85 (qd, J = 8.7, 2.8 Hz, 1H), 1.32 (t, J = 7.1 Hz, 3H).

[0357] Intermediate 5b: 2-chloro-4-((3-hydroxycyclobutyl)amino)pyrimidine-5-carboxylic acid [ka] Intermediate 5b was synthesized from intermediate 5a (67%) using the method used for intermediate 1f. 1 H NMR (400 MHz, (CD 3 ) 2 SO, mixture of rotamers) δ 13.82 (s, 1H), 8.70 (dd, J = 25.0, 7.1 Hz, 1H), 8.63 (s, 1H), 4.65 - 4.29 (m, 1H), 4.17 - 4.02 (m, 1H), 3.95 (p, J = 7.2 Hz, 1H), 2.74 (dh, J = 11.8, 3.1 Hz, 2H), 2.30 (t, J = 6.2 Hz, 1H), 1.88 (qd, J = 8.5, 2.8 Hz, 1H).

[0358] Intermediate 5c: 2-chloro-9-(3-hydroxycyclobutyl)-7,9-dihydro-8H-purin-8-one [ka] Intermediate 5c was synthesized from intermediate 5b using the method used for intermediate 1g. 1 H NMR (400 MHz, (CD 3 ) 2 SO, mixture of rotamers) δ 8.12 (d, J = 1.7 Hz, 1H), 7.29 - 7.13 (m, 1H), 4.26 (tt, J = 9.8, 7.5 Hz, 1H), 4.00 - 3.87 (m, 1H), 2.78 (dtd, J = 9.9, 8.1, 2.8 Hz, 2H), 2.59 - 2.52 (m, 2H).

[0359] Intermediate 5d: 2-chloro-9-(3-hydroxycyclobutyl)-7-methyl-7,9-dihydro-8H-purin-8-one [ka] Intermediate 5d was synthesized from intermediate 5c (61%) using the method used for intermediate 1h. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.32 (d, J = 2.4 Hz, 1H), 4.26 (tt, J = 9.8, 7.5 Hz, 1H), 3.98 - 3.85 (m, 1H), 3.31 (d, J = 2.4 Hz, 3H), 2.81 - 2.65 (m, 2H), 2.53 (ddt, J = 7.5, 4.1, 2.0 Hz, 2H).

[0360] Compound 5: 9-(3-hydroxycyclobutyl)-7-methyl-2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purin-8-one [ka] Compound 5 was synthesized from intermediate 5d and intermediate 1d using the method used for compound 1. 1H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.15 (s, 1H), 8.61 (s, 1H), 8.38 (s, 1H), 8.10 (s, 1H), 7.72 (s, 1H), 5.15 (d, J = 6.1 Hz, 1H), 4.26 - 4.17 (m, 1H), 3.94 (sextet, J = 6.8 Hz, 1H), 3.30 (s, 3H), 2.78 (qd, J = 8.3, 2.6 Hz, 2H), 2.61 - 2.54 (m, 2H), 2.41 - 2.39 (m, 3H). MS: 367.4 m / z [M+H].

[0361] Example 7 - Compound 6 Intermediate 6a: ethyl 6-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)nicotinate [ka] Intermediate 6a was synthesized from 4,6-dichloropyridine-3-carboxylate and tetrahydropyran-4-amine (46%) using the method used for intermediate 1e. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.61 (s, 1H), 8.13 (d, J = 7.9 Hz, 1H), 7.05 (s, 1H), 4.36 (q, J = 7.1 Hz, 2H), 3.90 (dt, J = 11.7, 3.8 Hz, 3H), 3.54 (td, J = 11.4, 2.2 Hz, 2H), 1.96 (dd, J = 12.6, 3.6 Hz, 2H), 1.52 (dtd, J = 12.7, 10.6, 4.3 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H).

[0362] Intermediate 6b: 6-chloro-4-((tetrahydro-2H-pyran-4-yl)amino)nicotinic acid [ka] Intermediate 6b was synthesized from intermediate 6a (74%) using the method used for intermediate 1f. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.57 (s, 1H), 8.36 (d, J = 8.0 Hz, 1H), 7.00 (s, 1H), 3.92 - 3.81 (m, 3H), 3.54 (td, J = 11.4, 2.2 Hz, 3H), 2.04 - 1.90 (m, 2H), 1.56 - 1.42 (m, 2H).

[0363] Intermediate 6c: 6-chloro-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Intermediate 6c was synthesized from intermediate 6b (76%) using the method used for intermediate 1g. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 11.32 (s, 1H), 7.94 (s, 1H), 7.44 (s, 1H), 4.38 (tt, J = 12.2, 4.2 Hz, 1H), 3.94 (dd, J = 11.5, 4.5 Hz, 2H), 3.42 (td, J = 11.9, 1.9 Hz, 2H), 2.31 (qd, J = 12.4, 4.6 Hz, 2H), 1.69 - 1.56 (m, 2H).

[0364] Intermediate 6d: 6-chloro-3-methyl-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Intermediate 6d was prepared by distillation of intermediate 6c in THF / H using the method used for intermediate 1h. 2O (2:1) solution (63%). 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.15 (s, 1H), 7.50 (s, 1H), 4.43 (tt, J = 12.1, 4.2 Hz, 1H), 3.94 (dd, J = 11.5, 4.5 Hz, 2H), 3.43 (td, J = 11.9, 1.9 Hz, 2H), 3.32 (s, 3H), 2.32 (qd, J = 12.4, 4.6 Hz, 2H), 1.63 (ddd, J = 12.2, 4.3, 1.9 Hz, 2H).

[0365] Compound 6: 3-Methyl-6-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Compound 6 was synthesized from intermediate 6d and intermediate 1f using the method used for compound 1. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 9.78 (s, 1H), 8.31 (s, 1H), 8.03 (s, 1H), 8.00 (s, 1H), 7.69 (s, 1H), 7.24 (s, 1H), 4.44 (d, J = 12.5 Hz, 1H), 4.04 (dd, J = MS: 380.4 (tt, J = 12.3, 7.0 Hz, 2H), 1.75 - 1.67 (m, 2H). MS: 380.4 m / z [M+H].

[0366] Example 8 - Compound 7 Intermediate 7a: 4,6-Dimethyl-5-nitropyridin-2-amine [ka] 4,6-Dimethylpyridin-2-amine (50 g, 1.0 equiv.) 2 SO 4 To the solution, add HNO 3 (3.25 equivalents) and H 2 SO 4 (2.3 equiv.) was added dropwise at -10°C. After addition, the mixture was stirred at this temperature for 1 h. The reaction mixture was diluted with NH 3 H 2 Quench by dropwise addition of HO at 0 °C. 2 O and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure to give a residue that was purified by column chromatography to give the product as a yellow solid (19%). 1 H NMR (400 MHz, CDCl 3 ) δ 7.70 (s, 1H), 2.53 (s, 3H), 2.45 (s, 3H).

[0367] Intermediate 7b: (E)-N'-(4,6-dimethyl-5-nitropyridin-2-yl)-N,N-dimethylformimidamide [ka] A solution of intermediate 7a (11.8 g, 1.0 equiv.) and DMF-DMA (1.1 equiv.) in toluene (0.7 M) was degassed and diluted with N 2 The mixture was purged with 3× with N 3 at 110° C. for 2 h. The reaction mixture was concentrated under reduced pressure to give a residue which was used directly in the next reaction without further purification.

[0368] Intermediate 7c: (E)-N'-(4,6-dimethyl-5-nitropyridin-2-yl)-N-hydroxyformimidamide [ka] A mixture of intermediate 7b (10 g, 1.0 equiv.) and hydroxylamine hydrochloride (2.0 equiv.) in MeOH (0.4–0.5 M) was degassed and diluted with N 2 Purge the mixture three times with N2 The mixture was stirred at 80° C. for 1 hour under atmospheric pressure. The reaction mixture was concentrated under reduced pressure, and the resulting residue was dissolved in NaHCO 3 It was diluted with aqueous solution and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give the product (19%) as a yellow solid. 1 H NMR (400 MHz, CDCl 3 ) δ 9.97 (d, J = 9.6 Hz, 1H), 8.27 (d, J = 9.5 Hz, 1H), 6.68 (s, 1H), 2.54 (s, 3H), 2.46 (s, 3H).

