Lipid nanoparticle formulation for delivery of nucleic acids to cells

Abstract

Provided are lipid nanoparticles (LNPs) and compositions thereof for delivery of nucleic acid molecules, e.g., ribonucleic acid (RNA), into cells, such as primary cells e.g., T cells, or induced cells, e.g., iPSCs and cells differentiated from iPSCs. Also provided are methods for formulating LNPs, and for delivering nucleic acid molecules into cells, primary cells or induced cells using LNP compositions, including in connection with modulating gene expression using DNA targeting systems.

Description

224742003040 LIPID NANOPARTICLE FORMULATION FOR DELIVERY OF NUCLEIC ACIDS TO CELLS Cross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No.63 / 660,446 filed June 14, 2024, the contents of which is incorporated by reference in its entirety. Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 224742003040SEQLIST.xml, created June 13, 2025, which is 54,887 bytes in size. The information in the electronic format of the Sequence Listing is herein incorporated by reference in its entirety. Field

[0003] The present disclosure relates in some aspects to lipid nanoparticles (LNPs) and compositions thereof for delivery of nucleic acid molecules, e.g., ribonucleic acid (RNA), into cells, such as primary cells, e.g., T cells, or induced cells, e.g., iPSCs or cells derived from iPSCs. Also provided are methods for formulating LNPs, and for delivering nucleic acid molecules into cells, such as primary cells or induced cells using LNP compositions, including in connection with modulating gene expression using DNA targeting systems. Background

[0004] Delivery of protein or nucleic acid cargo into cells is necessary to produce genetically edited cells for use in gene therapy applications. Various strategies for delivering protein and nucleic acid molecules into cells are available, including transfection- and transduction-based techniques. Among strategies for use in cell therapy and gene editing methods are viral-vector based techniques of introducing nucleic acids into cells. However, the production of viral vector-based compositions is labor intensive and resource-consuming and they may be limited in the size of the cargo (e.g., nucleic acid) they can deliver. Moreover, certain viral-based vectors are capable of integrating into the genome, which may not always be desirable. sf-6779026.8 1224742003040

[0005] While certain non-viral methods, such as lipid nanoparticles (LNPs), are capable of delivering larger cargo and are non-integrating, these methods can be toxic or have low efficiency particularly in many primary cell types, including T cells, and induced cells, such as iPSCs and cells derived from iPSCs. Improved non-viral compositions for delivering large nucleic acid cargo into cells, and methods of producing and using the same, are therefore needed. Summary

[0006] Provided herein is a lipid nanoparticle (LNP) comprising: i) from 30-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. In some aspects, the lipid nanoparticle (LNP) can be for delivery to a cell such as a primary cell or an iPSC or iPSC derived cell.

[0007] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: i) from 30-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

[0008] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to an iPSC or iPSC derived cell comprising: i) from 30-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

[0009] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising: i) from 30-40 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 20-30 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. In some sf-6779026.8 2224742003040 aspects, the lipid nanoparticle (LNP) can be for delivery to a cell such as a primary cell or an iPSC or iPSC derived cell.

[0010] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: i) from 30-40 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) from 20-30 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

[0011] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to an iPSC or iPSC derived cell comprising: i) from 30-40 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) from 20-30 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

[0012] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising: i) from 45-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 5-15 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. In some aspects, the lipid nanoparticle (LNP) can be for delivery to a cell such as a primary cell or an iPSC or iPSC derived cell.

[0013] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: i) from 45-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) from 5-15 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

[0014] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to an iPSC or iPSC derived cell comprising: i) from 45-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1- piperazinebutanamine linked to a tail; ii) from 5-15 mol % of a neutral lipid; iii) a polymer sf-6779026.8 3224742003040 conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

[0015] In some of any of the provided embodiments, the ionizable lipid has a 4-methyl-1- piperazinebutanamine head group that is linked via a degradable primary ester to an octyldodecyl tail. In some of any of the provided embodiments, the ionizable lipid is linked via two degradable primary esters to two octyldodecyl tails. In some of any of the provided embodiments, the ionizable lipid is characterized by 4 saturated alkyl tails with non-symmetric 8 and 10 carbon lengths. In some of any of the provided embodiments, the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24).

[0016] In some of any of the provided embodiments, the polymer conjugated lipid is present in a concentration ranging from 1 to 5 mol %. In some of any of the provided embodiments, the polymer conjugated lipid is present in a concentration ranging from 1 to 2 mol % or 1 to 3 mol%. In some of any of the provided embodiments, the polymer conjugated lipid is present in a concentration ranging from 2 to 5 mol %. In some of any of the provided embodiments, the steroid is present in a concentration ranging from 35 to 45 mol %.

[0017] In some of any of the provided embodiments, the neutral lipid is distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), palmitoyloleoyl- phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), 1-stearoyl-2- oleoyl phosphatidylcholine (SOPC), dioleoylphosphatidylcholine (DOPC), dioleoyl- phosphatidylethanolamine (DOPE) or 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9). In some of any of the provided embodiments, the neutral lipid is DSPC. In some of any of the provided embodiments, the polymer conjugated lipid is a polyethylene glycol (PEG)- conjugated lipid. In some of any of the provided embodiments, the polymer conjugated lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). In some of any of the provided embodiments, the steroid is cholesterol.

[0018] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising: (i) about 37.5 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4- methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 22.5 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at sf-6779026.8 4224742003040 least one messenger RNA (mRNA) molecule. In some aspects, the lipid nanoparticle (LNP) can be for delivery to a cell such as a primary cell or an iPSC or iPSC derived cell.

[0019] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: (i) about 37.5 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (C24); (ii) about 22.5 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule.

[0020] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to an iPSC or iPSC derived cell comprising: (i) about 37.5 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4- methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 22.5 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule.

[0021] In some aspects, provided herein is a lipid nanoparticle (LNP) comprising: (i) about 50 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4- methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 10 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule. In some aspects, the lipid nanoparticle (LNP) can be for delivery to a cell such as a primary cell or an iPSC or iPSC derived cell.

[0022] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: (i) about 50 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (C24); (ii) about 10 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; sf-6779026.8 5224742003040 and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule.

[0023] In some aspects, provided herein is a lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a an iPSC or iPSC cell comprising: (i) about 50 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (C24); (ii) about 10 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule.

[0024] In some of any of the provided embodiments, the at least one mRNA molecule is at least 3000 nucleotides in length. In some of any of the provided embodiments, the at least one mRNA molecule is from about 3,000 to 12,000 nucleotides in length, from about 4,500 to 9,000 nucleotides in length, or from about 4,800 to 7,500 nucleotides in length. In some of any of the provided embodiments the at least one mRNA molecule encodes a protein that is from about 1,000 to 4,000 amino acids in length, from about 1,500 to 3,000 amino acids in length, or from about 1,600 to 2,500 amino acids in length.

[0025] In some of any of the provided embodiments, the RNA cargo comprises a DNA- targeting system for modulating transcription of a gene. In some of any of the provided embodiments, the RNA cargo comprises a messenger RNA (mRNA) and a guide RNA (gRNA). In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 10:1 to 1:5, optionally 10:1, 5:1, 2:1, 1:1, 1:2, or 1:5. In some of any of the provided embodiments, the mRNA cargo has a mRNA:gRNA w / w ratio of 2:1 to 1:2. In some of any of the provided embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 1:1. In some of any of the provided embodiments, the LNP has an ionizable lipid nitrogen: oligonucleotide phosphate (N:P) molar ratio of 4:1 to 20:1. In some of any of the provided embodiments, the LNP has an N:P ratio of 8:1.

[0026] In some of any of the provided embodiments, the at least one mRNA encodes a fusion protein comprising a DNA-binding domain and an effector domain. In some of any of the provided embodiments, the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof. In some of any of the provided embodiments, the Cas protein or variant thereof is a nuclease-deactivated Cas (dCas) protein. In some of any of the provided embodiments, the dCas protein is a dCas9 protein. In some of any sf-6779026.8 6224742003040 of the provided embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

[0027] In some of any of the provided embodiments, the LNP is for delivery to a primary cell. In some of any of the provided embodiments, the LNP is for delivery to an iPSC or iPSC derived cell.

[0028] In some aspects, provided herein is a method of genetically engineering a population of cells, the method comprising transfecting an RNA cargo into a population of cells by delivering any of the LNPs provided herein into the population of cells. In some of any of the provided embodiments, the population of cells are primary cells. In some of any of the provided embodiments, the population of primary cells are T cells. In some of any of the provided embodiments, the population of primary cells are hepatocytes. In some of any of the provided embodiments, the population of primary cells are HPCs. In some of any of the provided embodiments, the T cell is CD4+. In some of any of the provided embodiments, the T cell is activated prior to transfection. In some of any of the provided embodiments, the T cells are activated by contacting the T cells with humanized CD3 and CD28 agonists. In some of any of the provided embodiments, the T cells are activated between at or about 24 hours to 168 hours prior to transfection. In some of any of the provided embodiments, the T cells are activated about 48 hours to about 72 hours prior to transfection. In some of any of the provided embodiments, the T cells are cultured in serum-free media. In some of any of the provided embodiments, the population of cells are induced pluripotent stem cell (iPSC) or an iPSC derived cells. In some of any of the provided embodiments, the population of iPSC derived cells are induced hematopoietic progenitor cells (iHPCs). In some of any of the provided embodiments, the iHPCs are CD34+. In some of any of the provided embodiments, the population of iPSC derived cells are emerging induced lymphoid progenitor cells. In some of any of the provided embodiments, the population of iPSC derived cells are induced lymphoid progenitor cells. In some of any of the provided embodiments, the induced lymphoid progenitors are induced T-cell progenitors. In some of any of the provided embodiments, the induced T-cell progenitors are double positive for CD4 and CD8 cell surface markers. In some of any of the provided embodiments, the population of iPSC derived cells are emerging induced T cells. In some of any of the provided embodiments, the population of iPSC derived cells are induced Natural Killer cells. In some of any of the provided embodiments, the population of iPSC derived cells are induced T cells. In some of any of the provided embodiments, the population of sf-6779026.8 7224742003040 iPSC derived cells are CD56+. In some of any of the provided embodiments, the population of iPSC derived cells are CD8+. In some of any of the provided embodiments, the LNP is complexed with human ApoE prior to transfection. In some of any of the provided embodiments, the LNP-ApoE complex is added to the cells at an ApoE concentration of between at or about 0.5 g / mL to 2 g / mL.

[0029] In some of any of the provided embodiments, the LNP is delivered at a dose of 50 ng to 10 g per 1x106cells. In some of any of the provided embodiments, the LNP is delivered at a dose of 100 ng to 5 g per 1x106cells. In some of any of the provided embodiments, the LNP is delivered at a dose of 0.5 g to 4 g per 1x106cells. In some of any of the provided embodiments, a single dose of the LNP is delivered into the cells. In some of any of the provided embodiments, more than one dose of the LNP is delivered into the cells.

[0030] In some aspects, provided herein is a method of genetically engineering a primary cell, the method comprising transfecting a primary cell with any of the LNPs provided herein. In some of any of the provided embodiments, the primary cell is a T cell. In some of any of the provided embodiments, the T cell is CD4+. In some of any of the provided embodiments, the T cell is activated prior to transfection. In some of any of the provided embodiments, the T cell is cultured in serum-free media. In some of any of the provided embodiments, the primary cell is an induced pluripotent stem cell (iPSC) or an iPSC derived cell. In some of any of the provided embodiments, the iPSC derived cell is a hematopoietic progenitor cell (HPC). In some of any of the provided embodiments, the HPC is CD34+. In some of any of the provided embodiments, the iPSC derived cell is CD56+. In some of any of the provided embodiments, the primary cell is a hepatocyte. Brief Description of the Drawings

[0031] FIG.1A-1F show GFP expression of primary human hepatocytes (PHHs) transfected with LNPs encapsulating GFP mRNA that were formulated with different ionizable lipids. FIG. 1A shows fluorescent microscopy images for assessing GFP expression of non-transfected (NT) control cells and PHHs transfected with LNPs comprising either a MC3, C24 or ATX-100 ionizable lipid. FIG.1B shows fluorescent microscopy images for assessing GFP expression of PHHs transfected with LNPs comprising either a SM-102, ALC-0315, L319 or ckk-E12 ionizable lipid. FIG.1C shows flow cytometry plots for assessing GFP expression of NT control cells and PHHs transfected with LNPs comprising either a MC3, C24 or ATX-100 sf-6779026.8 8224742003040 ionizable lipid. FIG.1D shows flow cytometry plots for assessing GFP expression of PHHs transfected with LNPs comprising either a SM-102, ALC-0315, L319 or ckk-E12 ionizable lipid. FIG.1E shows a histogram of GFP expression and cell count (as assessed by flow cytometry) for NT control cells and PHHs transfected with LNPs comprising either a MC3, C24 or ATX-100 ionizable lipid. FIG.1F shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) for NT control cells and PHHs transfected with LNPs comprising either a MC3, C24 or ATX-100 ionizable lipid.

[0032] FIG.2A-2C show GFP and PCSK9 expression in primary human hepatocytes (PHHs) after transfection with C24 LNPs encapsulating mRNA encoding a DNMT3AL- dSpCas9-KRAB fusion protein (SEQ ID NO: 1), a PCSK9 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. FIG.2A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post- transfection. FIG.2B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.2C shows PCSK9 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non- transfected (NT) cells.

[0033] FIG.3 shows quantified in vivo indel formation for PCSK9 (as assessed by sequencing) in liver-humanized mice after injection with C24 LNPs encapsulating mRNA encoding a Cas protein and a gRNA targeting mouse PCSK9. Control conditions included a mock (1x PBS) treatment. Results are shown for 48 hours post-injection.

[0034] FIG.4A-4E show GFP expression, cell viability, and CD45 expression in primary T cells after transfection with C24 LNPs encapsulating mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non-transfected (NT) cells. FIG.4A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.4B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post- transfection. FIG.4C shows percentage of live primary T cells 48 hours post-transfection. FIG. 4D shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.4E shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection.

[0035] FIG.5A-5C show CD45 expression and cell viability of primary T cells after transfection with increased doses of C24 LNPs encapsulating mRNA encoding a dSpCas9- sf-6779026.8 9224742003040 KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non-transfected (NT) cells. FIG.5A shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.5B shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post- transfection. FIG.5C shows percentage of live primary T cells 72 hours post-transfection.

[0036] FIG.6A-6C show CD45 expression in primary T cells after transfection with C24 LNPs encapsulating a CD45 targeting gRNA and either a mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3, “dCas”) or a mRNA encoding dSpCas9-KRAB-P2A-eGFP (SEQ ID NO: 2, “dCas-P2A-eGFP”). Control conditions included non-transfected (NT) cells. FIG.6A shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.6B shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG.6C shows CD45 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non- transfected (NT) cells.

[0037] FIG.7A-7E show GFP and CD45 expression in primary T cells after transfection with C24 LNPs encapsulating mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6) at various mRNA:gRNA ratios. Control conditions included non-transfected (NT) cells. FIG.7A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.7B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post- transfection. FIG.7C shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.7D shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG.7E shows CD45 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non-transfected (NT) cells.

[0038] FIG.8A-8D show CD45 expression and cell viability of primary T cells after transfection with C24 LNPs formulated with different neutral lipids. The C24 LNPs encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non- transfected (NT) cells. FIG.8A shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.8B shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG.8C shows CD45 sf-6779026.8 10224742003040 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non-transfected (NT) cells. FIG.8D shows percentage of live primary T cells 72 hours post-transfection.

[0039] FIG.9A-9D show GFP and CD45 expression in primary T cells after transfection with C24 LNPs formulated with different pegylated lipid (PEG-lipid) concentrations. The C24 LNPs encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non- transfected (NT) cells. FIG.9A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.9B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.9C shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.9D shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection.

[0040] FIG.10A-10F show GFP expression, CD45 expression, and cell viability of primary T cells after transfection with C24 LNPs formulated with different N:P lipid:mRNA molar ratios. The C24 LNPs encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non-transfected (NT) cells. FIG.10A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.10B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.10C shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.10D shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG.10E shows CD45 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non- transfected (NT) cells. FIG.10F shows percentage of live primary T cells 72 hours post- transfection.

[0041] FIG.11A-11F show GFP expression, CD45 expression, and cell viability of primary T cells after transfection with C24 LNPs formulated with different combinations of N:P ratios and PEG-lipid concentrations. The C24 LNPs encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non-transfected (NT) cells. FIG.11A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.11B sf-6779026.8 11224742003040 shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post- transfection. FIG.11C shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.11D shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG.11E shows CD45 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non-transfected (NT) cells. FIG.11F shows percentage of live primary T cells 72 hours post-transfection.

[0042] FIG.12A-12E show GFP expression, CD45 expression, and cell viability of primary T cells after transfection with C24 LNPs. T cells were either cultured in Optimizer media (“Old”) or were starved for 30 minutes before transfection and cultured in X-VIVO 15 media (“New”). The C24 LNPs encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6). Control conditions included non-transfected (NT) cells. FIG.12A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.12B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.12C shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.12D shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG.12E shows percentage of live primary T cells 24 hours post-transfection.

[0043] FIG.13A-13F show GFP expression, CD45 expression, and cell viability of primary T cells after transfection with C24 LNPs using either the C24 LNP 1.0 formulation or the C24 LNP 2.0 formulation (summarized in Table E3). Both formulations encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. FIG. 13A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.13B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.13C shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.13D shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection. FIG. 13E shows CD45 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non-transfected (NT) cells. FIG.13F shows percentage of live primary T cells 72 hours post-transfection. sf-6779026.8 12224742003040

[0044] FIG.14A-14D show GFP expression, cell viability, and IL2 expression in iPSC derived CD56+ cells after transfection with C24 LNPs (C24 LNP 1.0). The C24 LNPs encapsulated mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4), an IL2 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non- transfected (NT) cells. FIG.14A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.14B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.14C shows percentage of live CD56+ cells 48 hours post-transfection. FIG.14D shows IL2 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to non-transfected (NT) cells.

[0045] FIG.15A-15E show GFP expression, cell viability, and B2M expression in hematopoietic progenitor cells (HPCs) after transfection with C24 LNPs (C24 LNP 2.0). The C24 LNPs encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a B2M targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non- transfected (NT) cells and cells delivered with a dCas-effector and no gRNA (No gRNA). FIG. 15A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.15B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.15C shows percentage of live HPCs 48 hours post- transfection. FIG.15D shows live cell counts (live cells / mL) 48 hours post-transfection FIG. 15E shows B2M gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to the “No gRNA” control.

[0046] FIG.16A-16E show GFP expression, cell viability, and IL2 expression in hematopoietic progenitor cells (HPCs) after transfection with C24 LNPs (C24 LNP 2.0). The C24 LNPs encapsulated mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4), an IL2 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells and cells delivered with a dCas-effector and no gRNA (No gRNA). FIG.16A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.16B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.16C shows percentage of live HPCs 48 hours post-transfection. FIG.16D shows live cell counts (live cells / mL) 48 hours post-transfection FIG.16E shows IL2 gene expression as assessed by RT-qPCR 72 hours post-transfection. Results are shown as fold change in comparison to the “No gRNA” control. sf-6779026.8 13224742003040

[0047] FIG.17A shows quantified B2M expression (B2M MFI as assessed by flow cytometry) in iPSCs after using either C24 LNPs (C24 LNP 2.0) or electroporation (EP) to deliver an mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a B2M targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. Results are shown 48 hours post-transfection or post-electroporation.

[0048] FIG.17B shows flow cytometry plots for assessing GFP expression in iPSCs after transfection with C24 LNPs for delivery of an mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a B2M targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Results are shown 48 hours post-transfection.

[0049] FIG.17C shows flow cytometry plots for assessing GFP expression in iPSCs after electroporation for delivery of an mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a B2M targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Results are shown 48 hours post-electroporation.

[0050] FIG.17D shows quantified EGFR expression (EGFR MFI as assessed by flow cytometry) in iPSCs after using either C24 LNPs (C24 LNP 2.0) or electroporation (EP) to deliver an mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4), an EGFR targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non- transfected (NT) cells. Results are shown 48 hours post-transfection or post-electroporation.

[0051] FIG.17E shows flow cytometry plots for assessing GFP expression in iPSCs after transfection with C24 LNPs for delivery of an mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4), an EGFR targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Results are shown 48 hours post-transfection.

[0052] FIG.17F shows flow cytometry plots for assessing GFP expression in iPSCs after electroporation for delivery of an mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4), an EGFR targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Results are shown 48 hours post-electroporation.

[0053] FIG.17G shows the frequency of healthy iPSCs after using either C24 LNPs (LNP 2.0) or electroporation (EP) to deliver mRNA encoding a dCas-effector, gRNA and an eGFP RNA. Control conditions included non-transfected (NT) cells. Results are shown for Day 2 or Day 5 post-transfection or post-electroporation.

[0054] FIG.18A shows IL2 gene expression, as assessed by RT-qPCR, in iHPCs after transfection with C24 LNPs (C24 LNP 2.0). The C24 LNPs encapsulated mRNA encoding a sf-6779026.8 14224742003040 VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4) and an IL2 targeting gRNA, a TCF7 targeting gRNA, or both gRNAs. Control conditions included cells transfected with a dCas- effector and no gRNA (No gRNA). Results are shown for 72 hours post-transfection as fold change in comparison to the “No gRNA” control.

[0055] FIG.18B shows GATA2, GATA3, and LMO2 gene expression, as assessed by RT- qPCR, in iPSCs after transfection with C24 LNPs (C24 LNP 2.0). The C24 LNPs encapsulated mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4) and gRNAs targeting GATA2, GATA3 and LMO2. Control conditions included cells transfected with a dCas-effector and no gRNA (No gRNA) and a mock treatment. Results are shown for 72 hours post- transfection as fold change in comparison to the “No gRNA” control.

[0056] FIG.19A-19F show GFP expression, cell viability, and CD45 expression in primary T cells after transfection with C24 LNPs using different C24 LNP formulations (summarized in Table E4). Each formulation encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. FIG.19A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.19B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.19C shows percentage of live T cells 48 hours post-transfection. FIG.19D shows viable cell number 48 hours post-transfection. FIG.19E shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.19F shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection.

[0057] FIG.20A-20F show GFP expression, cell viability, and CD45 expression in primary T cells after transfection with C24 LNPs using different C24 LNP formulations (C24 LNP 2.0, 2.3, 2.6 and 3.0). Each formulation encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. FIG.20A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.20B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post- transfection. FIG.20C shows percentage of live T cells 48 hours post-transfection. FIG.20D shows viable cell number 48 hours post-transfection. FIG.20E shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post-transfection. FIG.20F shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post- sf-6779026.8 15224742003040 transfection.

[0058] FIG.21A-21E show GFP expression, cell viability, and CD45 expression in primary T cells after transfection with C24 LNPs using different C24 LNP formulations (summarized in Table E5). Each formulation encapsulated mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. FIG.21A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post-transfection. FIG.21B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.21C shows percentage of live T cells 48 hours post-transfection. FIG.21D shows quantified CD45 expression (% CD45+ cells as assessed by flow cytometry) 72 hours post- transfection. FIG.21E shows quantified CD45 expression (CD45 MFI as assessed by flow cytometry) 72 hours post-transfection.

[0059] FIG.22A-22D show GFP expression, cell viability, and MED12 expression in primary T cells after transfection with C24 LNPs using either the C24 LNP 2.0 formulation or the C24 LNP 3.0 formulation. Both formulations encapsulated mRNA encoding a DNMT3AL- dSpCas9-KRAB fusion protein (SEQ ID NO: 5), a MED12 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6). Control conditions included non-transfected (NT) cells. FIG.22A shows quantified GFP expression (% GFP+ cells as assessed by flow cytometry) 48 hours post- transfection. FIG.22B shows quantified GFP expression (GFP MFI as assessed by flow cytometry) 48 hours post-transfection. FIG.22C shows percentage of live T cells 48 hours post- transfection. FIG.22D shows MED12 gene expression as assessed by RT-qPCR 72 hours post- transfection. Results are shown as fold change in comparison to non-transfected (NT) cells.

[0060] FIGS.23A-23B show flow cytometry plots for assessing cell surface expression of IL7Ra and VCAM1 in iHPCs transfected with various doses of LNPs for delivery of a multiplex DNA-targeting system comprising mRNA encoding dSpCas9-2xVP64 and gRNAs targeting VCAM1, ILR7a, and RUNX3. Results are shown for 72 hours post-transfection. FIG.23A shows results for 72 hours post-transfection. FIG.23B shows results for 120 hours pos- transfection.

[0061] FIG.23C shows flow cytometry plots for assessing cell surface expression of VCAM1, Notch, or DLL4 in iHPCs transfected with LNPs for delivery of singleplex DNA- targeting systems comprising mRNA encoding dSpCas9-2xVP64 in combination with a guide targeting each gene. Results are shown for 120 hours post-transfection. sf-6779026.8 16224742003040

[0062] FIGS.24A-24D shows a quantification of %DLL4+ cells and %VCAM1 cells as assessed by flow cytometry for iHPCs transfected with LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and gRNAs targeting DLL4 and VCAM1 (DV) compared to a non-targeting gRNA (NT) control. Results are shown for D2, D7, D10 and D14 post-transfection. FIG.24A shows a quantification of % DLL4+ cells for iHPCs transfected with a single dose of LNPs at D0. FIG.24B shows a quantification of % VCAM1+ cells for iHPCs transfected with a single dose of LNPs at D0. FIG.24C shows a quantification of %DLL4+ cells for iHPCs transfected with a dose of LNPs at D0 and D8. FIG.24D shows a quantification of % VCAM1+ cells for iHPCs transfected with a dose of LNPs at D0 and D8.

[0063] FIG.25A shows a timeline of iHPC differentiation into CD8+ mature T cells, with iHPCs at D0, CD4+ / CD8+ T-cell progenitor cells at D21 and CD8+ mature T cells at D28.

[0064] FIG.25B shows a quantification of % DLL4+ cells as assessed via flow cytometry for iHPCs transfected with various doses of LNPs for delivery of mRNA encoding dSpCas9- 2xVP64 and a gRNA targeting DLL4 at D0. Results are shown for D2, D6, D9 and D14 post- transfection. Negative controls included cells transfected with LNPs encapsulating the dCas effector with a non-targeting guide RNA (NT).

[0065] FIGS.25C-25F show results for DLL4 expression and cell viability for iHPCs transfected with various doses of LNPs for delivery of mRNA encoding dSpCas9-2xVP64 and a gRNA targeting DLL4 at D0. Results are shown for D2, D6, and D10 post-transfection. Negative controls included cells transfected with LNPs encapsulating the dCas effector without a guide (Cas9) or with a non-targeting guide RNA (NT). FIG.25C shows a quantification of DLL4 surface protein expression as assessed by clow cytometry. FIG.25D shows a quantification of DLL4 gene expression as assessed by RT-qPCR. Results are shown normalized to the GAPDH housekeeping gene and the Cas only control. FIG.25E shows a quantification of live cells / mL as assessed by using a cell counter. FIG.25F shows a quantification of %viability as assessed by flow cytometry.