[0369] Intermediate 7d: 5,7-Dimethyl-6-nitro-[1,2,4]triazolo[1,5-a]pyridine [ka] To a mixture of intermediate 7c (2.2 g, 1.0 equiv.) in THF (0.5 M) was added TFAA (1.5 equiv.). The mixture was diluted with N 2 The mixture was stirred at 25° C. for 18 hours under atmospheric pressure. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was dissolved in NaHCO 3 It was diluted with aqueous solution and extracted three times with EtOAc. The combined organic layers were concentrated under reduced pressure and purified by column chromatography to give the product as a pale yellow solid (55%).

[0370] Intermediate 7e: 5,7-Dimethyl-[1,2,4]triazolo[1,5-a]pyridin-6-amine [ka] To a solution of intermediate 7d (1.1 g, 1.0 equiv.) in EtOH (0.5-0.6 M), NH 4 CO 2 H (1.0 equiv.) and Pd / C (10 w / w%, 1.0 equiv.) were added. The mixture was stirred at 105° C. for 2 h. The reaction mixture was filtered, concentrated under reduced pressure, and purified by column chromatography to give the product as a white solid (64%). 1 H NMR (400 MHz, (CD3 ) 2 SO) δ 8.15 (s, 1H), 7.38 (s, 1H), 4.77 (s, 2H), 2.58 (s, 3H), 2.28 (s, 3H).

[0371] Compound 7: 6-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-3-methyl-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Intermediate 7e (1.0 equivalent), Intermediate 6d (1.0 equivalent), BrettPhos Pd G3 (0.1 equivalent), Cs 2 CO 3 A solution of (2.0 equiv.) in DMF (0.15 M) was degassed with N 2 Purge the mixture three times with N 2 The mixture was stirred at 100° C. under atmospheric pressure for 18 hours. The reaction mixture was poured into water and extracted three times with DCM. The combined organic phase was washed with brine and diluted with anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the filtrate was concentrated in vacuo The residue was purified by column chromatography to give the product as a pale yellow solid. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.44 (s, 1H), 8.05 (s, 1H), 7.71 (s, 1H), 7.63 (s, 1H), 6.68 (s, 1H), 4.06 - 3.96 (m, 2H), 3.50 (d, J = 11.9 Hz, 2H), 3.26 (s, 3H), 2.60 (s, 3H), 2.29 (s, 5H), 1.70 (d, J = 11.5 Hz, 2H). MS: 394.4 m / z [M+H].

[0372] Example 9 - Compound 8 Compound 8: 2-((5,7-dimethyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7-methyl-9-(tetrahydro-2H-pyran-4-yl)-7,9-dihydro-8H-purin-8-one [ka] Intermediate 7e (1.0 equivalent), Intermediate 1h (1.0 equivalent), Cs 2 CO 3 (2.0 equivalent), Pd(dppf)Cl 2 Degas a mixture of XantPhos (0.2 equiv.) and XantPhos (0.4 equiv.) in DMF (0.1–0.2 M) and purify with N 2 Purge the mixture three times with N 2 The mixture was stirred at 130° C. under atmospheric pressure for 24 hours. The mixture was poured into water and extracted three times with DCM. The combined organic phase was washed with brine and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the filtrate was concentrated in vacuo The residue was purified by column chromatography to give the product as a brown solid. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.76 (s, 1H), 8.49 (s, 1H), 7.98 (s, 1H), 7.68 (s, 1H), 4.44 (s, 1H), 4.03 - 3.93 (m, 2H), 3.47 (d, J = 12.5 Hz, 2H), 3.32 (s, 3H), 2.64 (s, 3H), 2.34 (s, 3H), 1.71 (d, J = 12.3 Hz, 2H). MS: 395.4 m / z [M+H].

[0373] Example 10 - Compound 9 Intermediate 9a: Ethyl 4,6-dichloro-5-methylnicotinate [ka] A mixture of 4,6-dichloro-5-methyl-pyridine-3-carboxylic acid (1.8 g, 1.0 equiv.) in EtOH (0.4-0.5 M) was added to H 2 SO4 (1.0 equiv.) was added dropwise. The mixture was stirred at 80° C. for 12 h. The reaction mixture was diluted with NaHCO 3 The combined organic phase was washed with brine and anhydrous Na 2 SO 4 The mixture was dried at 40° C., filtered and the filtrate was concentrated in vacuo The residue was purified by column chromatography to give the product as a colorless oil (59%). 1 H NMR (400 MHz, CDCl 3 ) δ 8.55 (d, J = 4.2 Hz, 1H), 4.36 (pd, J = 6.9, 3.9 Hz, 2H), 2.49 (d, J = 4.3 Hz, 3H), 1.36 (td, J = 7.3, 4.0 Hz, 3H).

[0374] Intermediate 9b: ethyl 6-chloro-5-methyl-4-((tetrahydro-2H-pyran-4-yl)amino)nicotinate [ka] Intermediate 9b was synthesized from intermediate 9a (50%) using the method used for intermediate 1e. 1 H NMR (400 MHz, (CD 3 ) 2 SO) δ 8.44 (s, 1H), 7.43 (d, J = 9.3 Hz, 1H), 4.31 (q, J = 7.1 Hz, 2H), 3.79 (dt, J = 11.7, 3.8 Hz, 2H), 3.67 (tq, J = 9.7, 4.9, 4.1 Hz, 1H), 3.36 (dd, J = 11.5, 2.2 Hz, 2H), 2.30 (s, 3H), 1.84 - 1.75 (m, 2H), 1.41 (dtd, J = 17.5, 10.6, 9.6, 4.4 Hz, 2H), 1.31 (t, J = 7.1 Hz, 3H).

[0375] Intermediate 9c: 6-chloro-5-methyl-4-((tetrahydro-2H-pyran-4-yl)amino)nicotinic acid [ka] Intermediate 9c was synthesized from intermediate 9b (83%) using the method used for intermediate 1f.

[0376] Intermediate 9d: 6-chloro-7-methyl-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Intermediate 9c (0.45g, 1.0eq), Et 3 To a mixture of N (1.0 equiv.) in DMA solution (0.16 M) was added DPPA (1.0 equiv.). The mixture was 2 The mixture was stirred under atmosphere for 8 hours at 120° C. The reaction mixture was poured into water and the precipitate was collected by filtration, washed with water and dried under vacuum to give a residue which was used directly in the next reaction without further purification (67%).

[0377] Intermediate 9e: 6-chloro-3,7-dimethyl-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Intermediate 9e was synthesized from intermediate 9d (79%) using the method used for intermediate 1h. 1 H NMR (400 MHz, CDCl 3 ) δ 7.79 (s, 1H), 4.55 (tt, J = 12.0, 4.2 Hz, 1H), 4.08 (dd, J = 11.8, 4.7 Hz, 2H), 3.40 (td, J = 12.2, 2.0 Hz, 2H), 2.81 (qd, J = 12.5, 4.6 Hz, 2H), 1.66 (ddd, J = 12.5, 4.2, 1.9 Hz, 2H).

[0378] Compound 9: 3,7-dimethyl-6-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1,3-dihydro-2H-imidazo[4,5-c]pyridin-2-one [ka] Compound 9 was synthesized from intermediate 1d and intermediate 9e using the method used for compound 9. 1H NMR (400 MHz, DMSO) δ 8.63 (s, 1H), 8.30 (s, 1H), 7.72 (s, 1H), 7.64 (s, 1H), 7.54 (s, 1H), 4.65 - 4.56 (m, 1H), 3.95 (dd, J = 11.4, 4.5 Hz, 2H), 3.43 (t, J = 11.8 Hz, 2H), 3.22 (s, 3H), 2.69 - 2.52 (m, 2H), 2.50 (s, 3H), 2.21 (d, J = 1.1 Hz, 3H), 1.71 (d, J = 11.4 Hz, 2H). MS: 394.5 m / z [M+H].

[0379] Example 11 -TRAC LNP treatment of T cells with or without DNA-PK inhibitors T cells were thawed from liquid N2 storage and placed overnight in 2.5% human serum T proliferation activation medium (TCGM:CTS OpTmizer (Thermofisher #A3705001) containing 2.5% heat inactivated human AB serum (Gemini #100-512), 1x GlutaMAX (Thermofisher #35050061), 1% penicillin / streptomycin (Thermofisher #15140-122), 10 mM HEPES pH 7.4 (Thermofisher #15630080), IL-2 (200U / mL, Peptrotech #200-02), IL-7 (5ng / mL, Peptrotech #200-07), and IL-15 (5ng / mL, Peptrotech #200-15).

[0380] After overnight rest, T cells were activated with TransAct (1:100 dilution, Miltenyi) for 48 h before insertion. T cells were harvested, washed, and resuspended in TCGM without serum at 1.25 × 10 6 The concentration of cells / mL was adjusted to 100 μg / mL. LNP-ApoE solution was prepared at LNP concentration of 5 μg / mL in 5% human serum TCGM containing 1 μg / mL ApoE3 and incubated at 37°C for 10 minutes. LNP composition was formulated as described in Example 1 with mRNA encoding Cas9 (SEQ ID NO: 8) and sgRNA targeting human TRAC (G013006 SEQ ID NO: 1) in lipid molar ratios of component lipids lipid A, cholesterol, DSPC, and PEG2k-DMG at 50 / 38.5 / 10 / 1.5 or 35 / 47.5 / 15 / 2.5, respectively. The cargo weight ratio of sgRNA to Cas9 mRNA was 1:2. LNP-ApoE mix was mixed with T cells (50,000 cells / well) at a volume of 1:1. AAV encoding a homologous recombination repair template for GFP open reading frame (OFR) insertion into the TRAC locus (SEQ ID NO: 13) was added at 3×10 5 The cells were added at an MOI of viral genome / cell. DNA-PK inhibitors (compound 1, compound 2, compound 3, compound 4, compound 5, compound 6, compound 7, compound 8, or compound 9) were diluted in 2.5% serum TCGM and added to the cells to a final concentration of 1 μM, 0.25 μM, or 0.0625 μM. The next day, T cells were spun down in 96-well plates at 500 g for 5 min to remove the medium, washed once, resuspended in 2.5% serum TCGM, and grown for 5 days. During the 5-day growth, cells were split once into fresh 2.5% serum TCGM to prevent overgrowth on day 2.

[0381] Flow Cytometry Five days after editing, the phenotype of T cells was determined by flow cytometry to determine endogenous TCR knockout and GFP insertion. The T cell receptor alpha chain, encoded by TRAC, is required for the assembly and translocation of the T cell receptor / CD3 complex to the cell surface. Thus, disruption of the TRAC gene by genome editing causes CD3 protein loss at the cell surface of T cells. Briefly, edited T cells were stained with FACS buffer (PBS pH 7.4, 2% FBS, 1 mM EDTA) containing an antibody targeting CD3 (1:200) and incubated on ice protected from light for 30 minutes. Cells were then washed and resuspended in FACS buffer containing DAPI (1:5,000) and incubated on ice protected from light for 10 minutes. After staining, T cells were washed and resuspended in FACS buffer and analyzed using a CytoFLEX LX cytometer. T cells were gated for size, DAPI staining, and GFP and CD3 expression. The results are shown in Table 2 and Figure 1A. GFP positive cells were gated within the CD3 negative population as per Table 3 and Figure 1B. [Table 3-1] [Table 3-2] [Table 4-1] [Table 4-2]

[0382] Example 12 - Engineering functionally active TCR T cells using CRISPR / Cas9 and DNA-PK inhibitors The use of DNA-PK inhibitors to promote transgenic TCR (tgTCR) insertion in T cells without perturbing T cell proliferation, cytotoxicity, or cytokine release was evaluated.

[0383] 12.1. Isolation of T cells Healthy human donor apheresis was obtained commercially (HemaCare) from three donors (designated 007HD, 008HD, and 009HD). Cells were washed and resuspended in CliniMACS PBS / EDTA buffer (Miltenyi Cat. No. 130-070-525) in a LOVO device. T cells were isolated by positive selection using CD4 and CD8 magnetic beads (Miltenyi BioTec Cat. No. 130-030-401 / 130-030-801) using CliniMACS Plus and CliniMACS LS disposable kits. T cells were aliquoted into vials and cryopreserved in a 1:1 formulation of Cryostor CS10 (StemCell Technologies Cat. No. 07930) and Plasmalyte A (Baxter Cat. No. 2B2522X) for future use.

[0384] 12.2. T Cell Culture and Thawing T cells were thawed from liquid N2 storage and placed overnight in 2.5% human serum T cell activation medium (TCAM:CTS OpTmizer (Thermofisher #A3705001) containing 2.5% heat inactivated human AB serum (Gemini #100-512), 1x GlutaMAX (Thermofisher #35050061), 1% penicillin / streptomycin (Thermofisher #15140-122), 10 mM HEPES pH 7.4 (Thermofisher #15630080), IL-2 (200 U / mL, Peptrotech #200-02), IL-7 (5 ng / mL, Peptrotech #200-07), and IL-15 (5 ng / mL, Peptrotech #200-15)).

[0385] 12.3.T cell manipulation The resting T cells were counted and cultured at 2 × 10 in TCGM supplemented with TransAct Reagent at a dilution of 1:50. 6The T cells were resuspended at a density of 1×10 cells / mL. Meanwhile, 5 μg / mL TRBC-LNPs formulated with mRNA encoding Cas9 (SEQ ID NO: 8) and sgRNA targeting TRBC (G016239) (SEQ ID NO: 2) were incubated in TCAM containing 1 μg / mL recombinant human ApoE3, then mixed with T cells in a 1:1 volume and incubated at 37° C. for 48 hours. After 48 hours of activation, T cells were harvested, washed, and resuspended in TCAM to a concentration of 1×10 6 TRAC-LNP-ApoE solution was prepared at 5 μg / mL in TCAM containing 5 μg / mL ApoE3. TRAC-LNP was formulated with mRNA encoding Cas9 (SEQ ID NO: 8) and sgRNA targeting TRAC (G013006) (SEQ ID NO: 1). LNP-ApoE mix and T cells were mixed in a 1:1 volume. AAV encoding a homologous recombination repair template for insertion into the TRAC locus (SEQ ID NO: 13) of HD1, a WT1-specific TCR, was added at 3×10 5 The cells were added at an MOI of viral genomes / cell. DNA-PK inhibitors were added at a concentration of 0.25uM as indicated in Table 4. The next day, T cells were washed and resuspended in T cell growth medium (TCEM: as described for TCAM except with 5% human AB serum instead of 2.5%) and transferred to GREX plates (Wilson Wolf #80240M) and expanded for an additional 6 days, with cytokine supplementation every 2-3 days. Control samples were treated as above omitting any DNA-PK inhibitor treatment. Cells were harvested after expansion and counted using a Vi-CELL XR Cell Counter and characterized by flow cytometry. Fold expansion was determined by dividing the total cell count yield at the endpoint by the number of cells in each group at day 0 (i.e., starting material). No effect on T cell proliferation was observed with DNA-PK treatment with compounds 6, 7, and 8 (Table 4). T cells were cryopreserved in Cryostor CS10 cryopreservation medium for future analysis. [Table 5]

[0386] Flow Cytometry After manipulation and expansion, T cells were characterized for editing efficiency and memory phenotype using flow cytometry. Briefly, T cells were stained for 30 min at room temperature with an antibody cocktail targeting CD4, CD8, CD3ε, Vβ8, CD45RA, CD45RO, CD62L, and CCR7 diluted in FACS buffer (PBS pH 7.4, 2% FBS, 1 mM EDTA). The Vβ8 antibody recognizes the specific Vβ chain used by the WT1-TCR. After staining, T cells were washed and resuspended in FACS buffer and analyzed using a CytoFLEX LX cytometer. There was no effect of the inhibitors on the percentage of CD8+ T cells within each group (Figure 2A). A statistically significant increase (p<0.05, Student's t-test) in the percentage of CD8+ cells (CD3ε+, Vβ8+) with WT1-TCR insertion was observed in all DNA-PK inhibitor-treated groups compared to the untreated group (Table 5, Fig. 2C), and a trend toward increased endogenous TCR KO was also observed (Fig. 2B). [Table 6]