[0066] FIGS.26A-26E show the experimental timeline and results for cell surface marker expression and cell viability for emerging induced T cell progenitors transfected with various doses of either C243.0 LNPs that encapsulated an mRNA that encoded a VP64-dSpCas9-VP64 fusion protein and a guide RNA targeting DLL4 or C242.0 LNPs that encapsulated an mRNA that encoded a VP64-dSpCas9-VP64 fusion protein and a guide RNA targeting VCAM1. FIG. 26A shows a timeline of iHPC differentiation into CD8+ mature T cells with iHPCs at D0, sf-6779026.8 17224742003040 CD4+ / CD8+ T-cell progenitor cells at D21 and CD8+ mature T cells at D28. LNPs were delivered to the cells at D11 of differentiation. FIG.26B shows a flow plot demonstrating that ~70% of the total live cell population at D11 of the differentiation protocol expressed LDL-R, which mediates LNP uptake in concert with ApoE. FIG.26C shows a quantification of the percentage of cells positive for either DLL4 or VCAM1 at each LNP dose when cells were delivered with LNPs at D11. Protein expression of DLL4 and VCAM1 was assessed via flow cytometry 24 hours post-transfection. FIG.26D shows a quantification of live cells / mL as assessed by using a cell counter for each dose of the C243.0 and C242.0 LNPs compared to a no LNP control for cells transfected at D11. FIG.26E shows a quantification of %viability as assessed by flow cytometry for each dose of the C243.0 and C242.0 LNPs compared to a no LNP control for cells transfected at D11.

[0067] FIGS.27A-27C show the experimental timeline and results for cell surface marker expression and cell viability for emerging induced T cell progenitors transfected with various doses of either C243.0 LNPs that encapsulated an mRNA that encoded a VP64-dSpCas9-VP64 fusion protein and a guide RNA targeting DLL4 or C242.0 LNPs that encapsulated an mRNA that encoded a VP64-dSpCas9-VP64 fusion protein and a guide RNA targeting VCAM1. FIG. 27A shows a timeline of iHPC differentiation into CD8+ mature T cells with iHPCs at D0, CD4+ / CD8+ T-cell progenitor cells at D21 and CD8+ mature T cells at D28. LNPs were delivered to the cells at D14 of differentiation. FIG.27B shows a flow plot demonstrating that ~90% of the total live cell population at D14 of the differentiation protocol expressed LDL-R, which mediates LNP uptake in concert with ApoE. FIG.27C shows a quantification of the percentage of cells positive for either DLL4 or VCAM1 at each LNP dose when cells were delivered with LNPs at D14. Protein expression of DLL4 and VCAM1 was assessed via flow cytometry 24 hours post-transfection.

[0068] FIGS.28A-28B show the experimental timeline and results for DLL4 surface marker expression for double positive (DP) CD4+ / CD8+ iT cell progenitors transfected with C243.0 LNPs that encapsulated an mRNA that encoded a VP64-dSpCas9-VP64 fusion protein and a guide RNA targeting DLL4. FIG.28A shows a timeline of iHPC differentiation into CD8+ mature T cells with iHPCs at D0, CD4+ / CD8+ T-cell progenitor cells at D21, and CD8+ mature T cells at D28. LNPs were delivered to the cells at D24 of differentiation. FIG.28B shows flow plots assessing DLL4 expression at D2 and D10 post-transfection in cells transfected with C24 3.0 LNPs compared to non-transfected control cells (no LNP). Cells transfected with C243.0 sf-6779026.8 18224742003040 LNPs exhibited 96.8% DLL4+ cells at D2 post-transfection and 53.3% DLL4+ cells at D10 post-transfection.

[0069] FIGS.29A-29F show the experimental timeline and results for DLL4 surface marker expression for double positive (DP) CD4+ / CD8+ iT cell progenitors derived from two different iPSC cell lines that were transfected with various doses of C243.0 LNPs that encapsulated an mRNA that encoded a VP64-dSpCas9-VP64 fusion protein and a guide RNA targeting DLL4. FIG.29A shows a timeline of iHPC differentiation into CD8+ mature T cells with iHPCs at D0, CD4+ / CD8+ T-cell progenitor cells at D21 and CD8+ mature T cells at D28. LNPs were delivered to the cells at D21 of differentiation. FIG.29B shows flow plots assessing DLL4 expression in DP CD4+ / CD8+ progenitor cells derived from cell line A and cell line B for each dose 24 hours post-transfection. FIG.29C shows live cells / mL as assessed by using a cell counter for cells derived from cell line A for each dose 24 hours post-transfection. FIG.29D shows % viability for cells derived from cell line A for each dose 24 hours post-transfection. FIG.29E shows live cells / mL for cells derived from cell line B for each dose 24 hours post- transfection. FIG.29F shows % viability for cells derived from cell line B for each dose 24 hours post-transfection.

[0070] FIG.30 shows flow plots assessing GFP expression after 1, 3 or 7 days of activation prior to transfection with C241.0 LNPs encapsulating GFP mRNA compared to non-transfected control cells. Detailed Description

[0071] Provided herein are lipid nanoparticle (LNP) compositions for delivering cargo, such as nucleic acid molecules, including ribonucleic acid (RNA), into cells, such as a primary cell or induced cells (e.g, iPSCs or cells derived from iPSCs) and methods of producing and using the same. In any of the provided embodiments, the lipid nanoparticles contain an ionizable lipid with a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail, for example an ionizable lipid that is C24 (also referred to as “C24 ionizable lipid”), a neutral lipid, a polymer conjugated lipid (e.g., polyethylene glycol (PEG) lipid), a steroid, and a nucleic acid cargo (e.g., RNA). In some aspects, the nucleic acid cargo is RNA. In provided embodiments, the steroid is a cholesterol. In some embodiments, the RNA is an mRNA that encodes a sequence that can be made into a protein. Among exemplary types of RNA for encapsulation in a provided LNP is an mRNA molecule encoding a protein component of a DNA-targeting system for modulating sf-6779026.8 19224742003040 transcription of a target gene, a guide RNA (gRNA), or a combination of both an mRNA and gRNA.

[0072] Delivery of large nucleic acid cargo into certain primary cells, including primary T cells, remains challenging. In particular, electroporation is a common and very potent method of delivering nucleic acid to immune cells and other cell types but it typically causes severe toxicity. Further, methods like electroporation can only be used for in vitro or ex vivo cell engineering but not in vivo applications. In many cases, non-viral methods of gene delivery to primary cells, such as T cells, often suffer from low efficiency, toxicity, or both (Rahimmanesh et al., Res Pharm Sci (2020) 15(5):437-46).

[0073] Among non-viral delivery methods are the use of lipid nanoparticles (LNPs). While LNPs are already used or being investigated for delivery of RNA, drugs, antioxidants, and contrast agents, they can be plagued by issues with dose-limiting toxicities and reproducibility. For example, it has been shown that LNPs can be used to introduce RNA encoding machinery of the CRISPR-Cas9 system into cells, such as for gene editing (Finn et al., Cell Reports (2018) 22(9):2227-35; Miller et al. Angew Chem Int Ed Engl (2017) 56(4):1059-63). However, LNPs are not particularly efficient in delivering nucleic acid to most primary cell types, particularly immune cells. Furthermore, LNPs delivery to iPSCs or lymphoid or lymphoid progenitors derived therefrom is not always efficient. Many lipid nanoparticle applications require the lipid nanoparticles to incorporate targeting moieties to promote payload uptake into cells (e.g. cationic polymer-based delivery) (Smith et al. (2017) Nature Nanotech., 12:813-20).

[0074] The provided embodiments relate to particular LNP formulations that contain ionizable lipids, including the C24 ionizable lipid. Results herein demonstrate that LNPs and compositions thereof are suitable for delivery of nucleic acids, including RNA, into primary cells for use in targeted gene activation or repression.. In addition, results herein also show surprisingly high efficiency to hematopoietic progenitor cells (HSCs) including those derived from iPSCs as well as lymphoid progenitors derived therefrom. These results were observed without a targeting moiety indicating that even without a targeting moiety high efficiency delivery of cargo can be achieved In some aspects, the provided LNPs and compositions thereof deliver a ribonucleic acid (RNA) cargo comprising a mRNA molecule that encodes a CRISPR / Cas9 system and / or a guide RNA (gRNA) for targeted gene activation or repression. In some embodiments, the LNP compositions methods are advantageous by virtue of being nonviral, in that the time, labor, and potential safety risks associated with viral vector-based cell sf-6779026.8 20224742003040 engineering techniques are mitigated or avoided. Further, the LNP compositions provided herein exhibit high transfection and transduction efficiency, with limited cell toxicity observed.

[0075] For instance, results herein demonstrate that efficient delivery of large RNA cargo (e.g., mRNA encoding machinery of the CRISPR-Cas9 system) can be efficiently delivered to primary cells and induced cells, such as iPSCs and cells lymphoid cells or progenitors derived from iPSCs using an LNP with a C24 ionizable lipid compared to other ionizable lipids. The results show that the C24 LNP formulations are able to efficiently delivery cargo to certain cell types for which existing methods are unsatisfactory, including to primary T cells and hematopoietic progenitor cells (HPCs). In particular, larger payloads can be efficiently delivered into cells, including mRNA cargo that is at least 3000 nucleotides in length. Results herein demonstrate that in addition to improved delivery to cells in vitro or ex vivo, C24- containing LNPs also are able to efficiently deliver cargo to primary cells in vivo. Moreover, delivery could be further improved by the particulars of the formulation. For example, it is found herein that several attributes improved the formulation and efficiency of cargo delivery, such as a lower percentage of the C24 lipid of less than 50% (e.g., 30-40 mol %, such as 37.5 mol %), the mRNA:gRNA ratio (e.g., 1:1), a higher percentage of neutral lipid such as DSPC of greater than 10% (e.g., 20-30 mol %, such as 22.5 mol %), and / or particular amounts of a polymer conjugated lipid (e.g., PEG conjugated lipid, such as DMG-PEG2k). LNP compositions provided herein can be formulated reproducibly, stably, and without targeting moieties to genetically engineer primary cells and induced cells (e.g, iPSCs and cells derived from iPSCs) in a non-toxic manner. Further, observations herein demonstrate that an optimized formulation achieves high rates of transduction efficiency, while resulting in low or negligible cellular toxicity.

[0076] In some embodiments, among the provided embodiments are improved compositions for the delivery of large nucleic acid cargo (e.g. RNA encoding machinery of the CRISPR-Cas9 system) into T cells and other immune cells or precursor cells including HPCs. In some aspects, such cells are primary cells. In other aspects, such cells are induced cells, such as derived from iPSCs. In particular, the Examples herein illustrate that provided lipid particle formulations are highly effective in downregulating the mRNA and / or protein levels of target genes in such cells. Furthermore, the Examples herein illustrate that the presence of certain molar ratios of lipid components results in improved or enhanced activity of these lipid particle formulations. For instance, the C24 LNP 3.0 formulation described herein is an exemplary formulation of the sf-6779026.8 21224742003040 present invention that is particularly advantageous because it provides improved transfection efficacy, cell viability and targeted gene repression in primary T-cells and HPCs and other lymphoid cells or progenitors derived therefrom.

[0077] The lipid particles and compositions provided herein may be used for a variety of purposes, including the delivery of associated or encapsulated therapeutic agents to cells, both in vitro and in vivo. Accordingly, the present invention provides methods for treating diseases or disorders in a subject in need thereof, by contacting the subject with a lipid particle described herein comprising one or more suitable therapeutic agents.

[0078] Various exemplary embodiments of the lipid particles, as well as compositions and formulations comprising the same, and their use to deliver therapeutic agents and modulate target gene and protein expression, are described in further detail below.

[0079] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0080] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. I. LIPID NANOPARTICLES

[0081] Provided herein are LNPs and compositions containing the same, such as for delivering a nucleic acid cargo into a cell (e.g., a T cell). In some embodiments, the LNP comprises an ionizable lipid, such as C24. In some embodiments, the LNP further comprises a neutral lipid, a polyethylene glycol (PEG)-conjugated lipid, and cholesterol. Also provided herein are LNP compositions and uses thereof, such as in connection with cell therapy.

[0082] As used herein the term “lipid nanoparticle,” also referred to as LNP, refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm). In some embodiments, such lipid nanoparticles comprise, for example an ionizable lipid and one or more excipients selected from neutral lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid). In some embodiments, the LNPs include a nucleic acid, preferably RNA. In some embodiments, the nucleic acid, preferably RNA, or a portion thereof, is encapsulated in the lipid sf-6779026.8 22224742003040 portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response. In some embodiments, the RNA or a portion thereof is associated with the lipid nanoparticles.

[0083] In the context of the provided embodiments, lipid nanoparticles are not restricted to any particular morphology, and should be interpreted as to include any morphology generated when an ionizable lipid and optionally one or more further lipids are combined, e.g. in an aqueous environment and / or in the presence of a nucleic acid compound. For example, a liposome, a lipid complex, a lipoplex and the like are within the scope of a lipid nanoparticle.

[0084] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the mRNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease. As used herein, the mean diameter may be represented by the number-weighted average as determined by dynamic light scattering. A. Components

[0085] An LNP may comprise an ionizable lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. In further embodiments, the lipid nanoparticles also comprise one or more non-cationic lipids and a lipid conjugate. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. sf-6779026.8 23224742003040

[0086] Provided herein are LNPs comprising a nucleic acid cargo (e.g., RNA). In someembodiments, the LNPs comprise an ionizable lipid; a neutral lipid; a polyethylene glycol(PEG)-conjugated lipid; cholesterol; and one or more RNA molecules. In some embodiments,the LNPs further comprise GalNAc lipid conjugates. 1. Ionizable Lipid

[0087] In some embodiments, the LNP comprises an ionizable lipide containing a trivalenthead group 4-methyl-1-piperazinebutanamine linked to a tail. In some embodiments the 4-methyl-1-piperazinebutanamine head group is linked via two degradable primary esters tooctyldodecyl tails creating 4 saturated alkyl tails with non-symmetric 8 and 10 carbon lengths. In some embodiments, the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1- yl)butyl)azanediyl)dipropionate (C24).

[0088] In some embodiments, the C24 ionizable lipid has the structure:

[0089] In provided embodiments, the C24 lipid has a trivalent 4-methyl-1-piperazinebutanamine head group. The C24 lipid acts as an ionizable lipid because it is preferably cationizable, (i.e., it becomes protonated as the pH is lowered below the pKa of the ionizable group of the lipid), but is progressively more neutral at higher pH values. In certain embodiments, when positively charged, the lipid is then able to associate with negativelycharged nucleic acids. In some embodiments, the C24 lipid of a provided LNP is capable offorming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. C24 is a multiprotic ionizable lipid that produces multistage protonation in the LNP. In some embodiments, C24 exhibits multistageprotonation behavior resulting in greater endosomal protonation and more limitedbiodistribution (Alishetty et al. Research Square; 2021. DOI: 10.21203 / rs.3.rs-798453 / v1; published PCT application WO2022081750). In some embodiments, protonation of the ionizable lipid in the endosome can increase endosomal release of encapsulated nucleic acid. sf-6779026.8 24224742003040

[0090] In some embodiments, the mass fraction of the C24 ionizable lipid in the LNP is between about 10% and about 60%. In some embodiments, the mass fraction of the C24 ionizable lipid in the LNP is between about 35% and about 52%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, or about 52%.

[0091] In some embodiments, the mass fraction of the C24 ionizable lipid is about 35%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 35.5%. In some embodiments, the C24 mass fraction of the ionizable lipid is about 36%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 36.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 37%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 37.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 38%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 38.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 39%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 39.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 40%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 41.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 42%. In some embodiments, the mass fraction of the ionizable lipid is about C2442.5%. In some embodiments, the mass fraction of the ionizable lipid is about C2443%. In some embodiments, the mass fraction of the ionizable lipid is about C2443.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 44%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 44.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 45%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 45.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 46%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 46.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 47%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 47.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 48%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 48.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 49%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 49.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about sf-6779026.8 25224742003040 50%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 50.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 51%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 51.5%. In some embodiments, the mass fraction of the C24 ionizable lipid is about 52%. 2. Neutral Lipid

[0092] In some embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation. Suitable stabilizing lipids include neutral lipids and anionic lipids. The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH. Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

[0093] Exemplary neutral lipids include, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl- phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-lcarboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearioyl-2-oleoylphosphatidyethanol amine (SOPE), and 1,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE).

[0094] In some embodiments, the LNPs comprise a neutral lipid selected from the group consisting of distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) or 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9). sf-6779026.8 26224742003040

[0095] In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutrallipid has the structure of

[0096] In some embodiments, the neutral lipid is DLPC. In some embodiments, the neutrallipid has the structure of

[0097] In some embodiments, the neutral lipid is POPE. In some embodiments, the neutrallipid has the structure of

[0098] In some embodiments, the neutral lipid is POPC. In some embodiments, the neutrallipid has the structure of

[0099] In some embodiments, the neutral lipid is SOPC. In some embodiments, the neutrallipid has the structure of sf-6779026.8 27224742003040.

[0100] In some embodiments, the neutral lipid is DOPC. In some embodiments, the neutrallipid has the structure of.

[0101] In some embodiments, the neutral lipid is DOPE. In some embodiments, the neutrallipid has the structure of.

[0102] In some embodiments, the neutral lipid is 9A1P9. In some embodiments, the neutrallipid has the structure of

[0103] In some embodiments, the mass fraction of the neutral lipid in the LNP is betweenabout 5% and about 30%. In some embodiments, the mass fraction of the neutral lipid in the LNP is between about 8% and about 24%. In some embodiments, the mass fraction of the neutral lipid is about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, or about 24%. In some embodiments, the mass fraction of the neutral lipid is about 8%. In some embodiments, the mass fraction of the neutral lipid is about 8.5%. In some embodiments, the mass fraction of the neutral lipid is about 9%. In some embodiments, the mass sf-6779026.8 28224742003040 fraction of the neutral lipid is about 9.5%. In some embodiments, the mass fraction of the neutral lipid is about 10%. In some embodiments, the mass fraction of the neutral lipid is about 10.5%. In some embodiments, the mass fraction of the neutral lipid is about 11%. In some embodiments, the mass fraction of the neutral lipid is about 11.5%. In some embodiments, the mass fraction of the neutral lipid is about 12%. In some embodiments, the mass fraction of the neutral lipid is about 12.5%. In some embodiments, the mass fraction of the neutral lipid is about 13%. In some embodiments, the mass fraction of the neutral lipid is about 13.5%. In some embodiments, the mass fraction of the neutral lipid is about 14%. In some embodiments, the mass fraction of the neutral lipid is about 14.5%. In some embodiments, the mass fraction of the neutral lipid is about 15%. In some embodiments, the mass fraction of the neutral lipid is about 15.5%. In some embodiments, the mass fraction of the neutral lipid is about 16%. In some embodiments, the mass fraction of the neutral lipid is about 16.5%. In some embodiments, the mass fraction of the neutral lipid is about 17%. In some embodiments, the mass fraction of the neutral lipid is about 17.5%. In some embodiments, the mass fraction of the neutral lipid is about 18%. In some embodiments, the mass fraction of the neutral lipid is about 18.5%. In some embodiments, the mass fraction of the neutral lipid is about 19%. In some embodiments, the mass fraction of the neutral lipid is about 19.5%. In some embodiments, the mass fraction of the neutral lipid is about 20%. In some embodiments, the mass fraction of the neutral lipid is about 20.5%. In some embodiments, the mass fraction of the neutral lipid is about 21%. In some embodiments, the mass fraction of the neutral lipid is about 21.5%. In some embodiments, the mass fraction of the neutral lipid is about 22%. In some embodiments, the mass fraction of the neutral lipid is about 22.5%. In some embodiments, the mass fraction of the neutral lipid is about 23%. In some embodiments, the mass fraction of the neutral lipid is about 23.5%. In some embodiments, the mass fraction of the neutral lipid is about 24%. 3. PEG Lipid

[0104] In some embodiments, the LNPs comprise a polymer conjugated lipid which reduce particle aggregation and control particle size. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG) and the like. sf-6779026.8 29224742003040

[0105] In some embodiments, the LNPs comprise an additional, stabilizing-lipid which is apolyethylene glycol-lipid (pegylated lipid). Exemplary pegylated lipids include, for example, PEG-dilauroylglycerol, PEG-dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG- distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG-dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1- -(Cholest-5-en-3[beta]-oxy)carboxamido- -dioxaoctanyl]carbamoyl-[omega]-methyl-poly(ethylene glycol),PEG-DMB (3,4-ditetradecoxylbenzyl-[omega]-methyl-poly(ethylene glycol)ether), 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1,2-distearoyl-sn-glycerol, methoxypoly ethylene glycol (PEG2k-DSG), poly(ethylene glycol)-2000-dimethacrylate (PEG2k-DMA), and 1,2- distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DSA)

[0106] In some embodiments, the polyethylene glycol (PEG)-conjugated lipid is DMG-PEG2k. In some embodiments, the PEG-conjugated lipid has the structure of.

[0107] In some embodiments, the mass fraction of the PEG-conjugated lipid in the LNP isbetween about 0.5% and about 2.5%. In some embodiments, the mass fraction of the PEG- conjugated lipid is about 0.5%, about 1%, about 1.5%, about 2%, or about 2.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 0.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 1%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 1.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 2%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 2.5%. 4. Cholesterol

[0108] In various embodiments, the LNPs further comprise a steroid or steroid analogue.Steroids useful for the lipid nanoparticles described herein include, but are not limited to, cholestanes such as cholesterol, cholanes such as cholic acid, pregnanes such as progesterone, androstanes such as testosterone, and estranes such as estradiol. Further steroids include, but are not limited to, cholesterol (ovine), cholesterol sulfate, desmosterol-d6, cholesterol-d7, sf-6779026.8 30224742003040 lathosterol-d7, desmosterol, stigmasterol, lanosterol, dehydrocholesterol, dihydrolanosterol, zymosterol, lathosterol, zymosterol-d5, 14-demethyl-lanosterol, 14-demethyl-lanosterol-d6, 8(9)- dehydrocholesterol, 8(14)-dehydrocholesterol, diosgenin, DHEA sulfate, DHEA, lanosterol- d6, dihydrolanosterol-d7, campesterol-d6, sitosterol, lanosterol-95, Dihydro FF- MAS-d6, zymostenol-d7, zymostenol, sitostanol, campestanol, campesterol, 7- dehydrodesmosterol, pregnenolone, sitosterol-d7, Dihydro T-MAS, Delta 5-avenasterol, Brassicasterol, Dihydro FF-MAS, 24-methylene cholesterol, cholic acid derivatives, cholesteryl esters, and glycosylated sterols. In some embodiments, the LNPs comprise cholesterol.

[0109] In certain embodiments, the steroid or steroid analogue is cholesterol. In someembodiments, cholesterol has the structure of.

[0110] In some embodiments, the mass fraction of cholesterol in the LNP is between about30% and about 45% or between about 35% and 40%. In some embodiments, the mass fraction of cholesterol is about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, or about 45%. In some embodiments, the mass fraction of cholesterol is about 30%. In some embodiments, the mass fraction of cholesterol is about 31%. In some embodiments, the mass fraction of cholesterol is about 32%. In some embodiments, the mass fraction of cholesterol is about 33%. In some embodiments, the mass fraction of cholesterol is about 34%. In some embodiments, the mass fraction of cholesterol is about 35%. In some embodiments, the mass fraction of cholesterol is about 36%. In some embodiments, the mass fraction of cholesterol is about 37%. In some embodiments, the mass fraction of cholesterol is about 38%. In some embodiments, the mass fraction of cholesterol is about 39%. In some embodiments, the mass fraction of cholesterol is about 40%. In some embodiments, the mass fraction of cholesterol is about 41%. In some embodiments, the mass fraction of cholesterol is about 42%. In some embodiments, the mass fraction of cholesterol is about 43%. In some embodiments, the sf-6779026.8 31224742003040 mass fraction of cholesterol is about 44%. In some embodiments, the mass fraction of cholesterol is about 45%. 5. GalNAc Lipid

[0111] In certain embodiments, the LNPs further comprise a GalNAc lipid conjugate.Exemplary GalNAc lipid conjugates include, but are not limited to, those described in U.S.Patent Nos. 11,207,416 and 9,352,048. In some embodiments, the GalNAc lipid conjugate isGalNAc3-PEG2000-DSG. In some embodiments, the GalNAc lipid conjugate has the structure of

[0112] In some embodiments, the GalNAc lipid conjugate is Tri-GalNAc3-PEG2000-DSPE.In some embodiments, the GalNAc lipid conjugate has the structure of

[0113] In some embodiments, the mass fraction of the GalNAc lipid conjugate in the LNP isbetween about 0.25% and about 2% or between about 0.5% and 1%. In some embodiments, themass fraction of the GalNAc lipid conjugate is about 0.25%, about 0.50%, about 0.75%, about1%, about 1.25%, about 1.50%, about 1.75%, or about 2.0%. In some embodiments, the massfraction of the GalNAc lipid conjugate is about 0.25%. In some embodiments, the mass fractionof the GalNAc lipid conjugate is about 0.5%. In some embodiments, the mass fraction of theGalNAc lipid conjugate is about 0.75%. In some embodiments, the mass fraction of the GalNAcsf-6779026.8 32224742003040 lipid conjugate is about 1%. In some embodiments, the mass fraction of the GalNAc lipid conjugate is about 1.25%. In some embodiments, the mass fraction of the GalNAc lipid conjugate is about 1.75%. In some embodiments, the mass fraction of the GalNAc lipid conjugate is about 2%. 6. RNA

[0114] In some embodiments, the LNPs comprise a ribonucleic acid (RNA) cargo. In some embodiments, the RNA cargo comprises a DNA-targeting system for modulating transcription of a target gene. In some embodiments, the RNA cargo comprises at least one messenger RNA (mRNA). In some embodiments, the RNA cargo comprises one or more RNA molecules selected from the group consisting of a messenger RNA (mRNA) encoding a fusion protein comprising a DNA-binding domain that binds to the target site for the gene and at least one effector domain for modulating transcription of the gene, mRNA encoding for a fluorescent reporter (e.g., GFP (SEQ ID NO:6)), and guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a crispr RNA (crRNA) and a tracrRNA.

[0115] In some embodiments, the RNA cargo is mRNA encoding a fusion protein comprising a DNA-binding domain that binds to the target site for a gene and at least one effector domain for modulating transcription of the gene. In some embodiments, the DNA- binding domain comprises or is derived from a CRISPR associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), meganuclease, homing endonuclease, I-SceI enzyme, or variants thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g. nuclease-inactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a catalytically inactivated so that it is inactive for nuclease activity and is not able to cleave the DNA. In some embodiments, the RNA molecule is gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a crispr RNA (crRNA) and a tracrRNA.

[0116] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or variant thereof, such as a nuclease-inactive Cas or dCas (e.g. dCas9), and the RNA cargo comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and / or hybridizing to the target site of a gene. In some embodiments, the sf-6779026.8 33224742003040 gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site of a gene.

[0117] In some embodiments, the at least one effector domain is a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain may comprise a KRAB domain, ERF repressor domain, MXI1 domain, SID4X domain, MAD-SID domain, a DNMT family protein domain (e.g. DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g. DNMT3A / L, which comprises a fusion of DNMT3A and DNMT3L domains), LSD1, EZH2, a SunTag domain, a partially or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing

[0118] In some embodiments, the at least one effector domain is a transcriptional activator domain. In some embodiments, a transcriptional activator domain comprises a domain of a protein selected from among VP64, p65, Rta, p300, CBP, VPR, VPH, HSF1, a TET protein (e.g. TET1), a partially or fully functional fragment or domain thereof, or a combination of any of the foregoing.

[0119] In some embodiments, the mass fraction of the RNA cargo in the LNP is between about 0.25% and about 2% or between about 0.5% and 1%. In some embodiments, the mass fraction of the RNA cargo is about 0.25%, about 0.50%, about 0.75%, about 1%, about 1.25%, about 1.50%, about 1.75%, or about 2.0%. In some embodiments, the mass fraction of the RNA cargo is about 0.25%. In some embodiments, the mass fraction of the RNA cargo is about 0.5%. In some embodiments, the mass fraction of the RNA cargo is about 0.75%. In some embodiments, the mass fraction of the RNA cargo is about 1%. In some embodiments, the mass fraction of the RNA cargo is about 1.25%. In some embodiments, the mass fraction of the RNA cargo is about 1.75%. In some embodiments, the mass fraction of the RNA cargo is about 2%.