[0387] 12.5.WT1 TCR T cells were identified in 697 ALL cell line and K562-HLA-A * 02:01 Mediated cytotoxicity and cytokine release in response to CML cell lines. The ability of engineered WT1-TCR T cells from donors 007HD and 008HD to kill hematologic cancer cells expressing native levels of WT1 and to release cytokines was evaluated. TCR KO cells generated as above, without the addition of TRAC / AAV, were used as a non-killing negative control. Briefly, T cells were cultured with luciferase-expressing 697 ALL cells (697-luc2) or HLA-A in TCEM without the addition of IL-2, IL-7, or IL-15. * K562-luc2 cells transduced to express 02:01 (K5692-HLA-A *02:01-luc2) at various effector to target (E:T) ratios (2:1, 1:1, 0.5:1). Of note, effector to target ratios were normalized for the relative WT1 TCR insertion of each group, resulting in the same amount of absolute WT1 TCR expressing cells across inhibitor and untreated groups. After 24 hours of co-culture, supernatants from the 2:1 E:T group were collected and used in the MSD U / R-PLEX assay to quantify IL-2, TNFα, IFNγ, and Granzyme B according to the manufacturer's (Mesoscale Discovery) operating procedure. After 48 hours of co-culture, luciferase activity was quantified in relative light units (RLU) using the Bright-GLO Luciferase Assay System (Promega). Percent specific lysis was calculated using the formula: % specific lysis = 100-((RLU[experimental wells] / RLU[target only wells]) * 100) was determined using The results from the cytotoxicity assay are shown in Figures 3A-3D and Table 6, and cytokine release is reported in Table 7 and Figures 4A-4H. No significant differences were observed regarding T cell functionality in the groups treated with DNA-PK inhibitors. [Table 7] [Table 8]

[0388] Example 13 - Editing in B cells using DNA protein kinase inhibitors The effect of DNA protein kinase inhibitors (DNA-PKIs) on editing efficiency in B cells was evaluated.

[0389] B cells were isolated from leukopaks of healthy human donors by CD19 positive selection using the StraightFrom Leukopak CD19 MicroBead kit (Miltenyi, 130-117-021) on a MultiMACS Cell24 Separator Plus instrument. After MACS isolation, CD19+ B cells were activated in IMDM or Stemspan medium and frozen until needed. The basal medium was IMDM (Corning, 10-016-CV) or StemSpan SFEM (StemCell Technologies, 9650) supplemented with 1% penicillin / streptomycin (Corning, 30-002-CI), 50ng / ml hIL-2 (Peprotech, 200-02), 50ng / ml hIL-10 (Peprotech, 200-10), 10ng / ml hIL-15 (Peprotech, 200-15), 100ng / ml MEGACD40L, 1ug / ml CpG ODN 2006 (Invivogen, TLR-2006) and 10% fetal bovine serum (FBS). B cells were thawed and cultured in Stemspan medium supplemented with 1% penicillin / streptomycin (Corning, 30-002-CI), 50ng / ml hIL-2 (Peprotech, 200-02), 50ng / ml hIL-10 (Peprotech, 200-10), 10ng / ml hIL-15 (Peprotech, 200-15), 1ng / ml MEGACD40L, 1ug / ml CpG ODN 2006 (Invivogen, TLR-2006) and 5% human AB serum (Gemini Bio-Products, 100-512). After 2 days of culture, cells were harvested and resuspended at 100,000 cells / 100 μl in StemSpan medium containing 1% penicillin / streptomycin supplemented with 2x final concentrations of cytokines, 2ug / ml CpG ODN 2006 (Invivogen, TLR-2006) and 2ng / ml MEGACD40L prior to treatment with LNP compositions delivering mRNA encoding Cas9 (SEQ ID NO:8) and gRNA G000529 targeting B2M.

[0390] LNPs were generally prepared as described in Example 1 with a lipid composition of 50 / 38.5 / 10 / 1.5, expressed as the molar ratio of ionizable lipid / cholesterol / DSPC / PEG, respectively. LNPs were preincubated with 1.25 μg / ml ApoE4 (Peprotech, 350-04) at a concentration of 5 μg / ml total RNA cargo in StemSpan medium supplemented with 1% penicillin / streptomycin and 5% human AB serum (Gemini Bio-Products, 100-512) for approximately 15 minutes at 37° C. Preincubated LNPs were added to B cells at a final concentration of 2.5 ug / ml total RNA cargo, followed by the addition of DNAPK inhibitors Compound 1, Compound 3, or Compound 4 at 0.25 ug / ml.

[0391] On day 7 after LNP composition treatment, B cell phenotype was determined for the presence of B2M surface protein. In this case, B cells were incubated with an antibody targeting CD86 (Biolegend, 374216) and an antibody targeting B2M (Biolegend, 316312). Cells were then stained with a viability dye (Biolegend, 422801), washed, processed on a Cytoflex instrument (Beckman Coulter), and analyzed using the FlowJo software package. B cells were gated on size and viability status, and then gated on B2M expression against the total viable population. Percentage of B2M negative cells is shown in Table 8. An increased percentage of B2M negative B cells was observed in the presence of DNA-PKI compared to the absence of DNA-PKI, indicating increased gene editing. [Table 9]

[0392] 13.2. Editing in B Cells from Multiple Donors Using DNAPK Inhibitors B cells were isolated from PBMCs from three donors as described in Example 23.1. After MACS isolation, CD19+ B cells were activated in Stemspan medium containing 1 ug / ml CpG ODN 2006 (Invivogen, TLR-2006), 2.5% human AB serum (Gemini Bio-Products, 100-512), 1% penicillin-streptomycin (ThermoFisher, 15140122), 50 ng / ml IL-2 (Peprotech, 200-02), 50 ng / ml IL-10 (Peprotech, 200-10), and 10 ng / ml IL-15 (Peprotech, 200-15) and 1 ng / ml CD40L (Enzo Life Science, ALX-522-110-C010). Two days after activation, B cells were treated with an LNP composition delivering mRNA encoding Cas9 (SEQ ID NO:8) and gRNA G000529 targeting B2M. B cells were seeded in triplicate as shown in Table 9 at 50,000 cells per well in Stemspan complete medium as described above.

[0393] LNPs were generally prepared as in Example 1 with a lipid composition of 50 / 38.5 / 10 / 1.5, expressed as the molar ratio of ionizable lipid / cholesterol / DSPC / PEG, respectively. LNPs were preincubated with Stemspan medium containing 1 μg / mL CpG ODN 2006, 2.5% human AB serum, 1% penicillin-streptomycin, 50 ng / mL IL-2, 50 ng / mL IL-10, and 10 ng / ml IL-15, 1 ng / ml CD40L, and 1.25 μg / mL ApoE4 for 15 minutes at 37° C. The preincubated LNP composition was added to B cells at a final concentration of 2.5 μg / mL total RNA cargo, followed by the addition of DNAPK inhibitors Compound 1 or Compound 4 at 0.25 μg / mL. 72 hours after addition of the LNP composition, cells were washed and resuspended in Stemspan medium containing 1 μg / mL CpG ODN 2006, 2.5% human AB serum, 1% penicillin-streptomycin, 50 ng / mL IL-2, 50 ng / mL IL-10, and 10 ng / mL IL-15, and 100 ng / mL CD40L and transferred to a 48-well plate.

[0394] After 7 days of LNP composition treatment, the phenotype of cells was determined by flow cytometry. Briefly, B cells were incubated with antibodies targeting CD19 (Biolegend, 363010A), CD20 (Biolegend, 302322), CD86 (Biolegend, 374216) and B2M (Biolegend, 395806), followed by incubation with the viability dye DAPI (Biolegend, 422801). Cells were then washed, processed on a Cytoflex instrument (Beckman Coulter) and analyzed using the FlowJo software package. B cells were gated on size and viability, followed by B2M expression versus the total viable population. Table 9 and Figure 5 show the average B2M negative cell percentage after editing with DNAPK inhibitors. The addition of DNAPK inhibitors moderately improved the editing efficiency. [Table 10]

[0395] Example 14 - Insertion into NK cells using DNAPK inhibitors NK cells were evaluated for the effect of DNA protein kinase inhibitors (DNA-PKI) on indel and insertion rates. NK cells were treated with LNP compositions delivering mRNA encoding Cas9 (SEQ ID NO: 8) and gRNA G000562 targeting AAVS1 in the presence of DNA protein kinase inhibitors. A subset of samples was also treated with AAV (SEQ ID NO: 16) encoding a GFP coding sequence flanked by regions of homology to the AAVS1 editing site.