[0120] In some embodiments, the total RNA concentration of the RNA cargo in the LNP is between about 4 ug / mL and about 200 ug / mL, between about 5 ug / mL and about 50 ug / mL, or between about 50 ug / mL and about 150 ug / mL. In some embodiments, the total RNA concentration is about 4 ug / mL. In some embodiments, the total RNA concentration is about 4.5 ug / mL. In some embodiments, the total RNA concentration is about 5 ug / mL. In some embodiments, the total RNA concentration is about 6 ug / mL. In some embodiments, the total RNA concentration is about 6.5 ug / mL. In some embodiments, the total RNA concentration is about 7 ug / mL. In some embodiments, the total RNA concentration is about 7.5 ug / mL. In some embodiments, the total RNA concentration is about 8 ug / mL. In some embodiments, the total sf-6779026.8 34224742003040 RNA concentration is about 8.5 ug / mL. In some embodiments, the total RNA concentration is about 9 ug / mL. In some embodiments, the total RNA concentration is about 10 ug / mL. In some embodiments, the total RNA concentration is about 20 ug / mL. In some embodiments, the total RNA concentration is about 30 ug / mL. In some embodiments, the total RNA concentration is about 40 ug / mL. In some embodiments, the total RNA concentration is about 50 ug / mL. In some embodiments, the total RNA concentration is about 55 ug / mL. In some embodiments, the total RNA concentration is about 60 ug / mL. In some embodiments, the total RNA concentration is about 65 ug / mL. In some embodiments, the total RNA concentration is about 70 ug / mL. In some embodiments, the total RNA concentration is about 75ug / mL. In some embodiments, the total RNA concentration is about 80 ug / mL. In some embodiments, the total RNA concentration is about 85 ug / mL. In some embodiments, the total RNA concentration is about 90 ug / mL. In some embodiments, the total RNA concentration is about 95 ug / mL. In some embodiments, the total RNA concentration is about 100 ug / mL. In some embodiments, the total RNA concentration is about 125 ug / mL. In some embodiments, the total RNA concentration is about 150 ug / mL.

[0121] The LNPs can have different ratios between the positively charged amine groups of the biodegradable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid cargo to be encapsulated. This may be mathematically represented by the equation N / P. For example, the N / P ratio may be from about 0.5 to about 100, from about 1 to about 50, from about 1 to about 20, from about 1 to about 16, from about 1 to about 12, from about 8, or from about 4. The N / P ratio can also be from about 8 to about 16. In specific examples, the N / P ratio can be 8. B. Preparation of Lipid Nanoparticles

[0122] Provided herein are methods for producing LNPs for delivering a nucleic acid cargo into cell, such as a primary cell (e.g., a T cell) or induced cells (e.g., iPSCs or cells derived from iPSCs) and uses thereof, the lipid nanoparticles containing an ionizable lipid, a neutral lipid, a polyethylene glycol (PEG) lipid, cholesterol, and one or more RNA molecules. In some embodiments, the LNPs described herein may be produced by methods described herein.

[0123] The lipid particles of the present invention in which a therapeutic agent, such RNA encoding a DNA-targeting system, is encapsulated in a lipid bilayer and is protected from degradation, can be formed by any method known in the art including, but not limited to, a continuous mixing method or a direct dilution process. Methods of LNP preparation are disclosed in, for example, U.S. Patent Publication Nos.20040142025 and 20070042031. sf-6779026.8 35224742003040

[0124] In certain embodiments, the present invention provides for LNPs produced via a continuous mixing method, e.g., a process that includes providing an aqueous solution comprising a nucleic acid such as an interfering RNA in a first reservoir, providing an organic lipid solution in a second reservoir, and mixing the aqueous solution with the organic lipid solution such that the organic lipid solution mixes with the aqueous solution so as to substantially instantaneously produce a liposome encapsulating the nucleic acid cargo. This process and the apparatus for carrying this process are described in detail in U.S. Patent Publication No.20040142025, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

[0125] The action of continuously introducing lipid and buffer solutions into a mixing environment, such as in a mixing chamber, causes a continuous dilution of the lipid solution with the buffer solution, thereby producing a liposome substantially instantaneously upon mixing. As used herein, the phrase “continuously diluting a lipid solution with a buffer solution” (and variations) generally means that the lipid solution is diluted sufficiently rapidly in a hydration process with sufficient force to effectuate vesicle generation. By mixing the aqueous solution comprising a nucleic acid with the organic lipid solution, the organic lipid solution undergoes a continuous stepwise dilution in the presence of the buffer solution (i.e., aqueous solution) to produce a nucleic acid-lipid particle.

[0126] The LNPs formed using the continuous mixing method typically have a size of from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 nm to about 90 nm. The particles thus formed do not aggregate and are optionally sized to achieve a uniform particle size.

[0127] In some embodiments, the method includes (1) adding to an organic solvent comprising ethanol (a) an ionizable lipid, wherein the lipid is C24 or an analog thereof; (b) a neutral lipid; (c) a polyethylene glycol (PEG)-conjugated lipid; and (d) cholesterol, thereby generating an organic phase; (2) adding to an aqueous solvent having an acidic pH, a ribonucleic acid (RNA) molecule, thereby generating an aqueous phase; and (3) combining the organic phase and the aqueous phase by laminar flow mixing in a device, thereby generating a LNP containing RNA.

[0128] In some embodiments, the flow rate of the aqueous phase in the device is between about 14 mL / min and about 16 mL / min. In some embodiments, the flow rate of the aqueous phase in the device is about 14 mL / min. In some embodiments, the flow rate of the aqueous sf-6779026.8 36224742003040 phase in the device is about 15 mL / min. In some embodiments, the flow rate of the aqueous phase in the device is about 16 mL / min. In some embodiments, the flow rate of the organic phase in the device is between about 4 mL / min and about 6 mL / min. In some embodiments, the flow rate of the organic phase in the device is about 4 mL / min. In some embodiments, the flow rate of the organic phase in the device is about 5 mL / min. In some embodiments, the flow rate of the organic phase in the device is about 6 mL / min. In some embodiments, the ratio of the aqueous phase flow rate to the organic phase flow rate is about 3:1. In some embodiments, the flow rate of the aqueous phase is about 15 mL / min, and the flow rate of the organic phase is about 5 mL / min.

[0129] In some embodiments, the aqueous solvent is a citrate buffer. In some embodiments, the pH of the acetate buffer is between about 3.0 and about 4.5. In some embodiments, the pH of the acetate buffer is about 3.0. In some embodiments, the pH of the acetate buffer is about 3.5. In some embodiments, the pH of the acetate buffer is about 4.0. In some embodiments, the pH of the acetate buffer is about 4.5.

[0130] In some embodiments, the molarity of the citrate buffer is between about 10 mM and about 300 mM, between about 15 mM and about 275 mM, between about 20 mM and about 250 mM, between about 25 mM and about 200 mM, or between about 30 mM and about 150 mM. In some embodiments, the molarity of the citrate buffer is between about 10 mM and about 300 mM. In some embodiments, the molarity of the acetate buffer is between about 15 mM and about 275 mM. In some embodiments, the molarity of the acetate buffer is between about 20 mM and about 250 mM. In some embodiments, the molarity of the acetate buffer is between about 25 mM and about 200 mM. In some embodiments, the molarity of the acetate buffer is between about 30 mM and about 150 mM. In some embodiments, the molarity of the citrate buffer is about 500 mM.

[0131] In some embodiments, the method comprises collecting the generated LNPs from the device in the citrate buffer. In some embodiments, the method comprises washing the collected LNPs with an isotonic buffer. In some embodiments, isotonic buffer is phosphate buffered saline (PBS). In some embodiments, the pH of the isotonic buffer is about 7.4. In some embodiments, the method comprises dialysis of the LNPs in PBS. In some embodiments, the method comprises concentrating the LNPs using ultrafiltration. sf-6779026.8 37224742003040

[0132] In some embodiments, the method is carried out at about room temperature. In some embodiments, the method is carried out between about 20 degrees Celsius and about 25 degrees Celsius. In some embodiments, the method is carried out at about 20 degrees Celsius.

[0133] In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for at least about an hour, at least about 2 hours, at least about 4 hours, at least about 6 hours, at least about 8 hours, or at least about 12 hours. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for at least about an hour. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for at least about 2 hours. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for at least about 4 hours. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for at least about 6 hours. In some embodiments, the method includes storing the generated LNsP at about 4 degrees Celsius for at least about 8 hours. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for about 12 hours. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for about 18 hours. In some embodiments, the method includes storing the generated LNPs at about 4 degrees Celsius for about 24 hours. In some embodiments, the method includes storing the generated LNP sat about 12 degrees Celsius for between about 2 hours and about 12 hours. In some embodiments, the method includes storing the generated LNPs at about 12 degrees Celsius for at least about 2 hours.

[0134] In some embodiments, the method includes sterile filtering the LNPs, or a composition thereof. In some embodiments, the method includes sterile filtering the LNPs or a composition thereof following storage of the lipid nanoparticle composition at 4 degrees Celsius.

[0135] In some embodiments, the LNPs further comprise a GalNAc lipid conjugate. In some embodiments, the GalNAc lipid conjugate is added following LNP formulation. Methods of preparing LNPs comprising a GalNAc lipid conjugate are disclosed in, for example, U.S. Patent No.11,207,416. C. Exemplary Formulations

[0136] Provided herein are LNPs containing: (a) a C24 ionizable lipid or an analog thereof; (b) a neutral lipid that is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (c) a polyethylene glycol (PEG)-conjugated lipid that is DMG-PEG2k; (d) cholesterol; and (e) a ribonucleic acid (RNA) cargo. sf-6779026.8 38224742003040

[0137] In some embodiments, the ionizable lipid is C24 or an analog thereof. In some embodiments, the ionizable lipid is C24. In some embodiments, the mass fraction of C24 is between about 35% and about 55%. In some embodiments, the mass fraction of DSPC is between about 5% and about 25%. In some embodiments, the mass fraction of DMG-PEG2k is between about 0.5% and about 5%. In some embodiments, the mass fraction of cholesterol is between about 35% and about 45%. In some embodiments, the mass fraction of the ribonucleic acid (RNA) cargo is between about 0.25% and 2%.

[0138] In some embodiments, the LNP contains: (a) C24 with a mass fraction of between about 35% and about 55%; (b) DSPC with a mass fraction of between about 5% and about 25%; (c) DMG-PEG2k with a mass fraction of between about 0.5% and about 5%; (d) cholesterol with a mass fraction of between about 35% and about 45%; and (e) a ribonucleic acid (RNA) cargo.

[0139] In some embodiments, the ionizable lipid is C24 or an analog thereof. In some embodiments, the ionizable lipid is C24. In some embodiments, the mass fraction of C24 is between about 45% and about 55%. In some embodiments, the mass fraction of DSPC is between about 5% and about 15%. In some embodiments, the mass fraction of DMG-PEG2k is between about 1% and about 3%. In some embodiments, the mass fraction of cholesterol is between about 35% and about 40%. In some embodiments, the mass fraction of the ribonucleic acid (RNA) cargo is between about 0.25% and 1%.

[0140] In some embodiments, the LNP contains: (a) C24 with a mass fraction of between about 45% and about 55%; (b) DSPC with a mass fraction of between about 5% and about 15%; (c) DMG-PEG2k with a mass fraction of between about 1% and about 3%; (d) cholesterol with a mass fraction of between about 35% and about 40%; and (e) a ribonucleic acid (RNA) cargo. In some embodiments, the LNP contains: (a) C24 with a mass fraction of about 50%; (b) DSPC with a mass fraction of about 10%; (c) DMG-PEG2k with a mass fraction of about 1.5%; (d) cholesterol with a mass fraction of about 38%; and (e) a ribonucleic acid (RNA) cargo with a mass fraction of about 0.5%.

[0141] In some embodiments, the ionizable lipid is C24 or an analog thereof. In some embodiments, the ionizable lipid is C24. In some embodiments, the mass fraction of C24 is between about 30% and about 40%. In some embodiments, the mass fraction of DSPC is between about 15% and about 25%. In some embodiments, the mass fraction of DMG-PEG2k is between about 1% and about 3%. In some embodiments, the mass fraction of cholesterol is sf-6779026.8 39224742003040 between about 35% and about 40%. In some embodiments, the mass fraction of the ribonucleic acid (RNA) cargo is between about 0.25% and 1%.

[0142] In some embodiments, the LNP contains: (a) C24 with a mass fraction of between about 30% and about 40%; (b) DSPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2k with a mass fraction of between about 1% and about 3%; (d) cholesterol with a mass fraction of between about 35% and about 40%; and (e) a ribonucleic acid (RNA) cargo. In some embodiments, the LNP contains: (a) C24 with a mass fraction of about 37.5%; (b) DSPC with a mass fraction of about 22.5%; (c) DMG-PEG2k with a mass fraction of about 1.5%; (d) cholesterol with a mass fraction of about 38%; and (e) a ribonucleic acid (RNA) cargo with a mass fraction of about 0.5%. II. RIBONUCLEIC ACID (RNA) CARGO

[0143] Provided herein are LNPs and compositions containing the same, that are useful in the delivery of a ribonucleic acid (RNA) cargo to a mammalian subject or cell. In some embodiments, the RNA cargo comprises mRNA, such as mRNA encoding a protein component of a DNA-targeting system.

[0144] In some embodiments, the mRNA will comprise the base uracil (U), whereas DNA encoding the mRNA molecule will comprise the base thymine (T). A mRNA sequence herein may be defined by the DNA sequence encoding the mRNA, and / or the RNA sequence of the mRNA.

[0145] In some embodiments, the mRNA can be produced using methods known in the art such as in vitro transcription. In some embodiments of the method, the mRNA comprises a 5' cap. In some embodiments, the 5’ cap is an altered nucleotide on the 5’ end of primary transcripts such as messenger RNA. In some aspects, the 5’ caps of the mRNA improves one or more of RNA stability and processing, mRNA metabolism, the processing and maturation of an RNA transcript in the nucleus, transport of mRNA from the nucleus to the cytoplasm, mRNA stability, and efficient translation of mRNA to protein. In some embodiments, a 5’ cap can be a naturally-occurring 5’ cap or one that differs from a naturally-occurring cap of an mRNA. A 5’ cap may be any 5' cap known to a skilled artisan. In certain embodiments, the 5' cap is selected from the group consisting of an Anti-Reverse Cap Analog (ARCA) cap, a 7-methyl-guanosine (7mG) cap, a CleanCap® analog, a vaccinia cap, and analogs thereof. For instance, the 5’ cap may include, without limitation, an anti-reverse cap analogs (ARCA) (US7074596), 7-methyl- guanosine, CleanCap® analogs, such as Cap 1 analogs (Trilink; San Diego, CA), or sf-6779026.8 40224742003040 enzymatically capped using, for example, a vaccinia capping enzyme or the like. In some embodiments, the mRNA may be polyadenylated. The mRNA may contain various 5’ and 3’ untranslated sequence elements to enhance expression of the encoded protein and / or stability of the mRNA itself. Such elements can include, for example, posttranslational regulatory elements such as a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the mRNA comprises at least one nucleoside modification. The mRNA may contain modifications of naturally-occurring nucleosides to nucleoside analogs. Any nucleoside analogs known in the art are envisioned. Such nucleoside analogs can include, for example, those described in US 8,278,036. In certain embodiments of the method, the nucleoside modification is selected from the group consisting of a modification from uridine to pseudouridine and uridine to Nl- methyl pseudouridine. In particular embodiments of the method, the nucleoside modification is from uridine to Nl- methyl pseudouridine.

[0146] In some embodiments, the mRNA is least 1,500 nucleotides in length. In some embodiments, the mRNA is at least 3,000 nucleotides in length. In some embodiments, the mRNA is at least 4,500 nucleotides in length. nucleotides in length. In some embodiments, the mRNA is at least 6,000 nucleotides in length. In some embodiments, the mRNA is at least 7,5000 nucleotides in length.

[0147] In some embodiments, the mRNA is from about 1,500 to 15,000 nucleotides in length, from about 3,000 to 12,000 nucleotides in length, from about 4,500 to 9,000 nucleotides in length, or from about 4,800 to 7,500 nucleotides in length. In some embodiments, the mRNA is about 4,800 nucleotides in length. In some embodiments, the mRNA is about 4,800 nucleotides in length. In some embodiments, the mRNA is about 5,100 nucleotides in length. In some embodiments, the mRNA is about 5,400 nucleotides in length. In some embodiments, the mRNA is about 5,700 nucleotides in length. In some embodiments, the mRNA is about 6,000 nucleotides in length. In some embodiments, the mRNA is about 6,300 nucleotides in length. In some embodiments, the mRNA is about 6,600 nucleotides in length. In some embodiments, the mRNA is about 6,900 nucleotides in length. In some embodiments, the mRNA is about 7,200 nucleotides in length. In some embodiments, the mRNA is about 7,500 nucleotides in length.

[0148] In some embodiments, the mRNA encodes a protein that is from about 500 to about 5,000 amino acids in length, from about 1,000 to 4,000 amino acids in length, from about 1,500 to 3,000 amino acids in length, or from about 1,600 to 2,500 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 1,600 amino acids in length. In some sf-6779026.8 41224742003040 embodiments, the mRNA encodes a protein that is about 1,700 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 1,800 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 1,900 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 2,000 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 2,100 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 2,200 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 2,300 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 2,400 amino acids in length. In some embodiments, the mRNA encodes a protein that is about 2,500 amino acids in length.

[0149] In some embodiments, the RNA cargo comprises an mRNA and guide RNA (gRNA). In some embodiments, the gRNA is from about 85 to 125 nucleotides in length or from about 100 to 115 nucleotides in length. In some embodiments, the gRNA is about 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, or 115 nucleotides in length. In some embodiments, the gRNA is about 100 nucleotides in length. In some embodiments, the gRNA is about 101 nucleotides in length. In some embodiments, the gRNA is about 102 nucleotides in length. In some embodiments, the gRNA is about 103 nucleotides in length. In some embodiments, the gRNA is about 104 nucleotides in length. In some embodiments, the gRNA is about 105 nucleotides in length. In some embodiments, the gRNA is about 106 nucleotides in length. In some embodiments, the gRNA is about 107 nucleotides in length. In some embodiments, the gRNA is about 108 nucleotides in length. In some embodiments, the gRNA is about 109 nucleotides in length. In some embodiments, the gRNA is about 110 nucleotides in length. In some embodiments, the gRNA is about 111 nucleotides in length. In some embodiments, the gRNA is about 112 nucleotides in length. In some embodiments, the gRNA is about 113 nucleotides in length. In some embodiments, the gRNA is about 114 nucleotides in length. In some embodiments, the gRNA is about 115 nucleotides in length.

[0150] In some embodiments, the RNA cargo comprises multiple guide RNAs. In some embodiments, the RNA cargo comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or 8 or more guide RNAs. In some embodiments, the RNA cargo comprises 2 or more guide RNAs. In some embodiments, the RNA cargo comprises 3 or more guide RNAs. In some embodiments, the RNA cargo comprises 4 or more guide RNAs. In some embodiments, the RNA cargo comprises 5 or more guide RNAs. In some embodiments, the RNA cargo sf-6779026.8 42224742003040 comprises 6 or more guide RNAs. In some embodiments, the RNA cargo comprises 7 or more guide RNAs. In some embodiments, the RNA cargo comprises 8 or more guide RNAs.

[0151] In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4 or 1:5. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 10:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 9:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 8:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 7:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 6:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 5:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 4:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 3:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 2:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 1:1. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 1:2. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 1:3. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 1:4. In some embodiments, the RNA cargo has a mRNA:gRNA w / w ratio of 1:5. III. DNA-TARGETING SYSTEMS

[0152] In some embodiments, provided are LNPs and compositions containing the same, that are useful in the delivery of a ribonucleic acid (RNA) cargo comprising a DNA-targeting system. In some embodiments, the DNA-targeting system includes a fusion protein that comprises a DNA-binding domain that binds to the target site for a gene, and at least one effector domain for modulating transcription of the gene (e.g., a transcriptional activator or transcriptional repressor). In some embodiments, delivery of a DNA-targeting system by the provided LNPs increases or represses transcription of one or more target genes in a cell (e.g, a T cell). Exemplary components and features of DNA-targeting systems are provided below in the following subsections. A. Fusion Proteins

[0153] In some aspects, the provided LNPs and compositions containing the same include an RNA cargo comprising a DNA-targeting system. In some embodiments, the DNA-targeting system includes a fusion protein. In some embodiments, the RNA cargo comprises an mRNA encoding a fusion protein of a DNA-targeting system. sf-6779026.8 43224742003040

[0154] In some embodiments, the fusion protein comprises: (a) a DNA-binding domain capable of being targeted to a target site for one or more genes, and (b) at least one effector domain for modulating transcription of the one or more genes. In some embodiments, the at least one effector domain is a transcriptional activator domain for increasing transcription of one or more genes. In some, embodiments, the at least one effector domain is a transcriptional repressor domain for repressing transcription of one or more genes. In some aspects, the fusion protein is targeted to a target site in a gene or regulatory element thereof, and leads to increased or activated transcription of the gene. In some aspects, the fusion protein is targeted to a target site in a gene or regulatory element thereof, and leads to decreased or repressed transcription of the gene. In some aspects, the fusion protein is targeted to target sites in a combination of genes or regulatory elements thereof, and leads to increased or activated transcription of each of the genes. In some aspects, the fusion protein is targeted to target sites in a combination of genes or regulatory elements thereof, and leads to decreased or repressed transcription of each of the genes.

[0155] In some embodiments, the fusion protein comprises at least one of any of the DNA- binding domains described herein in Sections III.B and III.C and at least one of any of the effector domains described herein in Sections III.D and III.E. In some embodiments, the fusion protein contains a CRISPR / Cas-based DNA-binding domain, such as described in Section III.B, and at least one effector domain for transcriptional repression, as described in Section III.D. In some embodiments, the fusion protein contains a CRISPR / Cas-based DNA-binding domain, such as described in Section III.B., and at least one effector domain for transcriptional activation, as described in Section III.E.

[0156] In some embodiments, the DNA-binding domain and effector domain of the fusion protein are heterologous, i.e., the domains are from different species, or at least one of the domains is not found in nature. In some aspects, the fusion protein is an engineered fusion protein, i.e., the fusion protein is not found in nature.

[0157] In some embodiments, the at least one effector domain is fused to the N-terminus, the C-terminus, or both the N-terminus and the C-terminus, of the DNA-binding domain or a component thereof. The at least one effector domain may be fused to the DNA-binding domain directly, or via any intervening amino acid sequence, such as a linker sequence or a nuclear localization sequence (NLS). sf-6779026.8 44224742003040

[0158] In some embodiments, the fusion protein comprises, from N- to C-terminal order: a transcriptional activator domain and a DNA-binding domain. In some embodiments, the fusion protein comprises, from N- to C-terminal order: a DNA-binding domain and a transcriptional activator domain. In some embodiments, the fusion protein comprises from N- to C-terminal order: a first transcriptional activator domain, a DNA-binding domain, and a second transcriptional activator domain. In some embodiments, the first and second transcriptional activator domains are the same. In some embodiments, the first and second transcriptional activator domains are different.

[0159] In some embodiments, the fusion protein comprises, from N- to C-terminal order: a transcriptional repressor domain and a DNA-binding domain. In some embodiments, the fusion protein comprises, from N- to C-terminal order: a DNA-binding domain and a transcriptional repressor domain. In some embodiments, the fusion protein comprises from N- to C-terminal order: a first transcriptional repressor domain, a DNA-binding domain, and a second transcriptional repressor domain. In some embodiments, the first and second transcriptional repressor domains are the same. In some embodiments, the first and second transcriptional repressor domains are different.

[0160] In some embodiments, the fusion protein includes more than one effector domain. In some embodiments, the fusion protein includes 2, 3 or 4 effector domains, or more than 4 effector domains. In some embodiments, at least two of the effector domains of the fusion protein are different. In some embodiments, each of the effector domains of the fusion protein are different. In some embodiments, the fusion protein includes two effector domains. In some embodiments, the two effector domains are different. In some embodiments, the effector domains and the DNA-binding domain can be arranged in any order.

[0161] In some embodiments, the at least one effector domain of the fusion protein includes two different effector domains. In such embodiments, the two different effector domains and the DNA-binding domain can be arranged in any order. In some embodiments, each of the effector domains are N-terminal to the DNA-binding domain in which a first effector domain is fused to the N-terminus of the second effector domain and the second effector domain is fused to the N-terminus of the DNA-binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C- terminal order: a first effector domain, a second effector domain and the DNA binding domain. In some embodiments, each of the effector domains are C-terminal to the DNA-binding domain sf-6779026.8 45224742003040 in which a first effector domain is fused to the C-terminus of the DNA-binding domain and the second effector domain is fused to the C-terminus of the first effector domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a DNA-binding domain, a first effector domain, and a second effector domain. In some embodiments, the DNA-binding domain is between the effector domains, in which one effector domain is fused to the N-terminus of the DNA-binding domain and the other effector domain is fused to the C-terminus of the DNA- binding domain. In some embodiments, the fusion protein of a provided DNA-binding system, or a DNA-targeting module thereof, comprises from N- to C-terminal order: a first effector domain, a DNA-binding domain, and a second effector domain. In some embodiments, one or more of the components may be fused to each other directly, or via any intervening amino acid sequence, such as via a linker sequence or a nuclear localization sequence (NLS).

[0162] In some embodiments, the fusion protein comprises one or more linkers. In some embodiments, the linker is a peptide linker. In some embodiments, the one or more linkers connect the DNA-binding domain or a component thereof to the at least one effector domain. A linker may be included anywhere in the polypeptide sequence of the fusion protein, for example, between the effector domain and the DNA-binding domain or a component thereof. A linker may be of any length and designed to promote or restrict the mobility of components in the fusion protein. A linker may comprise any amino acid sequence of about 2 to about 100, about 5 to about 80, about 10 to about 60, or about 20 to about 50 amino acids. A linker may comprise an amino acid sequence of at least about 2, 3, 4, 5, 10, 15, 20, 25, or 30 amino acids. A linker may comprise an amino acid sequence of less than about 100, 90, 80, 70, 60, 50, or 40 amino acids. A skilled artisan can readily choose an appropriate linker for the connection of two domains. In some embodiments, the linker is a flexible linker. Flexible linkers are generally composed of small, non-polar or polar residues such as glycine, serine or threonine. A linker may include sequential or tandem repeats of an amino acid sequence that is 2 to 20 amino acids in length. Linkers may be rich in amino acids glycine (G), serine (S), and / or alanine (A). Linkers may include, for example, a GS linker. A linker may comprise repeats of a sequence, for example as represented by the formula (GGGGS)n, wherein n is an integer that represents the number of times the GGGGS sequence (SEQ ID NO: 7) is repeated (e.g. between 1 and 10 times). The number of times a linker sequence is repeated can be adjusted to optimize the linker length and achieve appropriate separation of the functional domains. For example, in some sf-6779026.8 46224742003040 embodiments, the linker is the (GGGGS)n linker, whereby n is an integer of 1 to 10. Appropriate linkers may be selected or designed based rational criteria known in the art, for example as described in Chen et al. Adv. Drug Deliv. Rev.65(10):1357-1369 (2013). In some embodiments, a linker comprises a linker described in WO 2021 / 247570.

[0163] In some embodiments, the fusion protein of the DNA-targeting system comprises one or more nuclear localization signals (NLS). In some embodiments, a fusion protein described herein comprises one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and / or in combination with one or more other NLSs present in one or more copies. Non- limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 8) the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 9)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 10) or RQRRNELKRSP (SEQ ID NO: 11); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 12); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 13) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 14) and PPKKARED (SEQ ID NO: 15) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 16) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 17) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 18) and PKQKKRK (SEQ ID NO: 19) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 20) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 21) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 22) of the human poly(ADP- ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 23) of the steroid hormone receptors (human) glucocorticoid. The NLS may comprise a portion of any of the foregoing. In general, the one or more NLSs are of sufficient strength to drive accumulation of the fusion protein in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the fusion protein, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the fusion protein, such that location within a cell may be visualized, such as in sf-6779026.8 47224742003040 combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of the fusion protein (e.g. an assay for altered gene expression activity in a cell transformed with the DNA-targeting system comprising the fusion protein), as compared to a control condition (e.g. an untransformed cell).

[0164] In some embodiments, the NLS is linked to the N-terminus or the C-terminus of the DNA-binding domain via a linker. In some embodiments, the NLS is linked to the N-terminus or the C-terminus of an effector domain via a linker. The linker may be any linker as described above.

[0165] In some configurations, the N- or C-terminus of the fusion protein can be linked to a moiety for detection and / or purification. In some aspects, the moiety is or includes a Flag tag DYKDDDDK (SEQ ID NO: 24) , a 3xFlag tag MDYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 25), an HA tag YPYDVPDYA (SEQ ID NO: 26) or a His tag, such as HHHHHH (SEQ ID NO:27).

[0166] In some embodiments, the fusion protein is a dCas-KRAB fusion protein, such as dSpCas9-KRAB. In some embodiments, the fusion protein is dSpCas9-KRAB. In some embodiments, the fusion protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 2 or a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 2.

[0167] In some embodiments, the fusion protein is a dCas-VP64 fusion protein, such as VP64-dSpCas9-VP64, which is a fusion of dSpCas9 fused to two copies of VP64. In some embodiments, the fusion protein is VP64-dSpCas9-VP64. In some embodiments, the fusion protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 4 or a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 4.

[0168] In some embodiments, the fusion protein is a dCas-KRAB-DNMT3AL fusion protein, such as DNMT3AL-dSpCas9-KRAB. In some embodiments, the fusion protein is DNMT3AL-dSpCas9-KRAB. In some embodiments, the fusion protein is encoded by the sf-6779026.8 48224742003040 nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 1 or a nucleotide sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the fusion protein is encoded by the nucleotide sequence set forth in SEQ ID NO: 5 or SEQ ID NO: 1.

[0169] In some embodiments, the DNA-targeting system targets to or binds to a target site in a gene, such as any described herein. In some embodiments, the target site is located in the gene and / or a regulatory DNA element of the gene. In some embodiments, the target site is located in a CpG island of the gene. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of the gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5’ UTR, or 3’ UTR of the gene. In some embodiments, the regulatory DNA element is a promoter. In some embodiments, a promoter is a nucleotide sequence to which RNA polymerase binds to begin transcription of the gene. In some embodiments, a promoter is a nucleotide sequence located within about 100bp, about 500bp, about 1000bp, or more, of a transcriptional start site of the gene. In some embodiments, a promoter is within 500bp of a transcriptional start site of the gene. In some embodiments the target site is located within a sequence of unknown or known function that is suspected of being able to control expression of a gene.

[0170] In some embodiments, the target site is located within about 50 base pairs (bp), about 100 bp, about 150 bp, about 200 bp, about 250 bp, about 300 bp, about 350 bp, about 400 bp, about 450 bp, about 500 bp, about 600b p, about 650 bp, about 700 bp, about 750 bp, about 800 bp, about 850 bp, about 900 bp, about 1000 bp, about 1050 bp, about 1100 bp, about 1200 bp, about 1250 bp, about 1300 bp, about 1350 bp about 1400 bp, about 1450 bp, or about 1500 bp of a transcription start site. B. CRISPR / Cas-Based DNA-Targeting Systems and DNA-Binding Domains

[0171] In some aspects, the provided LNPs and compositions containing the same include an RNA cargo comprising a DNA-targeting system based on CRISPR / Cas systems, i.e. CRISPR / Cas-based DNA-targeting systems, that are able to bind to a target site for a target gene, or to a combination of target sites, e.g. for a combination of target genes. In some embodiments, the CRISPR / Cas DNA-binding domain is nuclease inactive, such as includes a sf-6779026.8 49224742003040 dCas (e.g. dCas9) so that the system binds to the target site for a target gene without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA-targeting systems may be used to modulate expression of a target gene in a cell, such as a T cell. In some embodiments, the target gene may include any as described herein. In some embodiments, the CRISPR / Cas- based DNA-targeting system can include any known Cas enzyme, and generally a nuclease- inactive or dCas. In some embodiments, the CRISPR / Cas-based DNA-targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some embodiments, the effector domain decreases transcription of the one or more genes (e.g. the effector domain is a transcriptional repressor, such as any described in Section III.D). In some embodiments, the effector domain increases transcription of the one or more genes (e.g. the effector domain is a transcriptional activator, such as any described in Section III.E).

[0172] The CRISPR system (also known as CRISPR / Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers. Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a “memory” of past exposures. Spacers encode the DNA-targeting portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0173] In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The trans- activating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex.

[0174] Naturally occurring CRISPR / Cas systems, such as those with Cas9, have been engineered to allow efficient programming of Cas / RNA RNPs to target desired sequences in sf-6779026.8 50224742003040 cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013 / 176772, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), or Cong, L. et al. Science 339(6121):819-23 (2013). The spacer sequence of the gRNA can be chosen by a user to target the Cas / gRNA RNP complex to a desired locus, e.g. a desired target site in the target gene.

[0175] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. An exemplary deactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772, WO 2014 / 093661, Jinek, M. et al. Science 337(6096):816-21 (2012), and Qi, L. et al. Cell 152(5):1173-83 (2013). Exemplary dCas variants derived from the Cas12 system (i.e. Cpf1) are described, for example in WO 2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0176] dCas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator or transcriptional repressor. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP16, a 16 amino acid transactivation domain of the Herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Krüppel associated box) can result in robust repression of gene expression. A variety of dCas-fusion proteins with effector domains can be engineered for regulation of gene expression, for example as described in WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2021 / 247570, Adli, M. Nat. Commun.9, 1911 (2018), sf-6779026.8 51224742003040 Perez-Pinera, P. et al. Nat. Methods 10, 973–976 (2013), Mali, P. et al. Nat. Biotechnol.31, 833–838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977–979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nuñez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0177] In some aspects, the DNA-targeting system comprises a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector domain for increasing transcription or inducing transcriptional activation (i.e. a transcriptional activator) when targeted to a target gene in a cell (e.g. a T cell). In some embodiments, the dCas protein is any suitable dCas protein, such as any described herein. In some embodiments, the dCas protein is a dCas9 protein, such dSpCas9 or dSaCas9.

[0178] In some embodiments, the at least one effector domain is any suitable transcriptional repressor effector domain, such as any described in Section III.D, such as KRAB. In some embodiments, the at least one effector domain is KRAB. In some embodiments, the fusion protein is a dCas9-KRAB fusion protein, for example as described in Section III.A. In such embodiments, the DNA-targeting system also includes one or more gRNAs, provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA- targeting system to the target site of the target gene. In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to decrease or repress transcription of the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, wherein the effector domain mediates targeted epigenetic modification to decrease or repress transcription of the combination of target genes. Any of a variety of effector domains that decrease or repress transcription can be used as described further below.

[0179] In some embodiments, the at least one effector domain is any suitable transcriptional activator effector domain, such as any described in Section III.E, such as VP64. In some embodiments, the at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, for example as described in Section III.A. In such embodiments, the DNA-targeting system also includes one or more gRNAs, provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA- targeting system to the target site of the target gene. In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of sf-6779026.8 52224742003040 the target gene. In some embodiments, a combination of gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the combination of target genes. Any of a variety of effector domains that increase or activate transcription can be used as described further below.

[0180] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof. In particular embodiments here, the Cas protein is nuclease-inactive (i.e. is a dCas protein).

[0181] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system. In some embodiments, the Cas protein is derived from a Cas12 protein (i.e. Cpf1) or variant thereof, for example as described in WO 2017 / 189308 and Zetsche, B. et al. Cell.163(3):759-71 (2015). In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973–976 (2013), or Mali, P. et al. Nat. Biotechnol.31, 833–838 (2013). Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med.51, 1–11 (2019), Zhang, F. Q. Rev. Biophys.52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol.1311:47-75 (2015).

[0182] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes. sf-6779026.8 53224742003040

[0183] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str.657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0184] In some aspects, the Cas protein is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nuclease- inactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.

[0185] In some embodiments, the CRISPR / Cas-based DNA-targeting system includes a sf-6779026.8 54224742003040 fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas RNP complex to the target site of the target gene. In some embodiments, a target site of a gRNA may be referred to as a protospacer.

[0186] In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e. a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein.

[0187] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. composed of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two non- coding RNA sequences: a crRNA sequence and a tracrRNA sequence, for example as described in WO 2013 / 176772, or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0188] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target site to hybridize with the target site in the target gene and direct sequence-specific binding of a Cas / gRNA complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.

[0189] In some aspects, a gRNA targets a target site in double-stranded DNA. Thus, in some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer hybridizes to, or by the sequence complementary to the sequence that the gRNA spacer sf-6779026.8 55224742003040 hybridizes to. In some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer displaces in order to hybridize to the DNA. In some embodiments, the sequence of the target site is the sequence that the gRNA hybridizes to.

[0190] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt,18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt or 22 nt in length. In some embodiments, the gRNA spacer sequence is 20 nt in length.

[0191] A target site of a gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target a protospacer with a specific protospacer-adjacent motif (PAM), i.e. a sequence immediately adjacent to the protospacer that contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, S. pyogenes Cas9 uses the PAM 5’-NGG-, where N is any nucleotide. In some embodiments, S. aureus Cas9 uses the PAM 5’- NNGRRT-3’, where N is any nucleotide, and R is G or A. In some embodiments, Cas proteins may use or be engineered to use different PAMs from those listed above.

[0192] A spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0193] In some embodiments, the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, in some embodiments, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of the interaction of the gRNA molecule / Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence.

[0194] In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and / or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0195] Methods for designing gRNAs and exemplary targeting domains can include those sf-6779026.8 56224742003040 described in, e.g., International PCT Pub. Nos. WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, WO 2015 / 089427, WO 2016 / 049258, WO 2016 / 123578, WO 2021 / 076744, WO 2014 / 191128, WO 2015 / 161276, WO 2017 / 193107, and WO 2017 / 093969. C. Other DNA-Binding Domains and DNA-Targeting Systems

[0196] In some of any of the provided embodiments, the DNA-binding domain comprises a zinc finger protein (ZFP); a transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I-SceI enzyme or a variant thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing. In some embodiments, the fusion protein of the DNA-targeting system, or one or more DNA-targeting modules thereof, comprises a DNA-binding domain described herein, such as a DNA-binding domain that is an engineered zinc finger protein (eZFP) or a TALE.

[0197] In some embodiments, a ZFP, a zinc finger DNA binding protein, or zinc finger DNA binding domain, is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial, or engineered ZFPs (eZFPs), comprising ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides long, generated by assembly of individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix containing two invariant histidine residues coordinated through zinc with two cysteines of a single beta turn, and having two, three, four, five, or six fingers. Generally, sequence-specificity of a ZFP may be altered by making amino acid substitutions Thus, for example, the ZFP or ZFP-containing molecule is non-naturally occurring, e.g., is an eZFP that is engineered to bind to a target site of choice.

[0198] In some embodiments, zinc fingers are custom-designed (i.e. designed by the user), or obtained from a commercial source. Various methods for designing zinc finger proteins are available. For example, methods for designing zinc finger proteins to bind to a target DNA sequence of interest are described, for example in Liu, Q. et al., PNAS, 94(11):5525-30 (1997); Wright, D.A. et al., Nat. Protoc., 1(3):1637-52 (2006); Gersbach, C.A. et al., Acc. Chem. Res., 47(8):2309-18 (2014); Bhakta M.S. et al., Methods Mol. Biol., 649:3-30 (2010); and Gaj et al., sf-6779026.8 57224742003040 Trends Biotechnol, 31(7):397-405 (2013). In addition, various web-based tools for designing zinc finger proteins to bind to a DNA target sequence of interest are publicly available. See, for example, the Zinc Finger Tools design web site from Scripps available on the world wide web at scripps.edu / barbas / zfdesign / zfdesignhome.php. Various commercial services for designing zinc finger proteins to bind to a DNA target sequence of interest are also available. See, for example, the commercially available services or kits offered by Creative Biolabs (world wide web at creative-biolabs.com / Design-and-Synthesis-of-Artificial-Zinc-Finger-Proteins.html), the Zinc Finger Consortium Modular Assembly Kit available from Addgene (world wide web at addgene.org / kits / zfc-modular-assembly / ), or the CompoZr Custom ZFN Service from Sigma Aldrich (world wide web at sigmaaldrich.com / life-science / zinc-finger-nuclease- technology / custom-zfn.html).

[0199] In some embodiments, the fusion protein of the DNA-targeting system comprises an eZFP DNA-binding domain and an effector domain.

[0200] Transcription activator-like effectors (TALEs), are proteins naturally found in Xanthomonas bacteria. TALEs comprise a plurality of repeated amino acid sequences, each repeat having binding specificity for one base in a target sequence. Each repeat comprises a pair of variable residues in position 12 and 13 (repeat variable diresidue; RVD) that determine the nucleotide specificity of the repeat. In some embodiments, RVDs associated with recognition of the different nucleotides are HD for recognizing C, NG for recognizing T, NI for recognizing A, NN for recognizing G or A, NS for recognizing A, C, G or T, HG for recognizing T, IG for recognizing T, NK for recognizing G, HA for recognizing C, ND for recognizing C, HI for recognizing C, HN for recognizing G, NA for recognizing G, SN for recognizing G or A and YG for recognizing T, TL for recognizing A, VT for recognizing A or G and SW for recognizing A. In some embodiments, RVDs can be mutated towards other amino acid residues in order to modulate their specificity towards nucleotides A, T, C and G and in particular to enhance this specificity. Binding domains with similar modular base-per-base nucleic acid binding properties can also be derived from different bacterial species. These alternative modular proteins may exhibit more sequence variability than TALE repeats.

[0201] In some embodiments, a “TALE DNA binding domain” or “TALE” is a polypeptide comprising one or more TALE repeat domains / units. The repeat domains, each comprising a repeat variable diresidue (RVD), are involved in binding of the TALE to its cognate target DNA sequence. A single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in sf-6779026.8 58224742003040 length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins may be designed to bind to a target site using canonical or non-canonical RVDs within the repeat units. See, e.g., U.S. Pat. Nos. 8,586,526 and 9,458,205.

[0202] In some embodiments, the fusion protein of the DNA-targeting system comprises a TALE DNA-binding domain and an effector domain.

[0203] Zinc finger and TALE DNA-binding domains can be engineered to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger protein, by engineering of the amino acids in a TALE repeat involved in DNA binding (the repeat variable diresidue or RVD region), or by systematic ordering of modular DNA-binding domains, such as TALE repeats or ZFP domains. Therefore, engineered zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering zinc finger proteins and TALEs are design and selection. A designed protein is a protein not occurring in nature whose design / composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP or TALE designs (canonical and non-canonical RVDs) and binding data. See, for example, U.S. Pat. Nos. 9,458,205; 8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98 / 53058; WO 98 / 53059; WO 98 / 53060; WO 02 / 016536 and WO 03 / 016496. D. Effector Domains – Repressors

[0204] In some embodiments, provided herein are LNPs comprising an RNA cargos encoding a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in a gene or regulatory DNA element thereof, such as any DNA-binding domain described above in Sections III.B or III.C, and (b) at least one effector domain. In some aspects, the effector domain is capable of decreasing transcription of the gene, such as any of the genes described herein. In some aspects, the effector domain comprises a transcriptional repression domain. In some aspects, the effector domain comprises a domain that induces an epigenetic modification, such as heterochromatin formation.

[0205] In some aspects, the effector domain induces, catalyzes, or leads to repressed and / or reduced transcription of a gene when ectopically recruited to the gene or DNA regulatory element thereof. sf-6779026.8 59224742003040

[0206] In some embodiments, the effector domain induces, catalyzes or leads to transcription repression, transcription co-repression, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, heterochromatin formation, proteolysis, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, splicing, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain induces, catalyzes, or leads to transcription repression or transcription co-repression. In some embodiments, the effector domain induces transcription repression. In some embodiments, the effector domain has one of the aforementioned activities itself (i.e., acts directly). In some embodiments, the effector domain recruits and / or interacts with a protein or polypeptide domain that has one of the aforementioned activities (i.e., acts indirectly).

[0207] In some embodiments, the effector domain may comprise a KRAB domain, ERF repressor domain, MXI1 domain, SID4X domain, MAD-SID domain, a DNMT family protein domain (e.g. DNMT3A or DNMT3B), a fusion of one or more DNMT family proteins or domains thereof (e.g. DNMT3A / L, which comprises a fusion of DNMT3A and DNMT3L domains), LSD1, EZH2, a SunTag domain, a partially or fully functional fragment or domain of any of the foregoing, or a combination of any of the foregoing. For example, the fusion protein may be dCas9-KRAB, or dCas9-KRAB-DNMT3A / L.

[0208] In some embodiments, the effector domain comprises a transcriptional repressor domain described in WO 2021 / 226077.

[0209] In some embodiments, the effector domain comprises a KRAB domain, or a variant thereof. The KRAB-containing zinc finger proteins make up the largest family of transcriptional repressors in mammals. The Krüppel associated box (KRAB) domain is a transcriptional repressor domain present in many zinc finger protein-based transcription factors. The KRAB domain comprises charged amino acids and can be divided into sub-domains A and B. The KRAB domain recruits corepressors KAP1 (KRAB-associated protein-1), epigenetic readers such as heterochromatin protein 1 (HP1), and other chromatin modulators to induce transcriptional repression through heterochromatin formation. KRAB-mediated gene repression is associated with loss of histone H3-acetylation and an increase in H3 lysine 9 trimethylation (H3K9me3) at the repressed gene promoters. KRAB domains, including in dCas fusion proteins, have been described, for example, in WO 2017 / 180915, WO 2014 / 197748, US 2019 / 0127713, WO 2013 / 176772, Urrutia R. et al. Genome Biol.4, 231 (2003), Groner A. C. et al. PLoS sf-6779026.8 60224742003040 Genet.6, e1000869 (2010). In some embodiments, the effector domain comprises at least one KRAB domain or a variant thereof.

[0210] In some embodiments, the effector domain comprises at least one ERF repressor domain, or a variant thereof. ERF (ETS2 repressor factor) is a strong transcriptional repressor that comprises a conserved DNA-binding domain, and represses transcription via a distinct domain at the carboxyl-terminus of the protein. ERF repressor domains, including in dCas fusion proteins, have been described, for example, in WO2017180915, WO2014197748, WO2013176772, Mavrothalassitis, G., Ghysdael, J. Proteins of the ETS family with transcriptional repressor activity. Oncogene 19, 6524–6532 (2000). In some embodiments, the effector domain comprises at least one ERF repressor domain or a variant thereof.

[0211] In some embodiments, the effector domain comprises at least one MXI1 domain, or a variant thereof. The MXI1 domain functions by antagonizing the myc transcriptional activity by competing for binding to myc-associated factor x (MAX). MXI1 domains, including in dCas fusion proteins, have been described, for example, in WO2017180915, WO2014197748, US20190127713. In some embodiments, the effector domain comprises at least one MXI1 domain or a variant thereof.

[0212] In some embodiments, the effector domain comprises at least one SID4X domain, or a variant thereof. The mSin3 interacting domain (SID) is present on different transcription repressor proteins. It interacts with the paired amphipathic alpha-helix 2 (PAH2) domain of mSin3, a transcriptional repressor domain that is attached to transcription repressor proteins such as the mSin3 A corepressor. A dCas9 molecule can be fused to four concatenated mSin3 interaction domains (SID4X). SID domains, including in dCas fusion proteins, have been described, for example, in WO2017180915, WO2014197748, WO2014093655. In some embodiments, the effector domain comprises at least one SID domain or a variant thereof.

[0213] In some embodiments, the effector domain comprises at least one MAD domain, or a variant thereof. The MAD family proteins, Mad1, Mxi1, Mad3, and Mad4, belong to the basic helix-loop-helix-zipper class and contain a conserved N terminal region (termed Sin3 interaction domain (SID)) necessary for repressor activity. MAD-SID domains, including in dCas fusion proteins, have been described, for example, in WO2017180915, WO2014197748, WO2013176772. In some embodiments, the effector domain comprises at least one MAD-SID domain or a variant thereof.

[0214] In some embodiments, the effector domain comprises at least one DNMT3 domain, sf-6779026.8 61224742003040 or a variant thereof. The DNMT3A and DNMT3B are two DNA methyltransferases that catalyze de novo methylation, which depending on the site may be associated with transcriptional repression. DNMT3, including in dCas fusion proteins, have been described, for example, in US20190127713, Liu, X. S. et al. Cell 167, 233–247.e17 (2016), Lei, Y. et al. Nat. Commun.8, 16026 (2017). In some embodiments, the effector domain comprises at least one DNMT3 domain or a variant thereof.

[0215] In some embodiments, the effector domain comprises at least one DNMT3L domain, or a variant thereof. DNMT3L (DNA (cytosine-5)-methyltransferase 3-like) is a catalytically inactive regulatory factor of DNA methyltransferases that can either promote or inhibit DNA methylation depending on the context. DNMT3L is essential for the function of DNMT3A and DNMT3B; DNMT3L interacts with DNMT3A and DNMT3B and significantly enhances their catalytic activity. For instance, DNMT3L interacts with the catalytic domain of DNMT3A to form a heterodimer, demonstrating that DNMT3L has dual functions of binding an unmethylated histone tail and activating DNA methyltransferase. In some embodiments, the effector domain comprises a fusion of DNMT3A and DNMT3L (DNMT3A / L).

[0216] In some embodiments, the effector domain may comprise a LSD1 domain. LSD1 (also known as Lysine-specific histone demethylase 1A) is a histone demethylase that can demethylate lysine residues of histone H3, thereby acting as a coactivator or a corepressor, depending on the context. LSD1, including in dCas fusion proteins, has been described, for example, in WO 2013 / 176772, WO 2014 / 152432, and Kearns, N. A. et al. Nat. Methods. 12(5):401–403 (2015).

[0217] In some embodiments, the effector domain may comprise an EZH2 domain. EZH2 (also known as Histone-lysine N-methyltransferase EZH2) is a Catalytic subunit of the PRC2 / EED-EZH2 complex, which methylates 'Lys-9' (H3K9me) and 'Lys-27' (H3K27me) of histone H3, in some aspects leading to transcriptional repression of the affected target gene. EZH2, including in dCas fusion proteins, has been described, for example, in O’Geen, H. et al., Epigenetics Chromatin.12(1):26 (2019).

[0218] In some embodiments, the effector domain may comprise a SunTag domain. SunTag is a repeating peptide array, which can recruit multiple copies of an antibody-fusion protein that binds the repeating peptide. The antibody-fusion protein may comprise an additional effector domain, such as a transcription repression domain (e.g. KRAB), to reduce transcription of the target gene. SunTag, including in dCas fusion proteins for gene modulation have been described, sf-6779026.8 62224742003040 for example, in WO 2016 / 011070 and Tanenbaum, M. et al. Cell.159(3):635–646 (2014). In some embodiments, the SunTag effector domain recruits an antibody-fusion protein that comprises KRAB and binds the GCN4 peptide. E. Effector Domains – Activators

[0219] In some aspects, provided herein are LNPs comprising an RNA cargos encoding a DNA-targeting system comprising a fusion protein comprising: (a) a DNA-binding domain capable of being targeted to a target site in a gene or regulatory DNA element thereof, such as any DNA-binding domain described above in Sections III.B or III.C, and (b) at least one effector domain. In some aspects, the effector domain is capable of increasing transcription of the gene, such as any of the genes described herein. In some aspects, the effector domain comprises a transcriptional activation domain.

[0220] In some aspects, the effector domain activates, induces, catalyzes, or leads to increased transcription of a gene when ectopically recruited to the gene or DNA regulatory element thereof. In some embodiments, the effector domain activates, induces, catalyzes, or leads to: transcription activation, transcription co-activation, transcription elongation, transcription de-repression, transcription factor release, polymerization, histone modification, histone acetylation, histone deacetylation, nucleosome remodeling, chromatin remodeling, reversal of heterochromatin formation, proteolysis, ubiquitination, deubiquitination, phosphorylation, dephosphorylation, DNA methylation, DNA demethylation, histone methylation, histone demethylation, or DNA base oxidation. In some embodiments, the effector domain activates, induces, catalyzes or leads to transcription activation, transcription co- activation, or transcription elongation. In some embodiments, the effector domain induces transcription activation. In some embodiments, the effector domain has one of the aforementioned activities itself (i.e. acts directly). In some embodiments, the effector domain recruits and / or interacts with a polypeptide domain that has one of the aforementioned activities (i.e. acts indirectly).

[0221] Gene expression of endogenous mammalian genes, such as human genes, can be achieved by targeting a fusion protein comprising a DNA-binding domain, such as a dCas9, and an effector domain, such as a transcription activation domain, to mammalian genes or regulatory DNA elements thereof (e.g. a promoter or enhancer) via one or more gRNAs. Any of a variety of effector domains for transcriptional activation (e.g. transcription activation domains) are known and can be used in accord with the provided embodiments. Transcription activation sf-6779026.8 63224742003040 domains, as well as activation of target genes by Cas fusion proteins (with a variety of Cas molecules) and the transcription activation domains, are described, for example, in WO 2014 / 197748, WO 2016 / 130600 , WO 2017 / 180915, WO 2021 / 226555 , WO 2021 / 226077, WO 2013 / 176772 , WO 2014 / 152432, WO 2014 / 093661, Adli, M. Nat. Commun.9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973–976 (2013), Mali, P. et al. Nat. Biotechnol.31, 833–838 (2013), and Maeder, M. L. et al. Nat. Methods 10, 977–979 (2013).

[0222] In some embodiments, a transcriptional activation domain comprises a domain of a protein selected from among VP64, p65, Rta, p300, CBP, VPR, VPH, HSF1, a TET protein (e.g. TET1), a partially or fully functional fragment or domain thereof, or a combination of any of the foregoing.

[0223] In some embodiments, the transcriptional activation domain comprises a VP64 domain. For example, dCas9-VP64 can be targeted to a target site by one or more gRNAs to activate a gene. VP64 is a polypeptide composed of four tandem copies of VP16, a 16 amino acid transactivation domain of the Herpes simplex virus. VP64 domains, including in dCas fusion proteins, have been described, for example, in WO 2014 / 197748, WO 2013 / 176772, WO 2014 / 152432, and WO 2014 / 093661. In some embodiments, the transcriptional activation domain comprises at least one VP16 domain, or a VP16 tetramer (“VP64”) or a variant thereof.

[0224] In some embodiments, the transcriptional activation domain comprises a p65 activation domain (p65AD). p65AD is the principal transactivation domain of the 65kDa polypeptide of the nuclear form of the NF-KB transcription factor. An exemplary sequence of human transcription factor p65 is available at the Uniprot database under accession number Q04206. p65 domains, including in dCas fusion proteins, have been described, for example in WO 2017 / 180915 and Chavez, A. et al. Nat. Methods 12, 326–328 (2015).

[0225] In some embodiments, the transcriptional activation domain comprises an R transactivator (Rta) domain. Rta is an immediate-early protein of Epstein-Barr virus (EBV), and is a transcriptional activator that induces lytic gene expression and triggers virus reactivation. The Rta domain, including in dCas fusion proteins, has been described, for example in WO 2017 / 180915 and Chavez, A. et al. Nat. Methods 12, 326–328 (2015).