[0396] NK cells were isolated from commercially available leukopaks using EasySep Human NK Cell Isolation Kit (STEMCELL, Cat. No. 17955) according to the manufacturer's operating instructions. Human primary NK cells were activated in OpTmizer medium containing 5% human AB serum, 500U / mL IL-2, and 5ng / ml IL-15 and expanded for 3 days using K562-41BBL cells as feeder cells. NK cells were seeded in triplicate at 50,000 cells per well in OpTmizer supplemented as above with DNA-PKI at the concentrations shown in Tables 10 and 11. LNPs were pre-incubated with 10ug / ml APOE3 in OpTmizer medium containing 2.5% human AB serum, 500U / mL IL-2, and 5ng / ml IL-15 at 37°C for approximately 15 minutes. Pre-incubated LNP compositions were added to NK cells suspended in the same medium in triplicate at a final concentration of 10ug / ml total RNA cargo. In a subset of samples, AAV encoding GFP flanked by regions homologous to the AAVS1 editing site was added after editing at a multiplicity of infection (MOI) of 600,000 genome copies. Seven days after LNP composition treatment, cells were phenotyped by flow cytometry to measure GFP insertion rate. Briefly, NK cells were incubated with antibodies targeting CD3 (Biolegend, Cat. No. 317336) and CD56 (Biolegend, Cat. No. 318310). Cells were then washed, processed on a Cytoflex instrument (Beckman Coulter), and analyzed using the FlowJo software package. NK cells were gated for size, CD3 / CD56 status, and GFP expression. High GFP expressing cells were gated as targeted GFP insertion in the AAVS1 locus and low GFP expressing cells were gated as episomal retention. Cells were then harvested for NGS analysis as described in Example 1.4.

[0397] Tables 10 and 11 and Figures 6A and 6B show the percent editing following treatment with LNP compositions, AAV, and various concentrations of the DNAPK inhibitors Compound 1 and Compound 4. Both indel formation and insertions were increased in the presence of the DNAPK inhibitors. [Table 11] [Table 12]

[0398] Example 15 - Multiple editing with two insertions in T cells To demonstrate engineering of T cells with five distinct Cas9 edits, healthy donor cells were sequentially treated with five LNP compositions formulated with mRNA encoding Cas9 (SEQ ID NO:8) and either sgRNA targeting TRAC (G013006), TRBC (G016239), CIITA (G013676), HLA-A (G018995), or AAVS1 (G000562). Transgenic WT1 targeting the TCR was site-specifically integrated at the TRAC cleavage site by using AAV to deliver a homology-directed repair template (SEQ ID NO:14). As a proof of concept, GFP was also site-specifically integrated at the AAVS1 target site using a second homology-directed repair template (SEQ ID NO:15).

[0399] T cells were isolated from two healthy subjects with HLA-A *T cells were isolated from leukapheresis products (STEMCELL Technologies) from 02:01+ donors using the EasySep Human T cell Isolation Kit (STEMCELL Technologies, 17951) according to the manufacturer's operating instructions and cryopreserved using a Cryostor CS10 (STEMCELL Technologies, 07930). The day before the start of T cell editing, cells were thawed and placed overnight in T cell activation medium (TCAM:CTS OpTmizer (Thermofisher, A3705001) supplemented with 2.5% human AB serum (Gemini, 100-512), 1x GlutaMAX (Thermofisher, 35050061), 10 mM HEPES (Thermofisher, 15630080), 200 U / mL IL-2 (Peprotech, 200-02), 5 ng / mL IL-7 (Peprotech, 200-07), and 5 ng / mL IL-15 (Peprotech, 200-15).

[0400] LNP Composition Treatment and Expansion of T Cells LNPs were generally prepared as described in Example 1 with a lipid composition of 50 / 38.5 / 10 / 1.5, expressed as the molar ratios of ionizable lipid / cholesterol / DSPC / PEG, respectively. The LNP composition was pre-incubated in ApoE-containing medium immediately prior to exposure to T cells. The experimental design of the sequential editing steps and control group is described in Table 12. [Table 13]

[0401] Day 1: CIITA-targeted LNP compositions shown in Table 12 were incubated at a concentration of 5ug / mL in TCAM containing 5ug / mL rhApoE3 (Peprotech 350-02). T cells were harvested, washed, and plated at 2x10 in TCAM containing a 1:50 dilution of T Cell TransAct, human reagent (Miltenyi, 130-111-160). 6The T cells were resuspended at a density of 1000 cells / mL. Then, the T cells and LNP-ApoE solution were mixed in a 1:1 volume ratio, and the T cells were seeded overnight in a culture flask.

[0402] Day 2: HLA-A targeted LNP compositions as shown in Table 12 were incubated at a concentration of 25ug / mL in TCAM containing 20ug / mL rhApoE3 (Peprotech 350-02).The LNP-ApoE solution was then added to the appropriate cultures at a volume ratio of 1:10.

[0403] Day 3: TRAC-targeting LNP compositions were incubated at a concentration of 5ug / mL in TCAM containing 5ug / mL rhApoE3 (Peprotech 350-02). T cells were harvested, washed, and plated at 1x10 in TCAM. 6 The T cells were resuspended at a density of 3 × 10 cells / mL. The T cells and LNP-ApoE medium were mixed at a volume ratio of 1:1, and the T cells were seeded in a culture flask. Then, WT1 AAV was added at a density of 3 × 10 5 Compound 4, a DNA-PK inhibitor, was added to each group at a concentration of 0.25 μM.

[0404] Day 4: AAVS1-targeting LNP compositions were incubated at a concentration of 5ug / mL in TCAM containing 5ug / mL rhApoE3 (Peprotech 350-02), while T cells were harvested, washed, and plated at 1x10 in TCAM. 6 The T cells were resuspended at a density of 3 × 10 cells / mL. The T cells and LNP-ApoE medium were mixed at a volume ratio of 1:1, and the T cells were seeded in a culture flask. Then, GFP-AAV was added to the T cells at a density of 3 × 10 5 Compound 4, a DNA-PK inhibitor, was added to each group at a concentration of 0.25 μM.

[0405] Day 5: TRBC-targeted LNP compositions shown in Table 12 were incubated at a concentration of 5ug / mL in TCAM containing 5ug / mL rhApoE3 (Peprotech 350-02). T cells were harvested, washed, and plated at 1x10 in TCAM. 6 The cells were resuspended at a density of 1000 cells / mL. The LNP-ApoE solution was then added to the appropriate culture medium at a 1:1 volume ratio.

[0406] Days 6-11: T cells were transferred to 24-well GREX plates (Wilson Wolf, 80192) in T cell growth medium (TCEM: CTS OpTmizer (Thermofisher, A3705001) supplemented with 5% CTS Immune Cell Serum Replacement (Thermofisher, A2596101), 1x GlutaMAX (Thermofisher, 35050061), 10 mM HEPES (Thermofisher, 15630080), 200 U / mL IL-2 (Peprotech, 200-02), 5 ng / ml IL-7 (Peprotech, 200-07), 5 ng / ml IL-15 (Peprotech, 200-15)) and expanded according to the manufacturer's operating instructions. Briefly, T cells were expanded for 6 days with medium changes every other day.