[0226] In some embodiments, the transcriptional activation domain comprises a CREB- binding protein (CBP) domain or a p300 domain. In some aspects, CBP refers to the CREB- binding protein encoded by the human CREBBP gene. CBP is a coactivator that interacts with cAMP-response element binding protein (CREB). In some aspects, p300 refers to the Histone sf-6779026.8 64224742003040 acetyltransferase p300 protein encoded by the human EP300 gene, and is a coactivator closely related to CBP. CBP and p300 each interact with a variety of transcriptional activators to affect gene transcription (Gerritsen, M.E. et al. PNAS 94(7):2927-2932 (1997)). In some embodiments, the transcriptional activation domain comprises a p300 domain. p300 domains (such as the catalytic core of p300) including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016 / 130600, WO 2017 / 180915, and Hilton, I.B. et al., Nat. Biotechnol.33(5):510-517 (2015).

[0227] In some embodiments, the transcriptional activation domain comprises a HSF1 domain. In some aspects, HSF1 refers to the Heat shock factor protein 1 protein encoded by the human HSF1 gene. HSF1, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555, WO 2015 / 089427, and Konermann et al. Nature 517(7536):583-8 (2015).

[0228] In some embodiments, the transcriptional activation domain comprises the tripartite activator VP64-p65-Rta (also known as VPR). VPR comprises three transcription activation domains (VP64, p65, and Rta) fused by short amino acid linkers, and can effectively upregulate target gene expression. VPR, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555 and Chavez, A. et al. Nat. Methods 12, 326–328 (2015).

[0229] In some embodiments, the transcriptional activation domain comprises VPH. VPH is a tripartite activator polypeptide comprising VP64, mouse p65, and HSF1. VPH, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555.

[0230] In some embodiments, the transcriptional activation effector domain has demethylase activity. The effector domain may include an enzyme that remove methyl (CH3-) groups from nucleic acids, proteins (in particular histones), and other molecules. The effector domain may convert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. Alternatively, the transcriptional activation domain can convert the methyl group to hydroxymethylcytosine in a mechanism for demethylating DNA. The effector domain can catalyze this reaction. For example, the transcriptional activation domain that catalyzes this reaction may comprise a domain from a TET protein, for example TET1 (Ten-eleven translocation methylcytosine dioxygenase 1). In some aspects, TET1 refers to the Methylcytosine dioxygenase TET1 protein encoded by the human TET1 gene. TET1 catalyzes the conversion of the modified genomic base 5-methylcytosine (5mC) into 5- sf-6779026.8 65224742003040 hydroxymethylcytosine (5hmC) and plays a key role in active DNA demethylation. TET1, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2021 / 226555

[0231] In some embodiments, the effector domain may comprise a SunTag domain. SunTag is a repeating peptide array, which can recruit multiple copies of an antibody-fusion protein that binds the repeating peptide. The antibody-fusion protein may comprise an additional effector domain, such as a transcription activation domain (e.g. VP64), to induce increased transcription of the target gene. SunTag, including in dCas fusion proteins for gene activation, has been described, for example, in WO 2016 / 011070 and Tanenbaum, M. et al. Cell.159(3):635–646 (2014). In some embodiments, the SunTag effector domain recruits an antibody-fusion protein that comprises a transcriptional activator effector domain (e.g. VP64) and binds the GCN4 peptide, thereby activating transcription at the target site and acting as a transcriptional activator effector domain. F. DNA-Targeting Modules and Multiplexed DNA-Targeting Systems

[0232] In some embodiments, the DNA-targeting system contains at one or more DNA- targeting modules, where each DNA-targeting module of the system is a component of the DNA-targeting system that is independently capable of targeting one target site for a target gene. In some embodiments, each DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to the target site, and (b) an effector domain for modulating transcription of the gene. In some embodiments, the DNA-targeting system comprises a single DNA-targeting module for targeted transcriptional modulation of a single gene.

[0233] In some embodiments, a DNA-targeting module is a CRISPR / Cas-based DNA- targeting module. In such embodiments, the DNA-binding domain of the fusion protein is a Cas protein or variant thereof (e.g. a dCas protein, such as dCas9) and the DNA-targeting module further comprises a gRNA for targeting the DNA-binding domain to the target site.

[0234] In some embodiments, a DNA-targeting module is a zinc finger protein (ZFP) -based DNA-targeting module. In such embodiments the DNA-binding domain of the fusion protein is an engineered zinc finger protein (eZFP).

[0235] In some embodiments, a DNA-targeting module is a transcription activator-like effector (TALE) -based DNA-targeting module. In some embodiments, in a TALE-based DNA- targeting module, the DNA-binding domain of the fusion protein is an engineered TALE. sf-6779026.8 66224742003040

[0236] In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module targets a different target site. In some embodiments, one or more target sites are for different genes. In some embodiments, one or more target sites are for the same gene. In some embodiments, the DNA-targeting system is a multiplexed DNA-targeting system, i.e., is targeted to target sites for more than one gene. Hence, the term DNA-targeting system may include a multiplexed epigenetic-modifying DNA targeting system that includes more than one DNA-targeting module. In some embodiments, a multiplexed epigenetic-modifying DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA- targeting system comprises 2 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 3 DNA-targeting modules.

[0237] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise separate (i.e., non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein with a DNA-binding domain (e.g., a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein with a second DNA-binding domain (e.g., a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0238] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise shared (i.e., overlapping) components. For example, a DNA-targeting system may comprise: i) a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and an effector domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and ii) a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g., SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA-targeting modules.

[0239] In some aspects, the DNA-targeting system comprises a plurality of DNA-targeting sf-6779026.8 67224742003040 modules for modulating transcription of one or more genes. In some embodiments, the plurality of DNA-targeting modules comprises a first DNA-targeting module for modulating transcription of a first gene of the one or more genes, and a second DNA-targeting module for modulating transcription of a second gene of the one or more genes. In some embodiments, each DNA- targeting module comprises a fusion protein comprising: (a) a DNA-binding domain for targeting a target site of the target gene for the DNA-targeting module, and (b) at least one effector domain. In some embodiments, each DNA-targeting module comprises a transcriptional activator effector domain for increasing transcription of the one or more genes. IV. METHODS OF USE

[0240] In some embodiments, the LNPs, and compositions thereof, provided herein can be used to genetically engineer a cell or population of cells, such as primary cells (e.g., T cells) or induced cells, such as iPSCs and cells differentiated from iPSCs. Cells differentiated from iPSCs are also referred to herein as iPSC derived cells or cells derived from iPSCs. “Induced” cells are noted by the prefix “i” before the cell type. In provided embodiments, the “induced” or “i” population of cells exhibit a phenotype and function characteristic of such a cell type, such as present by a native (primary) population of such cells, for example a primary population of the cell type from a human subject, or a known population of cells differentiated by other accepted methods. In some embodiments, the cells differentiated from iPSCs are induced hematopoietic progenitor cells (iHPCs), induced lymphoid progenitor cells (iLPCs), induced common lymphoid progenitors (iCLPs), induced T-cell progenitors (iTCPs), induced NK (iNK) cells, or induced T (iT) cells.

[0241] In some embodiments, the cells are in the process of differentiation from one induced cell type to another induced cell type. These cells may exist in transitional states where they exhibit characteristics of both the preceding and succeeding cell types and may possess unique properties that are not present in fully differentiated cells. Cells that are in the process of differentiating and cannot be distinctly categorized as a specific cell type are referred to as “emerging” cells. These cells may be characterized by the expression of markers indicative of both the preceding and succeeding stages of differentiation. For example, cells may be emerging iTCPs, which are cells in the process of differentiating into T-cell progenitors from iHPCs (see e.g., Example 16, FIG.26A, and FIG.27A) or emerging iT cells, which are cells in the process of differentiating into mature T cells from iTCPs (see e.g., Example 17 and FIG.28 A). Over sf-6779026.8 68224742003040 the course of differentiation, emerging iTCPs transition from CD7+ cells to CD5+ / 7+ cells before becoming CD4+ / CD8+ iTCPs.

[0242] In some embodiments, the genetic engineering comprises introducing an RNA cargo into a cell(e.g., a T cell) or population of cells by transfection with any of the LNPs described herein. comprising an encapsuled nucleic acid, such as an RNA cargo. It is understood that reference to transfection with LNPs is referring to the process in which the LNP is used to deliver encapsulated nucleic acid (e.g., RNA cargo) to cells In some embodiments, the transfection comprises incubating the cell (e.g, primary cell or induced cell) or population of cells with any of the LNPs described herein. In some embodiments, the RNA cargo comprises a DNA-targeting system for modulating the transcription of one or more genes in the cell. The methods provided herein include use of one or more of the LNPs and compositions thereof provided herein (e.g. as described in Section I) for the delivery of an RNA cargo (e.g., as described in Section II) comprising a DNA targeting system (e.g., as described in Section III) to a cell or population of cells. In some embodiments, the methods increase the expression of one or more of target genes in the cell or population of cells. In some embodiments, the methods decrease the expression of one or more target genes in the cell or population of cells. In some embodiments, the provided methods include use of one or more of the LNPs and compositions thereof provided herein (e.g. as described in Section I) for the delivery of an RNA cargo (e.g., as described in Section II) comprising a DNA targeting system (e.g., as described in Section III) to a primary cell or a population of primary cells. In some embodiments, the methods increase the expression of one or more of target genes in the primary cell or population of primary cells. In some embodiments, the methods decrease the expression of one or more target genes in the primary cell or population of primary cells. In some embodiments, the provided methods include use of one or more of the LNPs and compositions thereof provided herein (e.g. as described in Section I) for the delivery of an RNA cargo (e.g., as described in Section II) comprising a DNA targeting system (e.g., as described in Section III) to an induced cell or a population of induced cells. In some embodiments, the methods increase the expression of one or more of target genes in the induced cell or population of induced cells. In some embodiments, the methods decrease the expression of one or more target genes in the induced cell or population of induced cells. Also provided herein is a population of genetically engineered cells (e.g., primary cells or induced cells) produced by any of the provided methods. sf-6779026.8 69224742003040

[0243] In some embodiments, the methods comprise contacting the LNP with exogenous apolipoprotein E (ApoE). In some embodiments, the contacting involves incubating the LNP with ApoE. In some embodiments, the contacting occurs prior to transfection of the cell or population of cells with the LNP. In some embodiments, the LNP is complexed with ApoE prior to transfection of the cell or population of cells with the LNP. In some embodiments, the cells are transfected with LNP complexed with ApoE. In some such embodiments, the LNP-ApoE complex is added to the cells at an ApoE concentration of between at or about 0.5 g / mL and 5 g / mL. In some embodiments the ApoE concentration is between at or about 1 g / mL and 3 g / mL. In some embodiments, the the ApoE concentration is at or about 1 g / mL, 1.5 g / mL, 2 g / mL, 2.5 g / mL, or 3 g / mL. In some embodiments, the ApoE concentration is at or about 2 g / mL.

[0244] In some embodiments, the methods can be carried out in vitro. In some embodiments, the methods can be carried out ex vivo on cells isolated from a subject. In some embodiments, the methods can be carried out in vivo in which the LNPs comprising the RNA cargo encoding a DNA-targeting system are administered to a subject. A. Cells

[0245] In some embodiments, provided are engineered cells, e.g., genetically engineered or modified cells, produced by any of the methods provided herein. In some embodiments, an RNA cargo comprising one or more polynucleotides, e.g., encoding a DNA-targeting system, such as any described herein, are introduced into a cell (e.g. a T cell or induced cell) or population of cells for engineering using one or more of the LNPs and compositions thereof provided herein (e.g. as described in Section I). In some aspects, the polynucleotides and / or portions thereof are heterologous, i.e., normally not present in a cell or sample obtained from the cell, such as one obtained from another organism or cell, which for example, is not ordinarily found in the cell being engineered and / or an organism from which such cell is derived. In some embodiments, the polynucleotides are nucleic acid sequences that are not naturally occurring, such as a nucleic acid sequences that are not found in nature or are modified from a nucleic acid sequences found in nature, including ones comprising chimeric combinations of nucleic acids.

[0246] The cells that are genetically engineered using the provided methods are generally eukaryotic cells, such as mammalian cells, and typically are human cells. In some embodiments, the cells are primary cells, such as those isolated directly from a subject and / or isolated from a sf-6779026.8 70224742003040 subject and frozen. In some embodiments, the cells are derived from the liver. In some embodiments, the cells are hepatocytes. In some embodiments, the cells are derived from the blood, bone marrow, lymph, or lymphoid organs, are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and / or NK cells. In some embodiments, the cells are T cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs) or hematopoietic progenitor cells (HPCs). In some embodiments, the cells are derived from iPSCs. In any of the provided embodiments, the cells are induced cells. In some embodiments, the cells are differentiated from iPSCs. In some embodiments, the cells are induced hematopoietic progenitor cells (iHPCs). In some embodiments, the cells are induced lymphoid progenitor cells (iLPCs). In some embodiments, the cells are induced common lymphoid progenitors (iCLPs). In some embodiments, the cells are induced T-cell progenitors (iTCPs). In some embodiments, the cells are induced NK (iNK) cells. In some embodiments, the cells are induced T (iT) cells. In some embodiments, the cells are emerging cells. In some embodiments the cells are emerging iHPCS. In some embodiments, the cells are emerging iLPCs. In some embodiments, the cells are emerging iCLPs. In some embodiments, the cells are emerging iTCPs. In some embodiments, the cells are emerging iNK cells. In some embodiments the cells are emerging iT cells.

[0247] In any of the provided embodiments, the cells are pluripotent stem cells. In some embodiments, the cells are induced pluripotent stem cells (iPSCs). In some embodiments, the stem are derived from, such as differentiated from, iPSCs. In some embodiments, the population of cells are or include hematopoietic progenitor cells (HPCs). In some embodiments, the population of HPCs are differentiated from iPSCs. In some embodiments, the population of cells are or include hematopoietic stem cells (HSCs). In some embodiments, the population of HSCs are differentiated from iPSCs.

[0248] In some embodiments, iPSC cells can be obtained by introducing a specific factor (nuclear reprogramming factor) into a mammalian somatic cell or an undifferentiated stem cell to reprogram them. A skilled artisan is familiar with various types of iPSCs and methods for obtaining the same. In some embodiments, iPSCs are established by introducing the four factors Oct3 / 4, Sox2, Klf4, and c-Myc into mouse fibroblasts, such as described by Yamanaka et al. (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676). In some embodiments, iPSC cells can be derived from human cells established by introducing the same four factors into human sf-6779026.8 71224742003040 fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872). In some embodiments, the iPSCs may be Nanog-iPS cells established by introducing the four factors, then selecting them by using the expression of Nanog as an index (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317). In some embodiments, iPS cells can be prepared by a method free of C-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106). In some embodiments, iPS cells can be established by introducing six factors by a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells.31(3): 458-66). In some embodiments, iPSCs can be established by introducing the four factors OCT3 / 4, SOX2, NANOG, and LIN28, such as described by Thomson et al. (Yu J., Thomson J A. et al., Science (2007) 318: 1917-1920). In some embodiments, iPSCs can be prepared by methods described in Daley et al. (Park I H, Daley G Q. et al., Nature (2007) 451: 141-146). In some embodiments, iPSCs can be prepared by methods described in Sakurada et al. (JP 2008-307007 A). Other methods for inducing or obtaining iPSCs are well known, including any described in any published literature (for example, Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol 3, Issue 5, 568-574; Kim J B., Scholer H R., et al., Nature, (2008) 454, 646-650; and Huangfu D., Melton, D A., et al., Nature Biotechnology, (2008) 26, No 7, 795-797), or patent (for example, JP 2008-307007 A, JP 2008-283972 A, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, and WO2009-007852). In some embodiments, the cells can be an iPSC cell line. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN- 2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's 253G1 line, 201B7 line, 409B2 line, 454E2 line, 606A1 line, 610B1 line, and 648A1 line.

[0249] In some embodiments, a hematopoietic stem cell (HSC) is a multipotent stem cell that can differentiate into blood cells, including lymphocytes. In some embodiments, a hematopoietic progenitor cell (HPC) is a cell that has the ability to differentiate into blood cells but does not have the ability to self-renew as much as a stem cell. In humans, HSCs and HPCs are mainly present in bone marrow, but are also present in peripheral blood and cord blood, and can be collected from each site. In provided embodiments, the hematopoietic stem cell may be a cell isolated from living tissues such as bone marrow, blood, or the like, or may be a cell prepared from an ES cell or an iPS cell. Both hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) are cells that are CD34-positive and CD3-negative as cell sf-6779026.8 72224742003040 the fact that it survives when transplanted and grafted to an animal, removed again, and then transplanted to another individual, which means that it has the ability to self-renew, in other words, that it is a “stem cell”, not a “progenitor cell”.

[0250] In some embodiments, the cells are HPCs differentiated from pluripotent stem cells (PSCs). In some embodiments, the cells are induced hematopoietic progenitor cells (iHPCs) differentiated from induced pluripotent stem cells (iPSCs). A skilled artisan is familiar with media and methods for iHPC differentiation from iPSCs. In some embodiments, the media or methods used to differentiate iHPCs from iPSCs may be commercially available media or kits. Exemplary commercially available kits include, for example, STEMdiff™ Hepatocyte Kit (STEMCELL Technologies), QualiStem® IPS Cell Hepatocyte Differentiation Kit (Creative Bioarray), Cellartis iPS Cell to Hepatocyte Differentiation System (Takara Bio), StemXVivo Hepatocyte Differentiation Kit (R&D Systems). In some embodiments, iHPCs are differentiated from iPSCs using epi-genetic editing, for example as described in WO 2025 / 059073, the disclosure of which is incorporated by reference in its entirety.

[0251] Both HPCs and iHPCs are cells that are positive for the CD34 cell surface marker (CD34+ cells). In some embodiments, the HPCs or iHPCs express CD34. In some embodiments, the HPCs or iHPCs express at least two markers from the group consisting of CD43, CD34, CD31, CD41, CD235 and CD45. In some embodiments, HPCs or iHPCs express one or more of the cell surface markers selected from the group consisting of CD34, CD43, CD7, DLL4, CD144, and CD235. In some embodiments, the HPCs or iHPCs express CD144, CD34, CD45, and CD7. In some embodiments, the HPCs or iHPCs express CD144, CD34, CD45, and CD7.

[0252] In some embodiments, the cells are lymphoid progenitor cells. In some embodiments the cells are induced lymphoid progenitor cells (iLPCs) differentiated from iHPCs. In some embodiments, the cells are common lymphoid progenitor cells. In some embodiments the cells are induced common lymphoid progenitors (iCLPs) differentiated from iHPCs.

[0253] In some embodiments, the cells are iLPCs, iCLPs, iT cells, or emerging cells of any of the forgoing generated using T cell differentiation media or methods, such as any described in Flippe et al., “Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells,” Front. Cell Dev. Biol., (2020) 8, Shukla et al., “Progenitor T-cell differentiation from hematopoietic stem cells using Delta-like-4 and VCAM-1,” Nature Methods, (2017) 14;531-538, Lai et al., “T and B lymphocyte differentiation from hematopoietic stem cell,” Semin. Immunol. (2008) 20(4); 207-212, Iriguchi et al., “In Vitro Differentiation of sf-6779026.8 73224742003040 T Cells: From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells,” Methods Mol. Biol. (2019) 2048;59-70, and Iriguchi et al., “A clinically applicable and scalable method to regenerate T-cells from iPSCs for off-the-shelf T-cell immunotherapy,” Nat. Commun., (2021) 12(1):430, all of which are incorporated herein in their entireties. In some embodiments, T cell differentiation media or methods are commercially available kits, such as StemSpan™ T Cell Generation Kit (STEMCELL Technologies).

[0254] In some embodiments, the cells are iLPCs, iCLPs, iNK cells, or emerging cells of any of the forgoing generated using NK cell differentiation media or methods, such as any described in Zhu et al., “An improved method to produce clinical scale natural killer cells from human pluripotent stem cells,” biorx (2019), Zhu et al., “Pluripotent stem cell-derived NK cells with high-affinity noncleavable CD18a mediate improved antitumor activity,” Blood (2020) 135(6):399-410, and Knorr et al., “Clinical-scale derivation of natural killer cells from human pluripotent stem cells for cancer therapy,” Stem Cells Transl Med. (2013) 4:274-83, all of which are incorporated herein in their entireties. In some embodiments, NK cell differentiation media or methods are commercially available kits, such as STEMdiff™ NK Cell Kit (STEMCELL Technologies).

[0255] In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence capacities, antigen-specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the T cells or iT cells express CD2, CD3, CD4, CD5, CD7, CD8b, or any combination thereof. In some embodiments, the iT cells express CD4 or CD8b. Induced T cell progenitors are double positive for the CD4 and C8 cell surface markers. In some embodiments, the iTCPs are CD4 / CD8 double positive cells. Mature iT cells derived from induced T cell progenitors are single positive for the CD8 cell surface marker (CD8+). In some embodiments, the iT cells are CD8 single positive T cells.

[0256] With reference to the subject to be treated, the cells may be allogeneic and / or autologous. Among the methods include off-the-shelf methods. In some aspects, such as for off-the-shelf technologies, the cells are pluripotent and / or multipotent, such as stem cells, such as iPSCs. In some embodiments, the methods include isolating cells from the subject, preparing, sf-6779026.8 74224742003040 processing, culturing, and / or engineering them, and re-introducing them into the same subject, before or after cryopreservation.

[0257] In some embodiments, the LNPs or compositions containing the same, as produced by the methods described herein, are used to deliver an RNA cargo into a T cell. In some embodiments, the T cells are activated prior to ex vivo delivery of the LNPs. In some embodiments, the T cells are activated between about 24 hours to 168 hours prior to transfection. In some embodiments, the T cells are activated about 24 hours, 72 hours, or 168 hours prior to transfection. In some embodiments, the T cells are activated by contacting the T cells with humanized CD3 and CD28 agonists, such as with an anti-CD3 antibody alone or in combination with an anti_CD28 antibody . In some embodiments, the T cells are activated by contacting the T cells with a commercially available reagent, such as T Cell TransAct™ (Miltenyi Biotec). In some embodiments, the T cells are not activated prior to ex vivo delivery of the LNPs. In some embodiments, delivering an RNA cargo into a T cell using a LNP described herein includes (a) incubating a composition containing T cells (“T cell composition”) under stimulating conditions; and (b) incubating the stimulated T cell composition with the LNPs, wherein the LNPs contain the RNA cargo. In some embodiments, the T cells are starved prior to delivery of the RNA cargo into the cell. In some embodiments, the T cells are starved for 10 minutes, 15, minutes, 20 minutes, 30 minutes, 45 minutes, or 60 minutes prior to delivery of the RNA cargo into the cell. In some embodiments, the T cells are starved for 30 minutes prior to delivery of the RNA cargo into the cell. In some embodiments, the T-cells are cultured in serum-free media. B. Genetic Engineering

[0258] In some embodiments, the provided LNPs can be used to deliver nucleic acid cargo to a cell or a population of cells. In some embodiments, the provided LNPs can be used to deliver an RNA cargo to a cell or a population of cells. In some embodiments, the provided LNPs can be used to a DNA cargo to a cell or a population of cells. In some embodiments, the provided nucleic acid cargo comprises a nucleic acid that is 1,200 to 12,000 nucleotides in length. In some embodiments, nucleic acid cargo comprises a nucleic acid that is at least 3,000 nucleotides in length, at least 4,500 nucleotides in length, is at least 6,000 nucleotides in length, or at least 7,5000 nucleotides in length.

[0259] In some embodiments, the provided LNPs can be used to deliver an RNA cargo encoding a DNA-targeting system to a cell (e.g., a T cell) or a population of cells. In some sf-6779026.8 75224742003040 embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of one or more target genes in the cell or population of cells. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the cell or population of cells.

[0260] In some embodiments, the DNA targeting system comprises a fusion protein comprising a DNA-binding domain (e.g., as described in Sections III.B and III.C) and an effector domain (e.g., as described in Sections III.D and III.E). In some embodiments, the effector domain is a transcriptional repressor domain (e.g., as described in Section III.D). In some embodiments, the effector domain is a transcriptional activator domain (e.g., as described in Section III.E). In some embodiments, the DNA-binding domain is a Cas (e.g., dCas) and the DNA targeting system further includes a gRNA. In some embodiments, the RNA cargo comprising the DNA targeting system comprises an mRNA encoding the fusion protein and a gRNA.

[0261] In some embodiments, the LNP is delivered to the cells one time or multiple times during a culture for differentiating cells. In some embodiments, a single dose of the LNP is delivered into the cells. In some embodiments, more than one dose of the LNP is delivered into the cells. In some such embodiments, a provided LNP is delivered to the cells intermittently several times during the culture of the cells. In some embodiments, the LNP is delivered at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times during a differentiation culture. In some embodiments, the LNP is delivered at least 2 individual times during a differentiation culture.

[0262] In some embodiments, the delivery of the LNP to the cells during a differentiation culture is performed according to a set interval of time. For example, in some embodiments, the the LNP is delivered to an induced hematopoietic progenitor cell or population of induced hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and then this is repeated once every day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, once every 7 days, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every 14 days, once every 15 days, once every 16 days, once every 17 days, once every 18 days, once every 19 days, or once every 20 days. In some embodiments, the LNP is delivered to induced hematopoietic progenitor cell or the population of induced hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and once every other day. In some embodiments, the LNP is sf-6779026.8 76224742003040 delivered to induced hematopoietic progenitor cell or the population of induced hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and one every three days. In some embodiments, the the LNP is delivered to induced hematopoietic progenitor cell or the population of induced hematopoietic progenitor cells at an initial or first time of culture (e.g. Day 0) and once every four day. In some embodiments, the delivery of the LNP to the cells is repeated 2-6 times, such as 2-4 times, during the culture. In some embodiments, the delivery of the LNP to the cells is repeated 2 times during the culture. In some embodiments, the delivery of the LNP to the cells is repeated 3 times during the culture.

[0263] In some embodiments, an individual delivery of the multiple deliveries of the LNP is performed dependent on the expression level of the product of one or more target genes of the DNA-targeting system. Target gene product expression levels may be monitored through any one of a number of methods known in the art. For example, if a product of a target gene is a protein expressed on the surface of a cell (e.g., DLL4 protein and VCAM1 protein), expression level may be monitored through flow cytometric analysis. In some embodiments, an individual delivery of the multiple deliveries of the LNP is performed following a decrease and / or loss of expression of the product of one or more target genes of the DNA-targeting system. In some embodiments, an individual delivery of the multiple deliveries of the LNP is performed dependent on the expression level of a marker and / or indicator of a differentiated population of cells. In some embodiments, the marker and / or indicator of a differentiated population of cells is a surface marker whose expression is associated with the differentiated population of cells. In some embodiments, an individual delivery of the multiple deliveries of the LNP is performed following an increase in expression level of a marker and / or indicator of a differentiated population of cells. In some embodiments, an individual delivery of the multiple deliveries of the LNP is performed following a decrease in expression level of a marker and / or indicator of a differentiated population of cells.

[0264] In some embodiments, the LNP is delivered at a dose of 50 ng to 10 g per 1x106cells. In some embodiments, the LNP is delivered at a dose of 100 ng to 5 g per 1x106cells. In some embodiments, the LNP is delivered at a dose of 0.5 g to 4 g per 1x106cells. In some embodiments, the LNP is delivered at a dose of 0.6 g to 2.4 g per 1x106cells. In some embodiments, the LNP is delivered at a dose of 80 ng per 1x106cells, 200 ng per 1x106cells, 400 ng per 1x106cells, 0.5 g per 1x106cells, 0.6 g per 1x106cells, 1 g per 1x106cells, 1.2 g per 1x106cells, 1.5 g per 1x106cells, 2 g per 1x106cells, 2.4 g per 1x106cells, 2.5 g sf-6779026.8 77224742003040 per 1x106cells, 3 g per 1x106cells, 3.5 g per 1x106cells, 4 g per 1x106cells, or a value between any of the forgoing. In some embodiments, the LNP is delivered at a dose of 80 ng per 1x106cells, 200 ng per 1x106cells, 400 ng per 1x106cells 0.5 g per 1x106cells, 0.6 g per 1x106cells, 1 g per 1x106cells, 1.2 g per 1x106cells, 1.5 g per 1x106cells, 2 g per 1x106cells, 2.4 g per 1x106cells, 2.5 g per 1x106cells, 3 g per 1x106cells, 3.5 g per 1x106cells, 4 g per 1x106cells. In some embodiments, the LNP is delivered at a dose of 0.5 g g per1x106 cells, 2 g per 1x106 cells, or 4 g per 1x106 cells. In some embodiments, the LNP isdelivered at a dose of 0.6 g g per 1x106cells, 1,2 g per 1x106cells, or 2.4 g per 1x106cells.