[0407] Quantification of T cell editing by flow cytometry and NGS After expansion, the edited T cells were then cloned into * Antibody targeting 02:01 (Biolegend 343307), antibody targeting HLA-DR-DP-DQ (Biolegend 361712), antibody targeting WT1-TCR (Vb8 + The cells were stained with antibodies targeting CD3e (Biolegend 348104), CD3e (Biolegend 300328), CD4 (Biolegend 317434), CD8 (Biolegend 301046), and Viakrome 808 Live / Dead (Cat. no. C36628). This cocktail was used to stain for HLA-A. *02:01 Knockout (HLA-A2 - ), HLA-DR-DP-DQ knockdown via CIITA knockout (HLA-DRDPDQ - ), WT1-TCR insert (CD3 + Vb8 + ), and the remaining endogenous TCR (CD3 + Vb8 - ) was determined. Insertion into the AAVS1 site was tracked by monitoring GFP expression. After antibody incubation, cells were washed and processed on a Cytoflex LX instrument (Beckman Coulter) and analyzed using the FlowJo software package. T cells were gated on size and CD4 / CD8 status before examining editing and insertion markers. Editing and insertion rates for CD8+ and CD4+ T cells can be found in Tables 13 and 14, respectively. Figures 7A-7F show total target editing rate graphs in CD8+ T cells. The percentage of T cells with all intended edits (i.e., WT1-TCR and GFP insertion combined with HLA-A and CIITA knockout) was compared with the percentage of T cells with all intended edits (i.e., WT1-TCR and GFP insertion combined with HLA-A and CIITA knockout) in CD3+ T cells. + Vb8 + GFP + HLA-A - HLA-DRDPDQ - High levels of HLA-A and CIITA knockout, and GFP and WT1-TCR insertion were observed in 5-fold edited samples from both donors, resulting in >75% fully edited CD8+ T cells and >85% fully edited CD4+ T cells. [Table 14] [Table 15]

[0408] Example 16 - LNP composition activity evaluated in serum media conditions To evaluate the editing efficacy of LNP compositions, LNP compositions were tested in vitro to evaluate the effect of alternative media conditions on insertion efficiency in CD3 positive T cells. T cells were treated with LNP compositions with various molar ratios of lipid components that encapsulate Cas9 mRNA and sgRNA targeting TRAC gene. AAV6 viral construct delivered a homologous recombination repair template (HDRT) that encodes a GFP reporter flanked by homology arms for site-specific integration into the TRAC locus (Vigene; SEQ ID NO: 17). TRAC gene disruption was evaluated by flow cytometry for loss of T cell receptor surface protein. Insertion was evaluated by flow cytometry for GFP emission.

[0409] The LNP composition was pre-incubated with ApoE3. An equal volume of ApoE3 medium was added to each well. Then, 100 μL of LNP-ApoE mix was added to each T cell plate. The final concentration of LNP at the highest dose was set to 5 μg / mL. The final concentration of ApoE3 at 5 μg / mL and T cells were at a final density of 0.5e6 cells / mL. The plates were incubated at 5% CO 2 The cells were incubated at 37° C. for 7 days and then harvested for flow cytometry analysis.

[0410] LNPs were generally prepared as described in Example 1 with the lipid compositions shown in Table 15, expressed as molar ratios of ionizable lipid A / cholesterol / DSPC / PEG, respectively. The LNP compositions delivered mRNA encoding Cas9 (SEQ ID NO:8) and sgRNA targeting and sgRNA targeting human TRAC (SEQ ID NO:1). The cargo weight ratio of sgRNA to Cas9 mRNA was 1:2. [Table 16]

[0411] T cells from a single donor (Lot No. W0106) were prepared as described in Example 1 with the following media modifications. T cells were seeded using media supplemented with either 2.5% human AB serum (HABS), 2.5% CTS Immune Cell SR (Gibco, Catalog No. A25961-01) serum replacement (SR), 5% serum replacement (SR), or a combination of 2.5% human AB serum and 2.5% serum replacement. T cells were activated 24 hours after thawing as described in Example 1.2. Two days after activation, T cells were transfected with LNP compositions at LNP concentrations of 0.31 μg / mL, 0.63 μg / mL, 1.25 μg / mL, and 2.5 μg / mL as described in Example 16.1. AAV6 (Vigene; SEQ ID NO: 13) encoding a homology-directed repair template (HDRT) encoding a GFP reporter flanked by homology arms for site-specific integration into the TRAC locus was delivered to 3 × 10 5 A multiplicity of infection (MOI) of viral particles / cell was added to each well. Compound 4, a small molecule inhibitor of DNA-dependent protein kinase, was added at 0.25 μM.

[0412] Five days after transfection, the phenotype of T cells was determined by flow cytometry analysis as described in Example 14 to evaluate the insertion efficiency of the LNP composition. Table 16 shows the percentage of CD3 negative cells. T cell receptor alpha chain encoded by TRAC is required for the assembly and translocation of T cell receptor / CD3 complex to the cell surface. Therefore, disruption of TRAC gene by genome editing causes CD3 protein loss on the cell surface of T cells. The average GFP positive T cell percentage of each medium condition is shown in Table 17 and Figures 8A and 8B. Cells expressing GFP protein indicate successful insertion into the genome. [Table 17] [Table 18]

[0413] 16.1. LNP transfection of T cells LNP dose-response curve (DRC) transfections were performed on a Hamilton Microlab STAR liquid handling system. The liquid handler was prepared with: (a) a 4x LNP dose of the highest desired dose in the top row of a 96 deep well plate, (b) ApoE3 diluted to 20 μg / mL in medium, (c) complete T cell expansion medium consisting of CTS OpTmizer Base Media as previously described in Example 1, and (d) 10 6 T cells were seeded at a density of 100 μg / mL. The liquid handler first performed eight 2-fold serial dilutions of LNP starting with a 4x dose of LNP in the deep-well plate. An equal volume of ApoE3 medium was then added to each well resulting in a 1:1 dilution of both LNP and ApoE3. 100uL of LNP-ApoE mix was then added to each T cell plate. The final concentration of LNP at the highest dose was set at 5μg / mL. ApoE3 at a final concentration of 5μg / mL and T cells were seeded at a final density of 0.5×10 6 The plates were incubated at 37 °C for 24 h at 5% CO 2 Activated or non-activated T cells were incubated at 37°C for 24 or 48 hours, respectively. After incubation, LNP-treated T cells were harvested and analyzed for on-target editing or Cas9 protein expression detection. The remaining cells were cultured for 7-10 days after LNP composition treatment, and protein surface expression was assessed by flow cytometry.

[0414] Example 17 - Comparison of off-target structural mutations of DNA-PKI by UnIT In this experiment, TRAC-targeting sgRNA (G013006) treated with Compound 3 or Compound 4 was assayed for off-target structural variation translocations compared to unedited or untreated TRAC sgRNA.

[0415] On the 8th day after editing, T cells from Example 12 from untreated, unedited, compound 3 guide and compound 4 guide samples were harvested, spun down and the pellets were used directly as input material for gDNA isolation using the "Zymo Quick DNA / RNA Mag Beads Kit" (Zymo Cat. No. R2131). The UnIT structural mutation characterization assay was applied to these gDNA samples. High molecular weight genomic DNA was simultaneously fragmented and sequence tagged ("tagmented") with Tn5 transposase and an adapter carrying a partial Illumina P5 sequence and a 12 bp unique molecular identifier (UMI). Two sequential PCRs were performed using primers for P5 and hemi-nested gene-specific primers (GSPs) giving Illumina P7 sequences (Illumina, ref. 15033624) to generate two Illumina-compatible NGS libraries per sample. Sequencing across both directions of the cut sites targeted by CRISPR / Cas9 using two libraries allows for the prediction and quantification of structural variation in DNA repair results after genome editing. If the two fragments are aligned to different chromosomes, the structural variation is classified as "interchromosomal translocation". The extent of structural variation is shown in Figure 9A and Table 18. The insertion percentage is shown in Figure 9B and Table 19. [Table 19] [Table 20]

[0416] Example 18 - Off-target analysis of TRAC guide and TRBC guide using DNA-PKI Screening was performed on T cells from Example 12 for validation of off-target genomic sites targeting TRAC and TRBC, performed according to Integrated DNA Technologies, IDT rhAmpSeq rhPCR protocol. In this experiment, two sgRNAs targeting TRAC and TRBC in combination with DNA-PKIs Compound 3 and Compound 4 were screened for off-target profile validation. The number of validated off-target sites for sgRNA targeting TRAC (G013006) and sgRNA targeting TRBC (G016239) are shown in Table 20. Off-target sites were validated when p-value was less than 0.05 indel percent. Of the 173 off-target sites identified for sgRNA targeting TRAC, 0 sites were validated. Of the 92 off-target sites identified for sgRNA targeting TRBC, 0 sites were validated. [Table 21]

[0417] Example 19 - SpyCas9-mediated insertion of immune receptors into TRAC with and without DNA-PK inhibitors 19.1. Preparation of T cells Apheresis of healthy human donors was obtained commercially (Hemacare, Cat. No. PB001F-2), cells were washed and resuspended in CliniMACS® PBS / EDTA buffer (Miltenyi Biotec Cat. No. 130-070-525) and processed with a MultiMACS™ Cell 24 Separator Plus device (Miltenyi Biotec). T cells were separated by positive selection using the Straight from Leukopak® CD4 / CD8 MicroBead Kit (Human) (Miltenyi Biotec Cat. No. 130-122-352). T cells were aliquoted and cryopreserved in a Cryostor® CS10 (StemCell Technologies Cat. No. 07930) for future use.

[0418] Upon thawing, T cells were cultured at 1.0 × 10 in T cell expansion medium (TCGM) consisting of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. No. A1048501), 5% human AB serum (GeminiBio, Cat. No. 100-512), 1× penicillin-streptomycin, 1× Glutamax, 10 mM HEPES, 200 U / mL recombinant human interleukin-2 (Peprotech, Cat. No. 200-02), 5 ng / mL recombinant human interleukin-7 (Peprotech, Cat. No. 200-07), and 5 ng / mL recombinant human interleukin-15 (Peprotech, Cat. No. 200-15). 6 T cells were seeded at a density of 10 ...

[0419] Example 19.2. T Cell Treatment and Expansion After 48 hours of activation, T cells were harvested, centrifuged at 500 g for 5 minutes, and cultured at 6.41 × 10 in T cell plating medium (TCPM) (a serum-free version of TCPM containing 400 U / mL recombinant human interleukin-2 (Peprotech, Cat. No. 200-02), 10 ng / mL recombinant human interleukin-7 (Peprotech, Cat. No. 200-07), and 10 ng / mL recombinant human interleukin-15 (Peprotech, Cat. No. 200-15)). 5 The T cells were resuspended at a concentration of 3.2 × 10 cells / mL in 50 μL of T cell-containing TCPM per treatment well of a flat-bottom 96-well plate. 4 T cells) were added.

[0420] LNPs were produced as described in Example 1 with a ratio of 50 / 38.5 / 10 / 1.5 (Lipid A / Cholesterol / DSPC / PEG2k-DMG). Prior to T cell treatment, two separate LNP mixes (hereafter referred to as Mix "A" and Mix "B") were prepared in T cell treatment medium (TCTM), a version of TCGM containing 20 μg / mL rhApoE3 in the absence of interleukin 2, 15, or 7.

[0421] Mix "A" consisted of LNPs containing G013006 (SEQ ID NO: 1) diluted to 13.36 μg / mL, and mix "B" consisted of LNPs containing Cas9 mRNA 60 (SEQ ID NO: 11) diluted to 13.36 μg / mL. LNP mixes "A" and "B" were incubated at 37°C for 15 minutes. Mix "A" was serially diluted 1:2 in TCTM and mixed 1:1 in volume with mix "B". The resulting solution was added in 25 μL to 3.2×10^4 T cells in a 96-well plate.

[0422] A repair template in the form of an adeno-associated virus (AAV) encoding the HD3 TCR (SEQ ID NO: 18) was then cultured at 3.84 × 10 11The resulting solution was added in 25 μL to the T cells treated with LNP in the previous step. To allow editing assessment by NGS without interference of the repair template, one group in this experiment received 25 μL of TCTM with or without compound 4 diluted to 2 μM in the absence of AAV.

[0423] After addition of LNPs, repair template, and compound 4, the T cells were incubated at 37° C. for 48 hours, at which time the T cells were centrifuged at 500 g for 5 minutes, resuspended in 200 μL of TCGM, and returned to the incubator.

[0424] Four days after treatment, cells that did not receive the AAV template were centrifuged at 500 g for 5 minutes and subjected to lysis, PCR amplification of each target locus and subsequent NGS analysis as described in Example 1. The percent indel results are shown in Table 21 and Figure 10A.

[0425] On day 4 of treatment, AAV-loaded cells were also mixed and subcultured in TCGM at a ratio of 1:4 (v / v). On day 7 of treatment, AAV-loaded cells were assessed by flow cytometry.

[0426] Example 19.3. Flow Cytometry On day 7 after LNP treatment, 50 μL of cells were transferred to a U-bottom 96-well plate and spun down at 500 g for 5 min. The supernatant was discarded and the cells were resuspended in 100 μL of FACS buffer containing Viakrome 808 (Beckman C., Catalog No. C36628) (1:100), PC5.5 anti-CD3 (Biolegend, Catalog No. 317336) (1:200), BV421 anti-CD4 (Biolegend, Catalog No. 317434) (1:100), BV785 anti-CD8 (Biolegend, Catalog No. 301046) (1:100), and anti-Vβ7.2 (Beckman C., IM3604) (1:50) and stained for 30 min at 4° C. in the dark. Cells were washed once with 200 μL FACS buffer, resuspended in 100 μL FACS buffer and processed on a Cytoflex LX flow cytometer. Percent HD3 TCR insertion results are shown in Table 22 and Figure 10B. [Table 22] [Table 23]

[0427] Additional sequence listing In the tables below and throughout, the terms "mA," "mC," "mU," or "mG" are used to indicate nucleotides that are 2'-O-Me modified.

[0428] In the table below, * " is used to indicate PS modification. * , C * , U * , or G * The term may be used to indicate a nucleotide that is linked to the next (eg, 3') nucleotide by a PS bond.

[0429] It is understood that when a DNA sequence (containing T) is referenced in relation to RNA, T should be replaced with U (which may be modified or unmodified, depending on the context), and vice versa.

[0430] In the table below, the one-letter code for amino acids is used to describe the peptide sequences. [Table 24-1] [Table 24-2] [Table 24-3] [Table 24-4] [Table 24-5] [Table 24-6] [Table 24-7] [Table 24-8] [Table 24-9] [Table 24-10] [Table 24-11] [Table 24-12] [Table 24-13] [Table 24-14]

Table 24-15

Table 24-16

Table 24-17

Table 24-18

Table 24-19

Table 24-20

Table 24-21

Table 24-22

Claims

1. Formula I: 【Chemistry 1】 or a salt thereof, During the ceremony, x １ is C-R ３ or N, R １ is C １ -C ３ is alkyl, R ２ is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R ６ is optionally replaced by R ３ is H or C １ -C ３ is alkyl, R ４ is H or C １ -C ３ is alkyl, R ５ is C １ -C ３ is alkyl, Each R ６ are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R ６ together with the atom(s) to which they are attached form a spiro or fused ring; and R ７ is H or C １ -C ３ Although it is an alkyl however, (a) x １ is C-R ３ That is, (b) R １ C ２ -C ３ is alkyl, (c) R ４ C １ -C ３ is alkyl, (d) R ２ is one R ６ is substituted with R ６ is the halo, (e) R 2 is substituted with two R 6 , which together with the atom(s) to which they are attached form a spiro or fused ring; and (f) R ２ However, one or more R ６ C optionally substituted with ３ -C ５ is cycloalkyl, The compound or a salt thereof, provided that at least one of the following conditions is met:

2. x １ is C-R ３ 2. The compound of claim 1,

3. R ３ The compound of claim 2, wherein is H or methyl.

4. x １ The compound of claim 1 , wherein is N.

5. R １ C ２ -C ３ The compound of claim 1 , wherein the aryl group is an alkyl group.

6. R １ The compound of claim 1 , wherein is selected from methyl and ethyl.

7. R ４ C １ -C ３ The compound of claim 1 , wherein the aryl group is an alkyl group.

8. R ４ The compound of claim 1 , wherein is H or methyl.

9. R ２ The compound of claim 1 , wherein is cycloalkyl.

10. R ２ The compound of claim 1 , wherein is heterocyclyl.

11. R ２ is independently selected from hydroxy, halo, methoxy, methyl, and cycloalkyl; ６ or two R 6 , taken together with the atom(s) to which they are attached, form a spiro or fused ring.