[0265] In some embodiments, the LNP is delivered at a dose of 6.25 ng to 1250 ng per 1.25x105cells. In some embodiments, the LNP is delivered at a dose of 12.5 ng to 625 per 1.25x105cells. In some embodiments, the LNP is delivered at a dose of 62.5 ng to 500 ng per 1.25x105cells. In some embodiments, the LNP is delivered at a dose of 75 ng to 300 ng per1.25x105 cells. In some embodiments, the LNP is delivered at a dose of 10 ng per 1.25x105 cells,25 ng per 1.25x105cells, 50 ng per 1.25x105cells, 75 ng per 1.25x105cells, 100 ng per 1.25x105cells, 125 ng per 1.25x105cells, 150 ng per 1.25x105cells, 175 ng per 1.25x105cells, 200 ng per 1.25x105cells, 250 ng per 1.25x105cells, 300 ng per 1.25x105cells, 350 ng per 1.25x105cells, 400 ng per 1.25x105cells, 450 ng per 1.25x105cells, 500 ng per 1.25x105cells or a value between any of the forgoing. In some embodiments, the LNP is delivered at a dose of 10 ng per 1.25x105cells, 25 ng per 1.25x105cells, 50 ng per 1.25x105cells, 75 ng per 1.25x105cells, 100 ng per 1.25x105cells, 125 ng per 1.25x105cells, 150 ng per 1.25x105cells, 175 ng per 1.25x105cells, 200 ng per 1.25x105cells, 250 ng per 1.25x105cells, 300 ng per 1.25x105cells, 350 ng per 1.25x105cells, 400 ng per 1.25x105cells, 450 ng per 1.25x105cells, 500 ng per 1.25x105cells. In some embodiments, the LNP is delivered at a dose of 62.5 ng per 1.25x105 cells, 250 ngper 1.25x105cells, or 500 ng per 1.25x105cells. In some embodiments, the LNP is delivered at a dose of 75 ng per 1.25x105cells, 150 ng per 1.25x105cells, or 300 ng per 1.25x105cells.

[0266] In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to a T cell. In some embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of one or more target genes in the T cell. In some embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of CD45. In some embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of MED12. sf-6779026.8 78224742003040

[0267] In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an iPSC. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the iPSC. In some embodiments, delivery of the DNA-targeting systems leads to increased or activated transcription of EGFR. In some embodiments, delivery of the DNA-targeting systems leads to increased or activated transcription of GATA2. In some embodiments, delivery of the DNA- targeting systems leads to increased or activated transcription of GATA3. In some embodiments, delivery of the DNA-targeting systems leads to increased or activated transcription of LMO2.

[0268] In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an HPC. In some embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of one or more target genes in the HPC. In some embodiments, delivery of the DNA-targeting systems leads to decreased or repressed transcription of B2M. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the HPC. In some embodiments, delivery of the DNA-targeting systems leads to increased or activated transcription of TCF7. In some embodiments, delivery of the DNA-targeting systems leads to increased or activated transcription of IL2.

[0269] In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an iHPC. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the iHPC. In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an emerging iTCP. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the emerging iTCP. In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an iTCP, In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the iTCP. In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an emerging iT cell. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more target genes in the emerging iT cell.

[0270] As an example, if the target gene is DLL4 and the cargo includes a DNA-targeting system for transcriptional activation (e.g., containing a DNA-targeting domain in Section III.B as a fusion protein with an effector domain in Section III.E and a gRNA targeting DLL4) , the sf-6779026.8 79224742003040 delivery of the DNA-targeting system leads to increased or activated transcription of DLL4. As another example, if the target gene is VCAM1 and the cargo includes a DNA-targeting system for transcriptional activation (e.g., containing a DNA-targeting domain in Section III.B as a fusion protein with an effector domain in Section III.E and a gRNA targeting VCAM1) , delivery of the DNA-targeting system leads to increased or activated transcription of VCAM1. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of DLL4 and VCAM1. The LNPs can be used for delivery of a DNA-targeting system for increasing or activating transcription, or in some cases reducing or pressing transcription, of any of a number of target genes into desired cell types as described. The exemplification of DLL4 and VCAM1 demonstrates the utility of the provided LNPs to mediate such delivery in primary cells, such as T cells, and in various induced cell types including iPSCs and cells derived from iPSCs.

[0271] In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to an iPSC derived CD56+ cell. In some embodiments, delivery of the DNA-targeting system leads to increased or activated transcription of one or more tamaryrget genes in the iPSC derived CD56+ cell. In some embodiments, delivery of the DNA-targeting systems leads to increased or activated transcription of IL2.

[0272] In some embodiments, the provided LNPs can be used to deliver a DNA-targeting system to a hepatocyte. In some embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of one or more target genes in the hepatocyte. In some embodiments, delivery of the DNA-targeting system leads to decreased or repressed transcription of PCSK9. V. DEFINITIONS

[0273] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art

[0274] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, “a” or “an” means “at least one” or “one or sf-6779026.8 80224742003040 more.” It is understood that aspects and variations described herein include “consisting” and / or “consisting essentially of” aspects and variations.

[0275] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0276] The term “about” as used herein refers to the usual error range for the respective value readily known. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X”. In some embodiments, “about” may refer to ±25%, ±20%, ±15%, ±10%, ±5%, or ±1%.

[0277] A “gene,” includes a DNA region encoding a gene product. Thus, the gene typically refers to coding and / or transcribed sequences. The sequence of a gene is typically present at a fixed chromosomal position or locus on a chromosome in the cell.

[0278] A “regulatory element” or “DNA regulatory element,” which terms are used interchangeably herein, in reference to a gene refers to DNA regions which regulate the production of a gene product, whether or not such regulatory sequences are adjacent to coding and / or transcribed sequences. Accordingly, a regulatory element includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions. sf-6779026.8 81224742003040

[0279] As used herein, a “target site” or “target nucleic acid sequence” is a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule (e.g. a DNA- binding domain disclosed herein) will bind, provided sufficient conditions for binding exist.

[0280] The term “expression” with reference to a gene or “gene expression” refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or can be a protein produced by translation of an mRNA. For instance, expression includes the transcription and / or translation of a particular nucleotide sequence drive by its promoter. Gene products also include RNAs which are modified, by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation. Hence, reference to expression or gene expression includes protein (or polypeptide) expression or expression of a transcribable product of or a gene such as mRNA. The protein expression may include intracellular expression or surface expression of a protein. Typically, expression of a gene product, such as mRNA or protein, is at a level that is detectable in the cell.

[0281] As used herein, the term “reduced expression” or “decreased expression” means any form of expression that is lower than the expression in an original or source cell that does not contain the modification for modulating a particular gene expression by a DNA-targeting system, for instance a wild-type expression level (which can be absence of expression or immeasurable expression as well). Reference herein to “reduced expression,” or “decreased expression” is taken to mean a decrease in gene expression relative to the level in a cell that does not contain the modification, such as the original source cell prior to contacting with, or engineering to introduce, the DNA-binding system into the cell, such as an unmodified cell or a wild-type cell. The decrease in expression can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the decrease in expression can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold or more.

[0282] As used herein, the term “reduced transcription” or “decreased transcription” refers to the level of transcription of a gene that is lower than the transcription of the gene in an original or source cell that does not contain the modification for modulating transcription by a DNA-targeting system, for instance a wild-type transcription level of a gene. Reference to sf-6779026.8 82224742003040 reduced transcription or decreased transcription can refer to reduction in the levels of a transcribable product of a gene such as mRNA. Any of a variety of methods can be used to monitor or quantitate a level of a transcribable product such as mRNA, including but not limited to, real-time quantitative RT (reverse transcriptase)- polymerase chain reaction (qRT-PCR), Northern Blot, microarray analysis, or RNA sequencing (RNA-Seq). The reduction in transcription can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the reduction in transcription can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold or more.

[0283] As used herein, the term “increased expression”, “enhanced expression” or “overexpression” means any form of expression that is additional to the expression in an original or source cell that does not contain the modification for modulating a particular gene expression by a DNA-targeting system, for instance a wild-type expression level (which can be absence of expression or immeasurable expression as well). Reference herein to “increased expression,” “enhanced expression” or “overexpression” is taken to mean an increase in gene expression relative to the level in a cell that does not contain the modification, such as the original source cell prior to contacting with, or engineering to introduce, the DNA-targeting system into the cell, such as an unmodified cell or a wild-type cell. The increase in expression can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the increase in expression can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-food, 500-fold, 1000-fold or more.

[0284] As used herein, the term “increased transcription” refers to the level of transcription of a gene that is additional to the transcription of the gene in an original or source cell that does not contain the modification for modulating transcription by a DNA-targeting system, for instance a wild-type transcription level of a gene. Reference to increased transcription can refer to an increase in the levels of a transcribable product of a gene such as mRNA. Any of a variety of methods can be used to monitor or quantitate a level of a transcribable product such as mRNA, including but not limited to, real-time quantitative RT (reverse transcriptase)- polymerase chain reaction (qRT-PCR), Northern Blot, microarray analysis, or RNA sequencing (RNA-Seq). The increase in transcription can be at least 5%, 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 100% or even more. In some cases, the increase in transcription sf-6779026.8 83224742003040 can be at least 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80- fold, 90-fold, 100-fold, 200-fold or more.

[0285] As used herein, the term “modification” or “modified” refers to any change or alteration in a cell that impacts gene expression in the cell. In some embodiments, the modification is an epigenetic modification that directly changes the epigenetic state of a gene or regulatory elements thereof to alter (e.g. increase) expression of a gene product. In some embodiments, a modification described herein results in increased expression of a target gene or selected polynucleotide sequence.

[0286] As used herein, an “epigenetic modification” refers to changes in the gene expression that are non-genetic modifications, i.e. not caused by changes in the DNA sequences, but are due to epigenetic changes such as events like DNA methylations or histone modifications. An epigenetic modification may result in a heritable change in gene activity and expression that occur without alteration in DNA sequence. For instance, epigenetic modifications include non- genetic modifications such as chemical modifications to the cytosine residues of DNA (DNA methylation) and histone proteins associated with DNA (histone modifications).

[0287] As used herein, a “fusion” molecule is a molecule in which two or more subunit molecules are linked, such as covalently. Examples of a fusion molecule include, but are not limited to, fusion proteins (for example, a fusion between a DNA-binding domain such as a ZFP, TALE DNA-binding domain or CRISPR-Cas protein and one or more effector domains, such as a transactivation domain). The fusion molecule also may be part of a system in which a polynucleotide component associates with a polypeptide component to form a functional system (e.g., a CRISPR / Cas system in which a single guide RNA associates with a functional domain to modulate gene expression). Fusion molecules also include fusion nucleic acids, for example, a nucleic acid encoding the fusion protein. Expression of a fusion protein in a cell can result from delivery of the fusion protein to the cell or by delivery of a polynucleotide encoding the fusion protein to a cell, where the polynucleotide is transcribed, and the transcript is translated, to generate the fusion protein.

[0288] The term “vector,” as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self- replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as sf-6779026.8 84224742003040 “expression vectors.” Among the vectors are viral vectors, such as adenoviral vectors or lentiviral vectors.

[0289] The term “expression vector" refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include, but are not limited to, cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0290] The term “isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

[0291] The term "polynucleotide" refers to a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomelic "nucleotides." The monomelic nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0292] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein's or peptide's sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, sf-6779026.8 85224742003040 substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

[0293] As used herein, “percent (%) amino acid sequence identity” and “percent identity” when used with respect to an amino acid sequence (reference polypeptide sequence) is defined as the percentage of amino acid residues in a candidate sequence (e.g., the subject antibody or fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various known ways, in some embodiments, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0294] In some embodiments, “operably linked” may include the association of components, such as a DNA sequence, (e.g. a heterologous nucleic acid) and a regulatory sequence(s), in such a way as to permit gene expression when the appropriate molecules (e.g. transcriptional activator proteins) are bound to the regulatory sequence. Hence, it means that the components described are in a relationship permitting them to function in their intended manner.

[0295] An amino acid substitution may include replacement of one amino acid in a polypeptide with another amino acid. The substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions may be introduced into a binding molecule, e.g., antibody, of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

[0296] Amino acids generally can be grouped according to the following common side- chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; sf-6779026.8 86224742003040 (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[0297] In some embodiments, conservative substitutions can involve the exchange of a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions can involve exchanging a member of one of these classes for another class.

[0298] As used herein, a composition refers to any mixture of two or more products, substances, or compounds, including cells. It may be a solution, a suspension, liquid, powder, a paste, aqueous, non-aqueous or any combination thereof.

[0299] As used herein, a “subject” or an “individual,” which are terms that are used interchangeably, is a mammal. In some embodiments, a “mammal” includes humans, non- human primates, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, monkeys, etc. In some embodiments, the subject or individual is human. In some embodiments, the subject is a patient that is known or suspected of having a disease, disorder or condition.

[0300] As used herein, the term “treating” and “treatment” includes administering to a subject an effective amount of a biological molecule, such as a therapeutic agent, so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results. For instance, a biological molecule may include cells (e.g. T cells), such as cells that have been modified by a DNA-targeting system or polynucleotide(s) encoding the DNA-targeting system described herein. For purposes of this technology, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the disease.

[0301] For purposes of this technology, beneficial or desired clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, diminishment of sf-6779026.8 87224742003040 extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.

[0302] The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term "therapeutically effective amount" includes that amount of a biological molecule, such as a compound or cells, that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the biological molecule, the disease and its severity and the age, weight, etc., of the subject to be treated.

[0303] As used herein, the term "autologous" is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[0304] "Allogeneic" refers to a graft derived from a different animal of the same species.

[0305] As used herein, a statement that a cell or population of cells is “negative” for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, wherein the staining is not detected by flow cytometry at a level substantially above the staining detected carrying out the same procedure with an isotype-matched control under otherwise identical conditions, and / or at a level substantially lower than that for cell known to be positive for the marker, and / or at a level substantially similar as compared to that for a cell known to be negative for the marker.

[0306] As used herein, the term “messenger RNA (mRNA)” refers to a polynucleotide that encodes at least one peptide, polypeptide or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, mRNA can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence sf-6779026.8 88224742003040

[0307] As used herein, the term “nucleic acid,” in its broadest sense, refers to any compound and / or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, “nucleic acid” encompasses RNA as well as single and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. VI. EXEMPLARY EMBODIMENTS

[0308] Among the provided embodiments are: 1. A lipid nanoparticle (LNP) comprising: i) from 30-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. 2. A lipid nanoparticle (LNP)) comprising: i) from 30-40 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 20-30 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. 3. A lipid nanoparticle (LNP) comprising: i) from 45-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 5-15 mol % of a neutral lipid; sf-6779026.8 89224742003040 iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. 4. The LNP of any of embodiments 1-3, wherein the ionizable lipid has a 4-methyl-1- piperazinebutanamine head group that is linked via a degradable primary ester to an octyldodecyl tail. 5. The LNP of any of embodiments 1-4, wherein the ionizable lipid is linked via two degradable primary esters to two octyldodecyl tails. 6. The LNP of any of embodiments 1-5, wherein the ionizable lipid is characterized by 4 saturated alkyl tails with non-symmetric 8 and 10 carbon lengths. 7. The LNP of any of embodiments 1-6, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24). 8. The LNP of any of embodiments 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 1 to 5 mol %. 9. The LNP of any of embodiments 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 1 to 2 mol % or 1 to 3 mol%. 10. The LNP of any of embodiments 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 2 to 5 mol %. 11. The LNP of any of embodiments 1-10, wherein the steroid is present in a concentration ranging from 35 to 45 mol %. 12. The LNP of any of embodiments 1-11, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) or 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9). 13. The LNP of any of embodiments 1-12, wherein the neutral lipid is DSPC. 14. The LNP of any of embodiments 1-13, wherein the polymer conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid. 15. The LNP of any of embodiments 1-14, wherein the polymer conjugated lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). sf-6779026.8 90224742003040 16. The LNP of any of embodiments 1-15, wherein the steroid is cholesterol. 17. A lipid nanoparticle (LNP) comprising: (i) about 37.5 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 22.5 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule. 18. A lipid nanoparticle (LNP) comprising: (i) about 50 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 10 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule. 19. The LNP of any of embodiments 1-18, wherein the at least one mRNA molecule is at least 3000 nucleotides in length. 20. The LNP of any of embodiments 1-19, wherein the at least one mRNA molecule is from about 3,000 to 12,000 nucleotides in length, from about 4,500 to 9,000 nucleotides in length, or from about 4,800 to 7,500 nucleotides in length. 21. The LNP of any of embodiments 1-20, wherein the at least one mRNA molecule encodes a protein that is from about 1,000 to 4,000 amino acids in length, from about 1,500 to 3,000 amino acids in length, or from about 1,600 to 2,500 amino acids in length. sf-6779026.8 91224742003040 22. The LNP of any of embodiments 1-21, wherein the RNA cargo comprises a DNA- targeting system for modulating transcription of a gene. 23. The LNP of any of embodiments 1-22, wherein the RNA cargo comprises a messenger RNA (mRNA) and a guide RNA (gRNA). 24. The LNP of embodiment 23, wherein the RNA cargo has a mRNA:gRNA w / w ratio of 10:1 to 1:5, optionally 10:1, 5:1, 2:1, 1:1, 1:2, or 1:5. 25. The LNP of embodiment 23, wherein the mRNA cargo has a mRNA:gRNA w / w ratio of 2:1 to 1:2. 26. The LNP of embodiment 23, wherein the RNA cargo has a mRNA:gRNA w / w ratio of 1:1. 27. The LNP of any of embodiments 1-26, wherein the LNP has an ionizable lipid nitrogen: oligonucleotide phosphate (N:P) molar ratio of 4:1 to 20:1. 28. The LNP of embodiment 27, wherein the LNP has an N:P ratio of 8:1. 29. The LNP of any of embodiments 1-28, wherein the at least one mRNA encodes a fusion protein comprising a DNA-binding domain and an effector domain. 30. The LNP of embodiment 29, wherein the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof. 31. The LNP of embodiment 30, wherein the Cas protein or variant thereof is a nuclease- deactivated Cas (dCas) protein. 32. The LNP of embodiment 31, wherein the dCas protein is a dCas9 protein. 33. The LNP of embodiment 32, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. 34. The LNP of any of embodiments 1-33, wherein the LNP is for delivery to a primary cell. 35. The LNP of any of embodiments 1-33, wherein the LNP is for delivery to an iPSC or iPSC derived cell. 36. A method of genetically engineering a population of cells, the method comprising transfecting an RNA cargo into a population of cells by delivering the LNP of any of embodiments 1-35 into the population of cells. 37. The method of embodiment 36, wherein the population of cells are primary cells. 38. The method of embodiment 37, wherein the population of primary cells are T cells. 39. The method of embodiment 37, wherein the population of primary cells are hepatocytes. sf-6779026.8 92224742003040 40. The method of embodiment 37, wherein the population of primary cells are HPCs. 41. The method of embodiment 38, wherein the T cells are CD4+. 42. The method of embodiment 38 or embodiment 41, wherein the T cells are activated prior to transfection. 43. The method of embodiment 42, wherein the T cells are activated by contacting the T cells with humanized CD3 and CD28 agonists. 44. The method of embodiment 42 or embodiment 43, wherein the T cells are activated between at or about 24 hours to 168 hours prior to transfection. 45. The method of any of embodiments 42-44, wherein the T cells are activated about 48 hours to about 72 hours prior to transfection. 46. The method of any of embodiments 38 or 41-45, wherein the T cells are cultured in serum-free media. 47. The method of embodiment 36, wherein the population of cells are induced pluripotent stem cells (iPSCs) or iPSC derived cells 48. The method of embodiment 47, wherein the population of iPSC derived cells are induced hematopoietic progenitor cells (iHPCs). 49. The method of embodiment 48, wherein the iHPCs are CD34+. 50. The method of embodiment 48 or embodiment49, wherein the population of iPSC derived cells are emerging induced lymphoid progenitor cells. 51. The method of embodiment 47, wherein the population of iPSC derived cells are induced lymphoid progenitor cells. 52. The method of embodiment 51, wherein the induced lymphoid progenitors are induced T-cell progenitors. 53. The method of embodiment 52, wherein the induced T-cell progenitors are double positive for CD4 and CD8 cell surface markers. 54. The method of embodiment 52 or embodiment 53, wherein the population of iPSC derived cells are emerging induced T cells. 55. The method of embodiment 47, wherein the population of iPSC derived cells are induced Natural Killer cells. 56. The method of embodiment 47, wherein the population of iPSC derived cells are induced T cells. sf-6779026.8 93224742003040 57. The method of embodiment 47 or embodiment 55, wherein the population of iPSC derived cells are CD56+. 58. The method of embodiment 47 or embodiment 56, wherein the population of iPSC derived cells are CD8+. 59. The method of any of embodiments 36-58, wherein the LNP is complexed with human ApoE prior to transfection. 60. The method of embodiment 59, wherein the LNP-ApoE complex is added to the cells at an ApoE concentration of between at or about 0.5 g / mL to 2 g / mL. 61. The method of any of embodiments 36-60, wherein the LNP is delivered at a dose of 50 ng to 10 g per million cells. 62. The method of any of embodiments 36-61, wherein the LNP is delivered at a dose of 100 ng to 5 g per million cells. 63. The method of any of embodiments 36-62, wherein the LNP is delivered at a dose of 0.5 g to 4 g per million cells. 64. The method of any of embodiments 36-63, wherein a single dose of the LNP is delivered into the cells. 65. The method of any of embodiments 36-63, wherein more than one dose of the LNP is delivered into the cells.

[0309] Further provided embodiments are: 1. A lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: i) from 30-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. 2. A lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: sf-6779026.8 94224742003040 i) from 30-40 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 20-30 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. 3. A lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: i) from 45-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 5-15 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule. 4. The LNP of any of embodiments 1-3, wherein the ionizable lipid has a 4-methyl-1- piperazinebutanamine head group that is linked via a degradable primary ester to an octyldodecyl tail. 5. The LNP of any of embodiments 1-4, wherein the ionizable lipid is linked via two degradable primary esters to two octyldodecyl tails. 6. The LNP of any of embodiments 1-5, wherein the ionizable lipid is characterized by 4 saturated alkyl tails with non-symmetric 8 and 10 carbon lengths. 7. The LNP of any of embodiments 1-6, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24). 8. The LNP of any of embodiments 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 1 to 5 mol %. 9. The LNP of any of embodiments 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 1 to 2 mol % or 1 to 3 mol%. 10. The LNP of any of embodiments 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 2 to 5 mol % sf-6779026.8 95224742003040 11. The LNP of any of embodiments 1-10, wherein the steroid is present in a concentration ranging from 35 to 45 mol %. 12. The LNP of any of embodiments 1-11, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), palmitoyloleoyl- phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), 1-stearoyl-2- oleoyl phosphatidylcholine (SOPC), dioleoylphosphatidylcholine (DOPC), dioleoyl- phosphatidylethanolamine (DOPE) or 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9). 13. The LNP of any of embodiments 1-12, wherein the neutral lipid is DSPC. 14. The LNP of any of embodiments 1-13, wherein the polymer conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid. 15. The LNP of any of embodiments 1-14, wherein the polymer conjugated lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k). 16. The LNP of any of embodiments 1-15, wherein the steroid is cholesterol. 17. A lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: (i) about 37.5 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 22.5 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule. 18. A lipid nanoparticle (LNP) for delivering a ribonucleic acid (RNA) cargo to a primary cell comprising: (i) about 50 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 10 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and sf-6779026.8 96224742003040 (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule. 19. The LNP of any of embodiments 1-18, wherein the at least one mRNA molecule is at least 3000 nucleotides in length. 20. The LNP of any of embodiments 1-19, wherein the at least one mRNA molecule is from about 3,000 to 12,000 nucleotides in length, from about 4,500 to 9,000 nucleotides in length, or from about 4,800 to 7,500 nucleotides in length. 21. The LNP of any of embodiments 1-20, wherein the at least one mRNA molecule encodes a protein that is from about 1,000 to 4,000 amino acids in length, from about 1,500 to 3,000 amino acids in length, or from about 1,600 to 2,500 amino acids in length. 22. The LNP of any of embodiments 1-21, wherein the RNA cargo comprises a DNA- targeting system for modulating transcription of a gene. 23. The LNP of any of embodiments 1-22, wherein the RNA cargo comprises a messenger RNA (mRNA) and a guide RNA (gRNA). 24. The LNP of embodiment 23, wherein the RNA cargo has a mRNA:gRNA w / w ratio of 10:1 to 1:5, optionally 10:1, 5:1, 2:1, 1:1, 1:2, or 1:5. 25. The LNP of embodiment 23, wherein the mRNA cargo has a mRNA:gRNA w / w ratio of 2:1 to 1:2. 26. The LNP of embodiment 23, wherein the RNA cargo has a mRNA:gRNA w / w ratio of 1:1. 27. The LNP of any of embodiments 1-26, wherein the LNP has an ionizable lipid nitrogen: oligonucleotide phosphate (N:P) molar ratio of 4:1 to 20:1. 28. The LNP of embodiment 27, wherein the LNP has an N:P ratio of 8:1. 29. The LNP of any of embodiments 1-28, wherein the at least one mRNA encodes a fusion protein comprising a DNA-binding domain and an effector domain. 30. The LNP of embodiment 29, wherein the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof. 31. The LNP of embodiment 30, wherein the Cas protein or variant thereof is a nuclease- deactivated Cas (dCas) protein. 32. The LNP of embodiment 31, wherein the dCas protein is a dCas9 protein. 33. The LNP of embodiment 32, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. sf-6779026.8 97224742003040 34. A method of genetically engineering a primary cell, the method comprising transfecting a primary cell with the LNP of any of embodiments 1-33. 35. The method of embodiment 34, wherein the primary cell is a T cell. 36. The method of embodiment 35, wherein the T cell is CD4+. 37. The method of embodiment 35 or embodiment 36, wherein the T cell is activated prior to transfection. 38. The method of any of embodiments 35-37, wherein the T cell is cultured in serum- free media. 39. The method of embodiment 34, wherein the primary cell is an induced pluripotent stem cell (iPSC) or an iPSC derived cell. 40. The method of embodiment 39, wherein the iPSC derived cell is a hematopoietic progenitor cell (HPC). 41. The method of embodiment 40, wherein the HPC is CD34+. 42. The method of embodiment 39, wherein the iPSC derived cell is CD56+. 43. The method of embodiment 34, wherein the primary cell is a hepatocyte. VII. EXAMPLES

[0310] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 : Hepatocyte Transfection using Lipid Nanoparticle Formulations with Varied Ionizable Lipids

[0311] Lipid nanoparticles formulated with various ionizable lipids were assessed for their ability to achieve LNP-mediated RNA delivery in primary human hepatocytes.

[0312] Briefly, LNPs each containing an ionizable lipid, a helper lipid, a polyethylene glycol (PEG)-conjugated lipid (“PEG lipid”), cholesterol, and mRNA encoding GFP were generated by microfluidic mixing and used to transfect primary human hepatocytes (PHH). The various LNP formulations were analyzed for their ability to deliver RNA by assessing GFP fluorescence of the transfected hepatocytes.

[0313] To generate the LNPs, the lipids were prepared in 100% ethanol as 50 mM stock solutions. An organic phase was prepared by diluting the ionizable lipid, the helper lipid, the PEG lipid, and the cholesterol to their specified concentrations in 100% ethanol. An aqueous phase was prepared by diluting the RNA to 0.1 mg / mL in 50 mM citrate buffer of pH 4.0. The sf-6779026.8 98224742003040 organic and aqueous phases were mixed at a volume ratio of 1:3 with a microfluidics mixer at a flow rate of 20 mL / min. After mixing, LNPs were dialyzed in 200x volume of 1xPBS overnight with 100K MWCO dialysis cassettes. LNPs were concentrated with 100K MWCO ultrafiltration tubes.

[0314] To assess how the identity of the ionizable lipid impacted the efficiency of LNP- mediated RNA delivery, LNPs were generated where only the ionizable lipid was varied. LNPs were formulated with one of the following ionizable lipids: MC3, C24, ATX-100, SM-102, ALC-0315, L319, and ckk-E12. Table E1 provides the specific composition of the LNP formulations with the varied ionizable lipids. The LNPs were formulated with an 8:1 N:P (lipid:mRNA) molar ratio. Table E1. LNP Formulation (C24 Formulation 1.0)

[0315] Primary human hepatocytes (PHH) were seeded in 48-well plates at 0.14x106viable cells / well and cultured overnight prior to transfection with the various LNPs encapsulating green fluorescent protein (GFP) mRNA. LNPs were pre-associated with human ApoE (0.1 mg / mL) for 5 minutes at 37 °C. Then, LNPs pre-complexed with ApoE were diluted in 200uL culture media with the final ApoE concentration of 2 ug / mL for 5 minutes at 37 °C prior to adding to cells. GFP fluorescence of the LNP transfected cells and non-transfected (“NT”) control cells was assessed 24 hours post-transfection using fluorescence microscopy (FIG.1A-1B) and flow cytometry (FIG.1C-1D). In preparation for imaging, cells were washed once with DPBS. Inpreparation for flow cytometry, cells were treated with 200 l / well trypsin for 5min, quenchedwith 150 l / well 10% FBS 1X PBS, spun down, washed twice with 200 l / well FACS buffer, andresuspended in 100 l FACS buffer.

[0316] As shown in FIG.1A, hepatocytes transfected with C24 LNPs exhibited substantially more GFP fluorescence than cells transfected with MC3 LNPs. Almost 50% of the cells transfected with C24 LNPs expressed GFP while less than 30% of the cells transfected with MC3 LNPs were GFP+ (FIG.1C). Cells transfected with ATX-100 LNPs also exhibited sf-6779026.8 99224742003040 stronger GFP fluorescence and more GFP+ cells than cells transfected with MC3 LNPs, though not to the same extent as cells transfected with C24 LNPs (FIGS.1A, 1C, 1E and 1F). In contrast, cells transfected with LNPs comprising the other ionizable lipids tested exhibited similar or decreased levels of GFP fluorescence and GFP+ cells compared to cells transfected with MC3 LNPs (FIGS.1B and 1D).

[0317] The results showed that LNPs comprising the C24 ionizable lipid can effectively deliver mRNA to hepatocytes. The results support the utility of C24 based LNP formulations for nucleic acid delivery to human hepatocytes. Example 2 : PCSK9 repression in hepatocytes using C24 lipid nanoparticles for DNA- targeting system delivery

[0318] Hepatocytes were transfected with C24-based lipid nanoparticles (LNPs) encapsulating a CRISPR / Cas-based DNA-targeting system for transcriptional repression of Proprotein convertase subtilisin / kexin type 9 (PCSK9) and assessed for targeted gene repression.

[0319] C24 LNPs were prepared generally as described in Example 1. Primary human hepatocytes (PHH) were transfected with C24 LNPs encapsulating mRNA encoding an exemplary DNMT3AL-dSpCas9-KRAB fusion protein (SEQ ID NO: 1), a PCSK9 targeting gRNA, and an eGFP mRNA (SEQ ID NO: 6) for assessing transection efficiency. The mRNA:gRNA ratio was 2:1 with 80% of the mRNA comprising the Cas fusion protein mRNA and the remaining 20% comprising the GFP mRNA. Cells were dosed at either 25 or 100 ng of C24 LNPs per 0.14 M cells (equivalent to 0.18 or 0.72 g per 1 M cells).

[0320] To determine transfection efficiency, transfected cells were assessed for GFP expression 48 hours post-transfection (FIGS.2A-2B). As shown in FIG.2A, ~60% of cells transfected with the C24 LNPs were GFP positive at both the 25 ng and 100 ng doses. As shown in FIG.2B, the 100ng dose resulted in a higher mean fluorescence intensity (MFI) than the 25 ng dose.

[0321] At 72 hours post-transfection, PCSK9 gene expression was assessed by RT-qPCR as fold change in comparison to the NT control cells. As shown in FIG.2C, cells transfected with the C24 LNPs delivering the DNA-targeting system for PCKS9 repression exhibited decreased levels of PCSK9 expression. Notably, cells transfected at 100 ng exhibited 90% PCSK9 repression compared to NT control cells. sf-6779026.8 100224742003040

[0322] The results showed that LNPs comprising the C24 ionizable lipid can effectively deliver a CRISPR / Cas-based DNA-targeting system to human hepatocytes. The results support the utility of C24 based LNP formulations for delivery of epi-editor DNA-targeting systems, including targeted gene repression in human hepatocytes. Example 3 : In vivo PSCKS9 repression in a humanized mouse model using C24 lipid nanoparticles for DNA-targeting system delivery

[0323] C24 LNP delivery of a CRISPR / Cas-based DNA-targeting system for gene-editing of PCSK9 was assessed for efficacy in vivo using a liver-humanized mouse model. Liver- humanized mice are Fah / Rag2 / Il2rg triple mutants (FRG KO) in which the liver is repopulated with human hepatocytes, for example as described in Azuma et al., Nat Biotechnol.25(8):903- 910 (2007).

[0324] C24 LNPs were prepared generally as described in Example 1. The C24 LNPs encapsulated mRNA encoding a Cas protein and a gRNA targeting mouse PCSK9. LNPs were delivered systemically via injection (e.g. into the tail vein).

[0325] To assess gene-editing efficiency, the PCSK9 gene was sequenced for indel formation 48 hours post-injection. As shown in FIG.3, 70% indel formation was observed using the C24 LNPs for DNA-targeting system delivery as compared to the mock (1xPBS) controls.

[0326] The results showed that LNPs comprising the C24 ionizable lipid can effectively deliver a CRISPR / Cas-based DNA-targeting system in vivo. The results support the utility of C24 based LNP formulations for targeted in vivo gene editing. Example 4 : CD45 repression in primary T cells using C24 lipid nanoparticles for DNA- targeting system delivery

[0327] Primary human T cells were transfected with C24 LNPs encapsulating a CRISPR / Cas-based DNA-targeting system for transcriptional repression of CD45 and assessed for targeted gene repression.

[0328] C24 LNPs were prepared generally as described in Example 1. Primary human T cells were cultured in T-cell medium (Optimizer media; ThermoFisher Scientific, Catalog No. A1048501) at 2x106cells / mL in G-Rex plates. For T-cell activation, the TransAct™ stimulation sf-6779026.8 101224742003040 to transection, cells were changed with fresh medium. LNPs were pre-associated with human ApoE (0.1 mg / mL) for 5 minutes at 37 °C. Then, LNPs pre-complexed with ApoE were added to cells with the final ApoE concentration of 2 ug / mL. T-cells were transfected 72 hours post- activation with C24 LNPs encapsulating mRNA encoding a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), a CD45 targeting gRNA, and an eGFP mRNA (SEQ ID NO:6) for assessing transection efficiency. The mRNA:gRNA ratio was 2:1 with 80% of the mRNA comprising the Cas fusion protein mRNA and the remaining 20% comprising the GFP mRNA. Cells were transfected at the following doses: 250 ng, 2 g, and 4 g per 1 M cells.

[0329] Transfected cells were assessed for GFP expression and viability by flow cytometry 48 hours post-transfection as compared to NT control cells. As shown in FIG 4A, the percentage of transfected cells expressing GFP increased with dose up to about 60% transfected cells at the highest (4 g) dose. Similarly, the MFI for GFP increased with dose (FIG.4B). Viability was assessed by diluting the cells with AO / PI dye at a volume ratio of 1:1 and quantifying live cell counts using a Nexcelom cell counter. As shown in FIG.4C, % cell viability remained relatively high, with the 4 g dose exhibiting ~80% viability.

[0330] At 72 hours post-transfection, CD45 marker expression was assessed by flow cytometry. As shown in FIG.4D, cells transfected with the C24 LNPs delivering the DNA- targeting system for CD45 repression exhibited a 15% decrease in CD45+ cells with the 4 g dose compared to the NT control cells. When CD45 expression was assessed as MFI, the 4 g dose resulted in a 24% decrease compared to the NT control cells (FIG.4E).

[0331] The results showed that LNPs comprising the C24 ionizable lipid using the formulation set forth in Table E1 can deliver a DNA-targeting system for CD45 repression to primary T cells. Example 5 : Generation of improved C24 lipid nanoparticles for nucleic acid delivery to primary T cells

[0332] Although the C24-based LNP as described in Example 4 was able to deliver nucleic acid into primary T cells, studies were carried out to improve the efficiency of delivery. The effects of C24 LNP formulation components and ratios, mRNA cargo, and transfection parameters on nucleic acid delivery in primary T-cells were assessed. In these experiments, various components of the initial C24 LNP formulation described in Example 1 (termed “C24 LNP 1.0”) were altered in their concentration, type of components and their ratios, and the sf-6779026.8 102224742003040 formulations were assessed for delivery of an encapsulated nucleic acid to primary T cells, generally as described in Example 4.

[0333] In these experiments, the exemplary encapsulated nucleic acid was a large nucleic acid mRNA cargo encoding for a DNA-targeted system containing a CRISPR-Cas repressor fusion protein and a gRNA for targeted gene repression of CD45. Specifically, the mRNA encoded the dSpCas9-KRAB fusion protein and was encapsulated into the LNP with a gRNA targeting CD45. Efficiency of delivery was monitored by assessing potency of repression of the target gene. a. Increased Dose

[0334] To assess if increased LNP dose would increase repression potency of the DNA- targeting system, primary T-cells were transfected with C24 LNPs (C24 LNP 1.0, as described in Example 1), generally as described in Example 4, at the following doses: 0.5 g, 1 g, 2 g, 5 g, 10 g, and 15 g per 1 million (1 M) cells. Percentage of CD45+ cells (FIG.5A), CD45 MFI (FIG.5B) and cell viability (FIG.5C) was assessed by flow cytometry 72 hours post- transfection. Increased LNP dose did not increase CD45 repression and resulted in decreased % cell viability. b. RNA Structure

[0335] Next, the effect of the dCas fusion mRNA structure on repression potency was assessed. Primary T-cells were transfected at either 0.5 or 2 g per 1 M cells with C24 LNPs using the dSpCas9-KRAB fusion mRNA construct used in Example 4 (SEQ ID NO: 3, “dCas”) or a version that included the coding sequence for eGFP and a P2A linker sequence (SEQ ID NO: 2,“dCas-P2A-eGFP”).

[0336] At 72 hours post-transfection, percentage of CD45+ cells (FIG.6A) and CD45 MFI (FIG.6B) was assessed by flow cytometry and the fold change in CD45 gene expression was assessed via RT-qPCR (FIG.6C). As shown in FIG.6C, the mRNA encoding the dCas mRNA resulted in higher repression of CD45 mRNA expression at both doses compared to the dCas- P2A-eGFP mRNA. c. mRNA:gRNA Ratios

[0337] The effect of varying the mRNA:gRNA ratio on repression potency was assessed. Primary T-cells were transfected at either 0.5 or 2 g per 1 M cells with C24 LNPs (C24 LNP sf-6779026.8 103224742003040 1.0, as described in Example 1), in which the features of the nucleic acid cargo were varied. In this experiment, the encapsulated nucleic acid cargo was an mRNA described in Example 4 that encoded a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. Of the total encapsulated mRNA, 80% was the mRNA encoding the fusion protein and 20% was the mRNA encoding GFP. Different total mRNA:gRNA ratios of the exemplary nucleic acid cargo were varied. To assess how the mRNA:gRNA ratio impacted transfection efficiency, GFP expression was assessed 48 hours post-transfection. As shown in FIGS.7A and 7B, GFP expression saturated at a mRNA:gRNA ratio of 1:1.

[0338] At 72 hours post-transfection, percentage of CD45 + cells (FIG.7C) and CD45 MFI (FIG.7D) was assessed by flow cytometry and the fold change in CD45 gene expression was assessed via RT-qPCR (FIG.7E) A mRNA:gRNA ratio of 1:1 resulted in the strongest repression of CD45, with a 65% decrease in the percentage of CD45+ cells (FIG.7C) and a 50% decrease in CD45 MFI (FIG.7D) at the 2 g dose compared to the NT control cells. At the mRNA level, all the ratios tested resulted in strong repression of CD45 expression at the 2 g dose with a mRNA:gRNA ratio of 1:1 resulting in >90% repression compared to the NT control cells (FIG.7E). d. Neutral Lipid Type

[0339] To assess the effect of the neutral lipid in the LNP formulation on nucleic acid delivery efficiency, C24 LNPs were formulated (C24 LNP 1.0, as described in Example 1), except where the type of neutral lipid was varied. The following eight neutral lipids were tested: DSPC, DLPC, POPE, POPC, SOPC, DOPC, DOPE and 9A1P9. In this experiment, the encapsulated nucleic acid cargo was an mRNA described in Example 4 that encoded a dSpCas9- KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. Primary T-cells were transfected with the different neutral lipid LNP formulations at either 0.5 or 2 g per 1 M cells. CD45 repression was assessed by flow cytometry (FIGS.8A and 8B) and RT-qPCR (FIG.8C) 72 hours post-transfection. Cell viability was also assessed 72 hours post-transfection (FIG.8D).

[0340] As shown in FIG.8A, DSPC LNPs resulted in a 30% decrease in the percentage of CD45+ cells and SOPC LNPs resulted in a 50% decrease at the 2 g dose compared to the NT control cells. A similar trend was observed for CD45 MFI (FIG.8B). As shown in FIG.8C, sf-6779026.8 104224742003040 DSPC LNPs resulted in a 75% decrease in CD45 mRNA expression at the 2 g dose compared to the NT control cells. There was no change in viability due to varying the identity of the neutral lipid (FIG.8D). e. Pegylated Lipid Concentration

[0341] Next, the effect of the pegylated lipid (PEG-lipid) concentration in the LNP formulation on transfection efficiency and repression potency was assessed. C24 LNPs were formulated (C24 LNP 1.0, as described in Example 1), except with the following PEG-lipid concentrations: 1%, 1.5%, 1.85%, 2%, 3.5%, and 5% (mol %). In this experiment, the encapsulated nucleic acid cargo was an mRNA described in Example 4 that encoded a dSpCas9- KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. Primary T-cells were transfected with C24 LNP formulations (with different PEG-lipid concentrations) at the following doses: 0.5, 2, or 4 g per 1 M cells. To assess how the concentration of the PEG-lipid impacted transfection efficiency, GFP expression was assessed 48 hours post-transfection.

[0342] As shown in FIGS.9A and 9B, GFP expression dropped substantially for PEG-lipid concentrations of 2% or higher. A PEG-lipid concentration of 1.5% at the 2 g dose resulted in the highest GFP MFI (FIG.9B).

[0343] Repression of CD45 was assessed by flow cytometry (FIGS.9C and 9D) 72 hours post-transfection. Consistent with the GFP expression data, 1.5% PEG-lipid resulted in the strongest repression of CD45 at each dose tested whereas PEG-lipid concentrations of 2% or higher had little effect on CD45 expression. f. N:P Lipid:mRNA Molar Ratios

[0344] To assess if increasing the N:P lipid:mRNA molar ratio (mole ratio of the ionizable lipid nitrogen to the oligonucleotide (mRNA) phosphate, or N:P) could increase transfection efficiency and gene repression potency, C24 LNPs were formulated (C24 LNP 1.0, as described in Example 1), except with the following N:P molar ratios: 4:1, 8:1, 12:1, 16:1 and 20:1. In this experiment, the encapsulated nucleic acid cargo was an mRNA described in Example 4 that encoded a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. Primary T-cells were transfected with C24 LNPs with varied N:P molar ratios at either 0.5 or 2 g per 1 M cells. GFP expression was assessed 48 sf-6779026.8 105224742003040 hours post transfection. As shown in FIGS.10A and 10B, a N:P molar ratio of 12:1 resulted in the strongest GFP expression at both doses.

[0345] At 72 hours post-transfection, CD45 repression and cell viability was assessed. At the protein level, CD45 repression increased with increased N:P molar ratio, with a 20:1 N:P ratio resulting in a 45% decrease in the percentage of CD45+ cells (FIG.10C) and a 60% decrease in CD45 MFI (FIG.10D) at the 2 g dose compared to the NT control cells. A similar trend was observed at the mRNA level for the 2 g dose, with a N:P of 20:1 resulting in 70% CD45 repression compared to the NT control cells (FIG.10E). As shown in FIG.10F, the percentage of live cells decreased as the N:P ratio increased, with the 20:1 N:P ratio resulting in less than 80% of live cells at both doses. g. N:P Ratio and PEG-Lipid Concentration

[0346] Next, C24 LNPs were formulated where both the N:P ratio and PEG-lipid concentrations were varied. Primary T-cells were transfected with C24 LNPs formulated (C24 LNP 1.0, as described in Example 1), except with different combinations of N:P ratios and PEG- lipid concentrations. Table E2 sets forth the combinations of N:P and PEG-lipid ratios tested. In this experiment, the encapsulated nucleic acid cargo was an mRNA described in Example 4 that encoded a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. Primary T-cells were transfected with C24 LNPs at doses of 0.5, 2 and 4 g and assessed for gene repression potency and cell toxicity. Table E2. C24 LNP Formulations with varied N:P ratios and PEG-lipid concentrations

[0347] GFP expression was assessed by flow cytometry 48 hours post-transfection (FIGS. 11A and 11B). As shown in FIG.11B, 1.5% PEG-lipid in combination with an 8:1 N:P ratio resulted in the highest GFP MFI followed by the 1.5% PEG-lipid and 16:1 N:P combination.

[0348] At 72 hours post-transfection, CD45 repression and cell viability was assessed. As shown in FIG.11C, 1.5% PEG-lipid in combination with either an 8:1 or 16:1 N:P ratio sf-6779026.8 106224742003040 decreased the percentage of CD45+ cells at all three doses, with the 4 g dose resulting in the strongest repression of CD45 protein expression. A similar trend was observed for CD45 MFI (FIG.11D). CD45 expression was also assessed at the mRNA level using RT-qPCR. As shown in FIG.11E, the 4 g dose with 1.5% PEG-lipid in combination with an 8:1 N:P ratio resulted in 82% CD45 repression while the 1.5% PEG-lipid and 16:1 N:P combination resulted in 88% CD45 repression, as compared to the NT control cells. As shown in FIG.11F, the 4 g dose with 1.5% PEG-lipid in combination with an 8:1 N:P ratio resulted in 80% viability while the 16:1 N:P ratio reduced viability by 50%, as compared to NT control cells. Thus, while the two N:P ratios had similar effects on repression at a PEG-lipid concentration of 1.5%, the higher N:P ratio of 16:1 negatively impacted cell viability. h. Cell culture conditions

[0349] Changes to the cell culture method were assessed for the capacity to enhance transfection efficiency and gene repression potency. Primary T cells were transfected with C24 LNPs (C24 LNP 1.0, as described in Example 1), at 0.5, 2, or 4 g per 1 M cells and cultured in Optimizer media (“Old”) or were starved for 30 minutes before transfection and cultured in X- VIVO 15 media (Lonza, Catalog No.02-060Q;“New”), which is a serum-free hematopoietic cell medium, with L-Glutamine, gentamicin and phenol red, xenofree. In this experiment, the encapsulated nucleic acid cargo was an mRNA described in Example 4 that encoded a dSpCas9- KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. GFP expression was assessed by flow cytometry 48 hours post transfection. CD45 repression was assessed by flow cytometry 72 hours post transfection.

[0350] As shown in FIGS.12A and12B, the new culture conditions resulted in increased GFP expression for all three doses compared to the old conditions. As shown in FIGS.12C and 12D, the new culture conditions resulted in decreased CD45 expression at the 2 g and 4 g doses as compared to the old culture conditions. While % viability at 24 hours post-transfection decreased as dose increased, there was no difference in viability between the old and new culture conditions (FIG.12E). i. Conclusion

[0351] Based on the experiments described above, an updated C24 formulation (designated “C24 LNP 2.0” formulation) was developed. In this experiment, the nucleic acid cargo for both formulations included the same mRNA described in Example 4 that encoded a dSpCas9-KRAB sf-6779026.8 107224742003040 fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO:6) and a guide RNA targeting CD45. The N:P ratio and mRNA:gRNA ratio were the same in both formulations. The features of the exemplary C24 LNP 2.0 compared to C24 LNP 1.0 are summarized in Table E3.

[0352] To directly compare the transfection efficiency and gene repression potency of the C24 LNP 2.0 formulation against the C24 LNP formulation 1.0, primary T cells were transfected with LNPs prepared using either the C241.0 or 2.0 LNP formulation at 0.5, 2 or 4 g per 1 M cells and assessed for GFP expression, CD45 expression and viability. Table E3. C24 LNP Formulations

[0353] GFP expression was assessed by flow cytometry 48 hours post transfection (FIGS. 13A and 13B). As shown in FIG.13A, the C24 LNP 2.0 formulation resulted in GFP expression for >80% of transfected cells for all three doses whereas the 1.0 formulation only reached ~60% at the 4 g dose. The C24LNP 2.0 formulation also resulted in substantially higher GFP MFI at all three doses compared to the C24 LNP 1.0 formulation (FIG.13B).

[0354] At 72 hours post-transfection, CD45 repression was assessed by flow cytometry (FIGS.13C and 13D) and RT-qPCR (FIG.13E). While the C24 LNP 1.0 formulation had a minimal effect on CD45 protein expression, the C24 LNP 2.0 formulation resulted in strong CD45 repression at both the 2 and 4 g doses. At the 4 g dose, the C24 LNP Formulation B resulted in a 65% decrease in the percentage of CD45+ cells (FIG.13C) and a 72% decrease in CD45 MFI (FIG.13D) compared to the NT control cells. At the mRNA level, the C24 LNP 2.0 formulation at the 4 g dose resulted in an 82% decrease in CD45 expression compared to the NT controls (FIG.13E). As shown in FIG.13F, the differences in cell viability between the C241.0 and 2.0 LNP formulations were minor with the C24 LNP 2.0 formulation maintaining 80% live cells at the 4 g dose.

[0355] The results showed that the C24 LNP 2.0 formulation can effectively deliver a DNA- targeting system for CD45 repression to primary T cells while maintaining high cell viability. sf-6779026.8 108224742003040 The results support the utility of C24 based LNP formulations for targeted gene repression in T cells. Example 6 : IL2 activation in iPSC derived CD56+ cells using C24 lipid nanoparticles for nucleic acid delivery

[0356] To assess the efficiency of C24 LNP-mediated RNA delivery, iPSC derived CD56+ cells were transfected with C24 LNPs (C24 LNP 1.0, as described in Example 1), in which the LNP was generated to encapsulate an mRNA encoding aCRISPR / Cas-based DNA-targeting fusion protein and an IL-2 targeting gRNA for transcriptional activation of Interleukin-2 (IL2). The mRNA encoding the CRISPR / Cas-based DNA fusion protein was VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4). A separate eGFP mRNA (SEQ ID NO:6) was co-encapsulated for assessing transfection efficiency. Prior to transfection, cells were changed with fresh medium. LNPs were pre-associated with human ApoE (0.1 mg / mL) for 5 minutes at 37 °C. Then, LNPs pre-complexed with ApoE were added to cells with the final ApoE concentration of 2 ug / mL . iPSC derived CD56+ cells were transfected with the C24 LNPs at doses of 0.25 g, 0.5 g, 1 g, 2 g, and 5 g C24 LNPs per 1 M cells. Efficiency of nucleic acid delivery was monitored by assessing targeted gene activation.

[0357] Transfected cells were assessed for GFP expression and viability by flow cytometry 48 hours post-transfection as compared to NT control cells. As shown in FIG.14A, ~100% of transfected cells were GFP+ at the 0.5 g dose and above. As shown in FIG.14B, the MFI for GFP increased with dose and saturated at the 2 g dose. As shown in FIG.14C, % cell viability began to dip at the 0.5 g dose and continued to decrease with increased dose.

[0358] At 72 hours post-transfection, IL2 gene expression was assessed by RT-qPCR as fold change in comparison to the NT control cells. As shown in FIG.14D, cells transfected with the C24 LNPs delivering the DNA-targeting system for IL2 activation exhibited activation of IL2 at all doses tested with a substantial increase in IL2 expression at the 5 g dose.

[0359] The results showed that LNPs comprising the C24 ionizable lipid can effectively deliver a DNA-targeting system for IL2 upregulation to iPSC-derived CD56+ cells. The results support the utility of C24 based LNP formulations for targeted gene activation in immune cells. sf-6779026.8 109224742003040 Example 7 : B2M repression in Hematopoietic Progenitor Cells (HPCs) using C24 lipid nanoparticles for DNA-targeting system delivery

[0360] To assess the efficiency of C24 LNP-mediated RNA delivery, hematopoietic progenitor cells (HPCs) were transfected with C24 LNPs (C24 LNP 2.0, as described in Example 5). In this experiment, the C24 LNPs encapsulated an mRNA encoding a CRISPR / Cas-based DNA-targeting system and a B2M targeting gRNA for transcriptional repression of Beta2-microglobulin (B2M) and assessed for targeted gene repression. The mRNA encoded a dSpCas9-KRAB fusion protein (SEQ ID NO: 3). A separate eGFP mRNA (SEQ ID NO:6) was co-encapsulated for assessing transfection efficiency. Prior to transfection, cells were changed with fresh medium. LNPs were pre-associated with human ApoE (0.1 mg / mL) for 5 minutes at 37 °C. Then, LNPs pre-complexed with ApoE were added to cells with the final ApoE concentration of 2 iPSC derived CD34+ HPCs were transfected with at one of the following doses: 12.5 ng, 25 ng, 50 ng, 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 1 g and 2 g per 1 M cells.

[0361] Transfected cells were assessed for GFP expression by flow cytometry 48 hours post- transfection as compared to NT control cells and cells transfected with mRNA encoding the dCas fusion protein (“No gRNA”) at 500 ng per 1 M cells. As shown in FIG.15A, the percentage of transfected cells expressing GFP increased with dose and saturated at 200 ng. In contrast, the MFI for GFP was negligible until the 100 ng dose (FIG.15B). Cell viability was also assessed 48 hours post-transfection. As shown in FIG.15C, % cell viability began to dip at the 100 ng dose and continued to decrease with increased dose. Live cell counts exhibited a similar trend (FIG.15D).

[0362] At 72 hours post-transfection, B2M gene expression was assessed by RT-qPCR as fold change in comparison to the “No gRNA” control. As shown in FIG.15E, cells transfected with the C24 LNPs delivering the DNA-targeting system for B2M repression exhibited decreased levels of B2M expression at all doses tested. Notably, cells transfected at 300 ng / 1 M cells exhibited 90% B2M repression compared to control cells transfected without a B2M gRNA. The cells transfected with the 300 ng dose also exhibited 97% cell transfection (FIG. 15A) and only a 19% reduction in cell viability (FIG.15C).