12. The compound of claim 11, wherein R 2 is substituted with two R 6 , and the two R 6 , together with the atom(s) to which they are attached, form a spiro ring or a fused ring.

13. R ５ The compound of claim 1 , wherein is methyl.

14. R ７ The compound of claim 1 , wherein is H or methyl.

15. 【Chemistry 2-1】 【Chemistry 2-2】 or a salt thereof.

16. 【Chemical 3】 、 or a salt thereof.

17. a) DNA protein kinase inhibitors (DNA-PKIs); b) DNA cutting substance; c) optionally, cells, and d) optionally donor DNA; wherein the DNA-PKI is of formula I 【Chemistry 4】 or a salt thereof, During the ceremony, x １ is C-R ３ or N, R １ is C １ -C ３ is alkyl, R ２ is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R ６ is optionally replaced by R ３ is H or C １ -C ３ is alkyl, R ４ is H or C １ -C ３ is alkyl, R ５ is C １ -C ３ is alkyl, Each R ６ are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R ６ together with the atom(s) to which they are attached form a spiro or fused ring; and R ７ is H or C １ -C ３ The composition wherein said alkyl is

18. x １ The composition of claim 17 , wherein is N.

19. R １ The composition of claim 17 or 18, wherein is methyl.

20. R ４ The composition of claim 17 or 18, wherein is H.

21. R ２ The composition of claim 17 or 18, wherein is cyclohexyl.

22. R ２ The composition of claim 17 or 18, wherein is tetrahydropyranyl or tetrahydrofuranyl.

23. R ２ is independently selected from hydroxy, methoxy, and methyl; ６ 19. The composition of claim 17 or 18, optionally substituted with

24. R ５ The composition of claim 17 or 18, wherein is methyl.

25. R ７ The composition of claim 17 or 18, wherein is H or methyl.

26. The DNA-PKI is 【Chemistry 5-1】 【Chemistry 5-2】 or a salt thereof.

27. The DNA-PKI is 【Chemistry 6】 or a salt thereof.

28. 19. The composition of claim 17 or 18, comprising a cell.

29. The composition of claim 28 , wherein the cell is a eukaryotic cell.

30. 29. The composition of claim 28, wherein the cells are useful in adoptive cell therapy (ACT).

31. The composition of claim 30 , wherein the cell is a stem cell or an immune cell.

32. The composition of claim 31, wherein the cell is an immune cell, the immune cell is a lymphocyte, and the lymphocyte is a T cell, a B cell, or a NK cell.

33. 19. The composition of claim 17 or 18, wherein the DNA cleavage agent comprises a CRISPR / Cas nuclease component and optionally a guide RNA component.

34. 34. The composition of claim 33, wherein the CRISPR / Cas nuclease component comprises a Cas nuclease or an mRNA encoding a Cas nuclease.

35. 35. The composition of claim 34, wherein the Cas nuclease is a class 2 Cas nuclease.

36. 36. The composition of claim 35, wherein the Cas nuclease is a Cas9 nuclease.

37. 37. The composition of claim 36, wherein the Cas nuclease is a S. pyogenes Cas9 nuclease or a N. meningitidis Cas9 nuclease.

38. 19. The composition of claim 17 or 18, comprising a modified RNA.

39. The composition described in claim 33, wherein the DNA cleaving agent comprises a guide RNA component, and the guide RNA component is a guide RNA nucleic acid such as a guide RNA.

40. 40. The composition of Claim 39, wherein the guide RNA nucleic acid is or encodes a dual guide RNA (dgRNA) or is or encodes a single guide (sgRNA).

41. The composition of claim 40, wherein the guide RNA nucleic acid is a gRNA, the gRNA is modified, and the modified gRNA includes a modification in one or more of the first five nucleotides at the 5' end or the 3' end.

42. 19. The composition of claim 17 or 18, comprising donor DNA.

43. 43. The composition of claim 42, wherein the donor DNA comprises a template comprising a protein-coding sequence, a regulatory sequence, or a sequence encoding a structural RNA.

44. 19. The composition of claim 17 or 18, wherein the DNA cleaving agent is present in a lipid nanoparticle (LNP) composition.

45. 1. An ex vivo method for performing targeted genome editing in a cell, for repairing double stranded DNA breaks in a cell, for inhibiting or suppressing repair of DNA breaks in a cell via a non-homologous end joining (NHEJ) pathway, or for targeted insertion of donor DNA into the genome of a cell, comprising contacting the cell with a DNA cleaving agent and a DNA-PKI, wherein the DNA-PKI is a compound represented by formula I. 【Chemistry 7】 or a salt thereof, During the ceremony, x １ is C-R ３ or N, R １ is C １ -C ３ is alkyl, R ２ is cycloalkyl or heterocyclyl, and cycloalkyl and heterocyclyl are each independently selected from one or more R ６ is optionally replaced by R ３ is H or C １ -C ３ is alkyl, R ４ is H or C １ -C ３ is alkyl, R ５ is C １ -C ３ is alkyl, Each R ６ are independently selected from hydroxy, halo, alkyl, alkoxy, cycloalkyl, amino, and cyano, or two R ６ together with the atom(s) to which they are attached form a spiro or fused ring; and R ７ is H or C １ -C ３ The method according to claim 1, wherein the aryl group is alkyl.

46. The method according to claim 45, wherein the DNA-PKI is a compound according to any one of claims 1 to 16.

47. The DNA-PKI is 【Chemistry 8】 or a salt thereof.

48. 46. ​​The method of claim 45, wherein the cells are useful in adoptive cell therapy (ACT).

49. 46. ​​The method of claim 45, wherein the cell is a stem cell or an immune cell.

50. 50. The method of claim 49, wherein the immune cell is a leukocyte or lymphocyte.

51. 46. ​​The method of claim 45, wherein the DNA cleaving agent is selected from a zinc finger nuclease, a TALE effector domain nuclease (TALEN), a CRISPR / Cas nuclease component, and combinations thereof.

52. 52. The method of Claim 51, wherein the CRISPR / Cas nuclease components comprise a Cas nuclease or an mRNA encoding a Cas nuclease.

53. 53. The method of claim 52, wherein the Cas nuclease is a class 2 Cas nuclease.

54. 54. The method of claim 53, wherein the Cas nuclease is a Cas9 nuclease.

55. 55. The method of claim 54, wherein the Cas nuclease is a S. pyogenes Cas9 nuclease or a N. meningitidis Cas9 nuclease.

56. 54. The method of claim 53, wherein the Cas nuclease is a Cas12a nuclease.

57. 46. ​​The method of claim 45, further comprising contacting the cell with a modified RNA.

58. 46. ​​The method of claim 45, further comprising contacting the cell with a guide RNA nucleic acid.

59. The method of claim 58, wherein the guide RNA nucleic acid is a modified gRNA, and the modified gRNA includes a modification in one or more of the first five nucleotides at the 5' end or the 3' end.

60. 46. ​​The method of claim 45, further comprising contacting the cell with donor DNA.

61. 61. The method of claim 60, wherein the donor DNA comprises a template comprising a protein-coding sequence, a regulatory sequence, or a sequence encoding a structural RNA.

62. 46. ​​The method of claim 45, further comprising contacting the cell with a vector.

63. Use of DNA-PKI in the manufacture of a pharmaceutical for performing targeted genome editing in a cell, for repairing double-stranded DNA breaks in a cell, for inhibiting or suppressing repair of DNA breaks in a cell via the non-homologous end joining (NHEJ) pathway, or for targeted insertion of donor DNA into the genome of a cell, comprising: The DNA-PKI is a compound according to any one of claims 1 to 16, The use, wherein performing targeted genome editing in the cell, repairing a double-stranded DNA break in the cell, inhibiting or suppressing repair of a DNA break in the cell via the non-homologous end joining (NHEJ) pathway, or the targeted insertion of donor DNA into the genome of the cell comprises contacting the cell with a DNA cleaving agent and DNA-PKI.

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