[0363] The results showed that LNPs comprising the C24 ionizable lipid can effectively deliver a DNA-targeting system for B2M repression to CD34+ HPCs. The results support the utility of C24 based LNP formulations for targeted gene repression in hematopoietic stem cells. sf-6779026.8 110224742003040 Example 8 : IL2 activation in Hematopoietic Progenitor Cells (HPCs) cells using C24 lipid nanoparticles for DNA-targeting system delivery

[0364] Hematopoietic Progenitor Cells (HPCs) were transfected with C24 LNPs (C24 LNP 2.0, as described in Example 5), in which the LNP was generated to encapsulate an mRNA encoding a CRISPR / Cas-based DNA-targeting system and an IL-2 targeting gRNA for transcriptional activation of Interleukin-2 (IL2). The mRNA encoding a CRISPR / Cas-based DNA-targeting system was VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4). A separate eGFP mRNA (SEQ ID NO:6) was co-encapsulated for assessing transfection efficiency. iPSC derived CD34+ HPCs were transfected with C24 LNPs at four different doses: 250 ng, 500 ng, 1 g, and 2 g per 1 M cells. Efficiency of nucleic acid delivery was monitored by assessing targeted gene activation.

[0365] Transfected cells were assessed for GFP expression by flow cytometry 48 hours post- transfection as compared to NT control cells and cells transfected with the mRNA encoding the dCas fusion protein (“No gRNA”) at 500 ng per 1 M cells. As shown in FIG 16A, close to 100% of cells were transfected at all four doses. As shown in FIG.16B, the MFI for GFP increased with dose. Cell viability was also assessed 48 hours post-transfection. As shown in FIG.16C, % cell viability began to dip at the 500 ng dose and continued to decrease with increased dose. Live cell counts exhibited a similar trend (FIG.16D).

[0366] At 72 hours post-transfection, IL2 gene expression was assessed by RT-qPCR as fold change in comparison to the “No gRNA” control. As shown in FIG.16E, cells transfected with the C24 LNPs delivering the DNA-targeting system for IL2 activation exhibited activation of IL2 at all doses tested, with ~5-fold upregulation of IL2 observed at the 500 ng dose.

[0367] The results showed that LNPs comprising the C24 ionizable lipid can effectively deliver a DNA-targeting system for IL2 upregulation to CD34+ HPCs. The results support the utility of C24 based LNP formulations for targeted gene activation in stem cells. Example 9 : Comparison of C24 LNP formulation and electroporation for DNA- targeting system delivery to iPSCs

[0368] The efficiency of RNA delivery by a C24 LNP-mediated formulation (C24 LNP 2.0, as described in Example 5) vs electroporation in iPSCs was compared. Specifically, iPSCs were transfected with an mRNA encoding a CRISPR / Cas-based DNA-targeting system and a gRNA targeting B2M for transcriptional repression of Beta2-microglobulin (B2M) using these systems. sf-6779026.8 111224742003040 The mRNA encoded a dSpCas9-KRAB fusion protein (SEQ ID NO: 3). A separate eGFP mRNA (SEQ ID NO:6) was co-delivered for assessing transfection efficiency. LNP-mediated delivery was assessed at the following doses of total RNA: 30 ng, 100 ng, 300 ng, and 1 g. Electroporation-mediated delivery was assessed at the following doses of total RNA: 150 ng, 15 g, and 4.6 g. At 48 hours post-transfection or post-electroporation, B2M MFI and GFP expression were assessed via flow cytometry.

[0369] As shown in FIG.17A, LNP-mediated delivery resulted in more potent repression of B2M at lower doses compared to electroporation. iPSCs transfected with the 100 ng dose of LNPs exhibited ~50% reduction in B2M expression compared to the NT control cells. In contrast, cells electroporated at 150 ng showed almost no reduction of B2M expression while the cells electroporated at 1.5 g exhibited similar levels of repression as the LNP transfected cells at 100 ng. LNP-transfected cells also exhibited higher transfection efficiency than the electroporated cells as assessed by GFP expression. LNP transfected cells exhibited 99.7% GFP+ cells (FIG.17B) while the electroporated cells exhibited 78.5% GFP+ cells (FIG.17C).

[0370] A similar experiment was carried out to assess delivery of an alternative nucleic acid. Specifically, iPSCs were transfected with an mRNA encoding a CRISPR / Cas-based DNA- targeting system and a gRNA targeting Epidermal Growth Factor Receptor (EGFR) for transcriptional activation of EGFR using either C24 LNP or EP. The mRNA encoding a CRISPR / Cas-based DNA-targeting system was VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4). A separate eGFP mRNA (SEQ ID NO:6) was co-delivered for assessing transfection efficiency. LNP-mediated delivery was assessed at the following doses: 30 ng, 100 ng, 300 ng, and 1 g. Electroporation-mediated delivery was assessed at the following doses: 150 ng, 15 g, and 4.6 g. At 48 hours post-transfection or post-electroporation, EGFR MFI and GFP expression were assessed via flow cytometry.

[0371] As shown in FIG.17D, iPSCs transfected with the 100 ng dose of LNPs exhibited substantial activation EGFR expression compared to the NT control cells. In contrast, cells electroporated at 150 ng showed a small increase in EGFR expression while the cells electroporated at 4.6 g exhibited similar levels of repression as the LNP transfected cells at 100 ng. LNP-transfected cells also exhibited higher transfection efficiency than the electroporated cells as assessed by GFP expression. LNP transfected cells exhibited 99.7% GFP+ cells (FIG. 17E) while the electroporated cells exhibited 65.6% GFP+ cells (FIG.17F). sf-6779026.8 112224742003040

[0372] The frequency of healthy cells for the LNP-transfected and electroporated cells at each dose was assessed at day 2 and day 5 days post-transfection or electroporation. As shown in FIG.17G, the C24 LNPs resulted in a higher frequency of healthy cells compared to the electroporated cell at day 5.

[0373] The results showed that the C24 LNP formulation can effectively deliver a DNA- targeting system to iPSCs for targeted gene repression and activation with increased transfection efficiency and cell viability compared to electroporation. The results support the utility of C24 based LNP formulations for targeted gene repression and activation in iPSCs and the potential for achieving a higher yield of engineered cells. Example 10 : Delivery of a mRNA and gRNA multiplexed system via LNP-mediated RNA delivery

[0374] To assess the efficiency of delivery of a multiplexed gRNA system using C24-based LNPs, mRNA encoding a CRISPR / Cas-based DNA-targeting systems and gRNAs targeting two different genes was encapsulated in the LNPs. Specifically, C24 LNPs (C24 LNP 2.0 as described in Example 5) were generated encapsulating mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4) and gRNAs targeting IL2 and TCF7 alone or in combination. The LNPs were generated to transfect iHPCs with the IL2 gRNA (gIL2), the TCF7 gRNA (gTCF7), or both gIL2 and gTCF7 at the following ratios: 0.75 gIL2:0.25 TCF7, 0.5 gIL2:0.5 gTCF7, and 0.25 gIL2: 0.75 TCF7.

[0375] Prior to transfection, cells were changed with fresh medium. LNPs were pre- associated with human ApoE (0.1 mg / mL) for 5 minutes at 37 °C. Then, LNPs pre-complexed with ApoE were added to cells with the final ApoE concentration of 2 ug / mL. iHPCs were transfected with the LNPs at either 50 ng or 300 ng. At 72 hours post-transfection, IL2 gene expression was assessed by RT-qPCR as fold change in comparison to the “No gRNA” control. As shown in FIG.18A, IL2 expression increased proportional to increasing gIL2 ratio. At the 300 ng dose, the lowest gIL2 ratio (0.25 gIL2: 0.75 gTCF7) resulted in ~1000 fold increase in IL2 expression compared to the no gRNA control.

[0376] In another experiment, iPSCs were transfected with C24 LNPs (C24 LNP 2.0 as described in Example 5) encapsulating mRNA encoding a VP64-dSpCas9-VP64 fusion protein (SEQ ID NO: 4) and gRNAs targeting GATA2, GATA3, and LMO2. LNPs were pre-associated with human ApoE (0.1 mg / mL) for 5 minutes at 37 °C. Then, LNPs pre-complexed with ApoE sf-6779026.8 113224742003040 were diluted in 200uL culture media with the final ApoE concentration of 2 ug / mL for 5 minutes at 37 °C prior to adding to cells. Cells were dosed at 100, 300, or 500 ng. At 72 hours post- transfection, gene expression of the three target genes was assessed by RT-qPCR as fold change in comparison to the “No gRNA” control. As shown in FIG.18B, there was a dose dependent increase in both GATA2 and GATA3 expression starting at the 300 ng dose.

[0377] Results showed that the C24-based LNP formulation can effectively deliver a DNA- targeting system to iHPCs and iPSCs for multiplexed engineering as exemplified by delivery of systems for multiplexed targeted transcriptional activation of multiple genes. The results support the utility of C24 based LNP formulations for multi-gene engineering in stem cells. Example 11 : Assessment of molar ratios of C24, cholesterol and neutral lipids in C24- based lipid nanoparticle formulation on nucleic acid delivery

[0378] The effects of C24 LNP formulation component ratios on transfection efficiency, cell viability and targeted gene repression potency in primary T-cells were assessed. In these studies, the nucleic acid cargo included an mRNA described in Example 4 that encoded a dSpCas9- KRAB fusion protein (SEQ ID NO:3), an mRNA that encoded eGFP (SEQ ID NO: 6) and a gRNA targeting CD45.

[0379] As an initial screen, C24 LNPs were formulated where the molar ratios of the C24 ionizable lipid, cholesterol and DSPC neutral lipid were varied while keeping the ratio of the DMG-PEG2k lipid constant. Table E4 sets forth the compositions of the various C24 LNP formulations tested. Table E4. C24 LNP formulations with varied ionizable lipid, cholesterol, and neutral lipid ratiossf-6779026.8 114224742003040

[0380] Primary T-cells were transfected with the varied C24 LNP formulations at either 1 g or 4 g. Transfected cells were assessed for GFP expression and viability by flow cytometry 48 hours post-transfection. Cells transfected with the C24 LNP 3.0 formulation (C24 / Chol / DSPC / DMG-PEG2000 = 37.5 / 38 / 22.5 / 1.5) at the 4 g dose exhibited higher levels of GFP+ cells (FIG.19A) and GFP MFI (FIG.19B) than cells transfected with the C24 LNP 2.0 (C24 / Chol / DSPC / DMG-PEG2000 = 50 / 38 / 10 / 1.5). As shown in FIGS.19C and 19D, the C24 LNP 3.0 formulation resulted in similar levels of viable cells as the C24 LNP 2.0 formulation.

[0381] At 72 hours post-transfection, CD45 marker expression was assessed by flow cytometry (FIGS.19E and 19F). While there was no change in the percentage of CD45+ cells regardless of the formulation used (FIG.19E), the C24 LNP 3.0 formulation reduced CD45 MFI compared to the C24 LNP 2.0 formulation (FIG.19F).

[0382] FIGS.20A-20F compare GFP expression, viability and CD45 expression of the formulations where only the ratios of the C24 ionizable lipid and DSPC neutral lipid were allowed to vary while the cholesterol and the PEG-lipid were held constant (C24 formulations 2.0, 2.3, 2.6, and 3.0). The C24 LNP 3.0 formulation comprising 37.5% C24 ionizable lipid resulted in increased GFP expression (FIGS.20A-B), improved viability (FIGS.20C-D) and increased repression of CD45 MFI (FIG.20F) compared to the C24 LNP 2.0 formulation comprising 50% C24.

[0383] To test if the C24 LNP 3.0 formulation could be further improved by increasing the amount of the DSPC neutral lipid, additional formulations were generated where the C24 and PEG-lipid ratios were held constant and the levels of the DSPC lipid were gradually increased with a corresponding decrease in cholesterol level. Table E5 sets forth the compositions of these additional formulations. Similar to above, the nucleic acid cargo included an mRNA described in Example 4 that encoded a dSpCas9-KRAB fusion protein (SEQ ID NO: 3), an mRNA encoding eGFP (SEQ ID NO: 6) and a gRNA targeting CD45. Table E5. C24 LNP formulations with increasing DSPC ratiossf-6779026.8 115224742003040

[0384] Primary T-cells were transfected with the above C24 LNP formulations at the following doses: 1 g, 2.5 g or 4 g. Transfected cells were assessed for GFP expression and viability by flow cytometry 48 hours post-transfection. The C24 LNP 3.0 formulation resulted in a higher percentage of GFP+ cells (FIG.21A) and GFP MFI (FIG.21B) at every dose compared to the C24 LNP 2.0 formulation. The other formulations tested resulted in reduced or similar GFP expression as the C24 LNP 2.0 formulation. As shown in FIG.21C, improved viability was observed for all C24 LNP formulations a lower molar ratio amount of C24 (e.g., 37.5) as compared to C24 formulation 2.0.

[0385] At 72 hours post-transfection, CD45 marker expression was assessed by flow cytometry. As shown in FIG.21D, the C24 LNP 3.0 formulation resulted in a dose dependent decrease in CD45+ cells while the C24 LNP 2.0 and 3.0 variants exhibited no decrease at any of the doses tested compared to the NT control cells. Similarly, the 3.0 formulation exhibited an improved dose dependent decrease in CD45 MFI compared to the 2.0 formulation (FIG.21E).

[0386] The results showed that the C243.0 LNP formulation can effectively deliver a DNA- targeting system for CD45 repression to primary T cells while maintaining high cell viability. The results support the utility of C24 based LNP formulations for delivery of epi-editor DNA- targeting systems, including targeted gene repression, in T cells. Example 12 : Comparison of C242.0 and 3.0 LNP formulations and for targeted repression of the exemplary target MED12 in primary T cells

[0387] To assess the ability to transfect primary T cells and delivery a DNA-targeting system, primary T cells were assessed for delivery of a CRISPR / Cas-based DNA-targeting system with a gRNA directed against an exemplary target gene using the LNPs generated in prior Examples. Specifically, primary T cells were transfected with C242.0 or C243.0 LNPs encapsulating an mRNA encoding a CRISPR / Cas-based DNA-targeting system and a gRNA targeting MED12 for transcriptional repression of Mediator Complex Subunit 12 (MED12) and assessed for targeted gene repression. In this experiment, the mRNA encoded DNMT3AL- dSpCas9-KRAB fusion protein (SEQ ID NO: 5). A separate eGFP mRNA (SEQ ID NO:6) was co-encapsulated for assessing transfection efficiency. sf-6779026.8 116224742003040

[0388] Primary CD4+ T cells were transfected with C24 LNP 2.0 or C24 LNP 3.0 encapsulating the mRNA and a MED 12 targeting gRNA at either 2 g or 4 g per 1 M cells.

[0389] Transfected cells were assessed for GFP expression and viability by flow cytometry 48 hours post-transfection as compared to NT control cells. As shown in FIG 22A, the C24 LNP 3.0 formulation resulted in a higher percentage GFP+ cells at the 2 g dose compared to the C24 LNP 2.0 formulation. As shown in FIG.22B, the C24 LNP 3.0 formulation resulted in increased GFP MFI at both doses compared to the C24 LNP 2.0 formulation. As shown in FIG.22C, the C24 LNP 3.0 formulation resulted in slightly better % cell viability at the 4 g dose.

[0390] At 72 hours post-transfection, MED12 gene expression was assessed by RT-qPCR as fold change in comparison to NT control cells. As shown in FIG.22D, the C24 LNP 3.0 formulation resulted in increased MED12 repression at both doses compared to the C24 LNP 2.0 formulation.

[0391] The results showed that the C24 LNP 3.0 formulation can effectively deliver a DNA- targeting system for MED12 repression to primary T cells. The results support the utility of C24 based LNP formulations for delivery of epi-editor DNA-targeting systems, including targeted gene repression, in T cells. Example 13: Multiplex and singleplex gene activation in iHPCs using C24 lipid nanoparticles for DNA-targeting system delivery

[0392] Induced hematopoietic progenitor cells (iHPCs) were transfected with C24 LNPs (C24 LNP 2.0, as described in Example 5)- encapsulating an exemplary multiplex or singleplex CRISPR / Cas-based DNA-targeting system and assessed for activation of surface protein expression via flow cytometry.

[0393] To assess delivery of a multiplex DNA-targeting system, separate C24 were generated to individually encapsulate an mRNA encoding a VP64-dSpCas9-VP64 fusion protein and gRNAs targeting VCAM, ILR7a, and RUNX3.

[0394] Prior to transfection, the LNPs were pre-associated with human ApoE (0.1 mg / mL) for 5 minutes at 37º C. The cell culture media was changed with fresh medium and the LNPs pre-complexed with ApoE were added to the cells at a final ApoE concentration of 2 ug / mL. HPCs with lymphoid potential were induced from iPSCs (iHPCs). The iHPCs were co- transfected with the separate C242.0LNPs encapsulating the fusion protein and each guide. Cells were transfected with each LNP at doses of 150 ng, 75 ng, 37.5 ng, and 18.75 ng C24 sf-6779026.8 117224742003040 LNPs per 1.25x105cells. Efficiency of nucleic acid delivery was monitored by assessing protein expression by flow cytometry at 72 hours post-transfection (FIG.23A) and 120 hours post- transfection (FIG.23B).

[0395] As shown in FIG.23A, iHPCs transfected with the multiplex DNA targeting system by C242.0 LNP delivery exhibited an increase of both VCAM1 and IL7Ra surface protein expression at 72 hours post-transfection. The 75 ng dose resulted in the highest percentage of VCAM / IL7Ra double positive cells at 83.4% while the 18.75 ng dose resulted in 49.7% double positive cells. As shown in FIG.23B, the percentage of VCAM / IL7Ra double positive cells remained high at 120 hours post-transfection, with the 75 ng resulting in 66.8% double positive cells and the 150 ng dose resulting in 77.7% double positive cells.

[0396] In another experiment, C24 LNP 2.0 delivery of a single plex DNA-targeting system to iHPCs was assessed.LNPs were generated encapsulating an mRNA encoding a VP64- dSpCas9-VP64 fusion protein in combination with a gRNA targeting either VCAM, Notch, or DLL4. The LNPs were pre-complexed with ApoE and added to the cells at a final ApoE concentration of 2 ug / mL. iHPCs were transfected at a dose of 75 ng per 1.25x105cells. Surface protein expression was assessed 120 hours post-transfection by flow cytometry. As shown in FIG.23C, at 120 hours post-transfection each protein was detectable in high abundance relative to the non-transfected (0 ng) control. C24 LNP delivery of the effector with a VCAM guide resulted in 97.9% VCAM+ cells, delivery with the Notch guide resulted in 89.2% Notch positive cells, and delivery with the DLL4 guide resulted in 96.9% DLL4 positive cells,

[0397] Results showed that C24 LNPs can effectively deliver a DNA-targeting system to iHPCs for targeted transcriptional activation of multiple or single genes. Abundant surface protein expression resulting from the targeted single and multi-gene activation was detectable 120 hours post transfection. The results support the utility of C24 based LNP formulations for single and multi-gene engineering for sustained surface protein expression in stem cells at lower doses, which can minimize toxicity. Example 14: Assessment of multiple C242.0 LNP doses for multiplex DNA-targeting system delivery on durability of DLL4 and VCAM1 activation in iHPCs

[0398] The effect of multiple doses of C24 LNPs (C242.0 LNP, as described in Example 5), encapsulating a multiplex DNA-targeting system on targeted activation of multiple genes in iHPCs was assessed. In these studies, the nucleic acid cargo included an mRNA that encoded a sf-6779026.8 118224742003040 VP64-dSpCas9-VP64 fusion protein, a guide RNA targeting DLL4, and a guide RNA targeting VCAM1. Durability of the gene activation following LNP delivery of the multiplex DNA- targeting system was assessed by monitoring DLL4 and VCAM 1 protein expression by flow cytometry over time.

[0399] C242.0 LN...

Claims

224742003040 CLAIMS 1. A lipid nanoparticle (LNP) comprising: i) from 30-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

2. A lipid nanoparticle (LNP)) comprising: i) from 30-40 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 20-30 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

3. A lipid nanoparticle (LNP) comprising: i) from 45-55 mol % of an ionizable lipid, wherein the ionizable lipid comprises a trivalent head group 4-methyl-1-piperazinebutanamine linked to a tail; ii) from 5-15 mol % of a neutral lipid; iii) a polymer conjugated lipid; iv) a steroid; and v) an RNA cargo encapsulated within or associated with the lipid nanoparticle comprising at least one messenger RNA (mRNA) molecule.

4. The LNP of any of claims 1-3, wherein the ionizable lipid has a 4-methyl-1- piperazinebutanamine head group that is linked via a degradable primary ester to an octyldodecyl tail. sf-6779026.8 149224742003040 5. The LNP of any of claims 1-4, wherein the ionizable lipid is linked via two degradable primary esters to two octyldodecyl tails.

6. The LNP of any of claims 1-5, wherein the ionizable lipid is characterized by 4 saturated alkyl tails with non-symmetric 8 and 10 carbon lengths.

7. The LNP of any of claims 1-6, wherein the ionizable lipid is bis(2-octyldodecyl) 3,3'-((4- (4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24).

8. The LNP of any of claims 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 1 to 5 mol %.

9. The LNP of any of claims 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 1 to 2 mol % or 1 to 3 mol%.

10. The LNP of any of claims 1-7, wherein the polymer conjugated lipid is present in a concentration ranging from 2 to 5 mol % 11. The LNP of any of claims 1-10, wherein the steroid is present in a concentration ranging from 35 to 45 mol %.

12. The LNP of any of claims 1-11, wherein the neutral lipid is distearoylphosphatidylcholine (DSPC), dilauroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), 1-stearoyl-2-oleoyl phosphatidylcholine (SOPC), dioleoylphosphatidylcholine (DOPC), dioleoyl-phosphatidylethanolamine (DOPE) or 2-(dioctylamino)ethyl nonyl hydrogen phosphate (9A1P9).

13. The LNP of any of claims 1-12, wherein the neutral lipid is DSPC.

14. The LNP of any of claims 1-13, wherein the polymer conjugated lipid is a polyethylene glycol (PEG)-conjugated lipid. sf-6779026.8 150224742003040 15. The LNP of any of claims 1-14, wherein the polymer conjugated lipid is 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k).

16. The LNP of any of claims 1-15, wherein the steroid is cholesterol.

17. A lipid nanoparticle (LNP) comprising: (i) about 37.5 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 22.5 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule.

18. A lipid nanoparticle (LNP) comprising: (i) about 50 mol % of an ionizable lipid, wherein the ionizable lipid is bis(2- octyldodecyl) 3,3'-((4-(4-methylpiperazin-1-yl)butyl)azanediyl)dipropionate (C24); (ii) about 10 mol % of a neutral lipid; wherein the neutral lipid is DSPC; (iii) about 1.5% mass fraction of a polyethylene glycol (PEG)-conjugated lipid; wherein the PEG-conjugated lipid is DMG-PEG2k; (iv) about 38% mass fraction of cholesterol; and (v) about 0.5% mass fraction of an RNA cargo comprising at least one messenger RNA (mRNA) molecule.

19. The LNP of any of claims 1-18, wherein the at least one mRNA molecule is at least 3000 nucleotides in length. sf-6779026.8 151224742003040 20. The LNP of any of claims 1-19, wherein the at least one mRNA molecule is from about 3,000 to 12,000 nucleotides in length, from about 4,500 to 9,000 nucleotides in length, or from about 4,800 to 7,500 nucleotides in length.

21. The LNP of any of claims 1-20, wherein the at least one mRNA molecule encodes a protein that is from about 1,000 to 4,000 amino acids in length, from about 1,500 to 3,000 amino acids in length, or from about 1,600 to 2,500 amino acids in length.

22. The LNP of any of claims 1-21, wherein the RNA cargo comprises a DNA-targeting system for modulating transcription of a gene.

23. The LNP of any of claims 1-22, wherein the RNA cargo comprises a messenger RNA (mRNA) and a guide RNA (gRNA).

24. The LNP of claim 23, wherein the RNA cargo has a mRNA:gRNA w / w ratio of 10:1 to 1:5, optionally 10:1, 5:1, 2:1, 1:1, 1:2, or 1:

5.

25. The LNP of claim 23, wherein the mRNA cargo has a mRNA:gRNA w / w ratio of 2:1 to 1:

2.

26. The LNP of claim 23, wherein the RNA cargo has a mRNA:gRNA w / w ratio of 1:

1.

27. The LNP of any of claims 1-26, wherein the LNP has an ionizable lipid nitrogen: oligonucleotide phosphate (N:P) molar ratio of 4:1 to 20:

1.

28. The LNP of claim 27, wherein the LNP has an N:P ratio of 8:

1.

29. The LNP of any of claims 1-28, wherein the at least one mRNA encodes a fusion protein comprising a DNA-binding domain and an effector domain.

30. The LNP of claim 29, wherein the DNA-binding domain is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof. sf-6779026.8 152224742003040 31. The LNP of claim 30, wherein the Cas protein or variant thereof is a nuclease- deactivated Cas (dCas) protein.

32. The LNP of claim 31, wherein the dCas protein is a dCas9 protein.

33. The LNP of claim 32, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

34. The LNP of any of claims 1-33, wherein the LNP is for delivery to a primary cell.

35. The LNP of any of claims 1-33, wherein the LNP is for delivery to an iPSC or iPSC derived cell.

36. A method of genetically engineering a population of cells, the method comprising transfecting an RNA cargo into a population of cells by delivering the LNP of any of claims 1-35 into the population of cells.

37. The method of claim 36, wherein the population of cells are primary cells.

38. The method of claim 37, wherein the population of primary cells are T cells.

39. The method of claim 37, wherein the population of primary cells are hepatocytes.

40. The method of claim 37, wherein the population of primary cells are HPCs.

41. The method of claim 38, wherein the T cells are CD4+.

42. The method of claim 38 or claim 41, wherein the T cells are activated prior to transfection.

43. The method of claim 42, wherein the T cells are activated by contacting the T cells with humanized CD3 and CD28 agonists. sf-6779026.8 153224742003040 44. The method of claim 42 or claim 43, wherein the T cells are activated between at or about 24 hours to 168 hours prior to transfection.

45. The method of any of claims 42-44, wherein the T cells are activated about 48 hours to about 72 hours prior to transfection.

46. The method of any of claims 38 or 41-45, wherein the T cells are cultured in serum-free media.

47. The method of claim 36, wherein the population of cells are induced pluripotent stem cells (iPSCs) or iPSC derived cells 48. The method of claim 47, wherein the population of iPSC derived cells are induced hematopoietic progenitor cells (iHPCs).

49. The method of claim 48, wherein the iHPCs are CD34+.

50. The method of claim 48 or claim 49, wherein the population of iPSC derived cells are emerging induced lymphoid progenitor cells.

51. The method of claim 47, wherein the population of iPSC derived cells are induced lymphoid progenitor cells.

52. The method of claim 51, wherein the induced lymphoid progenitors are induced T-cell progenitors.

53. The method of claim 52, wherein the induced T-cell progenitors are double positive for CD4 and CD8 cell surface markers.

54. The method of claim 52 or claim 53, wherein the population of iPSC derived cells are emerging induced T cells. sf-6779026.8 154224742003040 55. The method of claim 47, wherein the population of iPSC derived cells are induced Natural Killer cells.

56. The method of claim 47, wherein the population of iPSC derived cells are induced T cells.

57. The method of claim 47 or claim 55, wherein the population of iPSC derived cells are CD56+.

58. The method of claim 47 or claim 56, wherein the population of iPSC derived cells are CD8+.

59. The method of any of claims 36-58, wherein the LNP is complexed with human ApoE prior to transfection.

60. The method of claim 59, wherein the LNP-ApoE complex is added to the cells at an ApoE concentration of between at or about 0.5 g / mL to 2 g / mL.

61. The method of any of claims 36-60, wherein the LNP is delivered at a dose of 50 ng to 10 g per 1x106cells.

62. The method of any of claims 36-61, wherein the LNP is delivered at a dose of 100 ng to 5 g per 1x106cells.

63. The method of any of claims 36-62, wherein the LNP is delivered at a dose of 0.5 g to 4 g per 1x106cells.

64. The method of any of claims 36-63, wherein a single dose of the LNP is delivered into the cells.

65. The method of any of claims 36-63, wherein more than one dose of the LNP is delivered into the cells. sf-6779026.8 155

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