Lipid nanoparticles for delivery of nucleic acids and related methods and uses

Abstract

The present disclosure relates to compositions comprising lipid nanoparticles for delivering nucleic acid molecules into cells. Also included are methods for producing and using such compositions.

Description

LIPID NANOPARTICLES FOR DELIVERY OF NUCLEIC ACIDS AND RELATEDMETHODS AND USESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from U.S. provisional application No. 63 / 617,414 filed January 3, 2024, entitled “LIPID NANOPARTICLES FOR DELIVERY OF NUCLEIC ACIDS AND RELATED METHODS AND USES”, the contents of which are incorporated by reference in its entirety.FIELD

[0002] The present disclosure relates in some aspects to lipid nanoparticles (LNPs) and compositions thereof for delivery of nucleic acid molecules, e.g., deoxyribonucleic acid (DNA), into lymphocytes, e.g., T cells. Also provided are methods for formulating LNPs, and for delivering nucleic acid molecules into lymphocytes, e.g., T cells, using LNP compositions, including in connection with gene editing and cell therapy.BACKGROUND

[0003] Viral vector-based delivery of nucleic acid, such as nucleic acid encoding a recombinant receptor (e.g., a CAR), into T cells can be effective in the production of T cells expressing the recombinant receptor, and such recombinant receptor-expressing T cells can be used in adoptive T cell therapies. The engineered expression of recombinant receptors, such as chimeric antigen receptors (CARs), on the surface of T cells enables the redirection of T cell specificity. In clinical studies, CAR-T cells, for example anti-CD19 CAR-T cells, have produced durable, complete responses in both leukemia and lymphoma patients (Porter et al. (2015) Sci Transl Med., 7:303ral39; Kochenderfer (2015) J. Clin. Oncol., 33: 540-9; Lee et al. (2015) Lancet, 385:517-28; Maude et al. (2014) N Engl J Med, 371:1507-17). Similarly, viral-vector based delivery of nucleic acid, such as nucleic acid encoding molecular gene editing components, into cells can be effective in the production of genetically edited cells for use in gene therapy applications.

[0004] Various strategies for delivering nucleic acid molecules into cells are available, including transfection- and transduction-based techniques. Among strategies for use in cell therapy are viral- vector based techniques of introducing nucleic acids into cells. Similarly, viral vectors are commonly used to effect gene editing. However, the production of viral vector-based compositions is labor intensive and resource-consuming. Improved non-viral compositions for delivering nucleic acid molecules into cells, and methods of producing and using the same, are therefore needed.

[0005] In certain contexts, viral-based methods of engineering cells (e.g., T cells) may not always be entirely satisfactory. Viral vectors, such as lentiviral vectors, are commonly used to genetically engineer T cells to express recombinant receptors (e.g., CARs), as well as to geneticallyedit cells for use in gene therapy applications. Such viral vectors must be of consistently high quality to ensure predictable genetic engineering of cells. In addition, viral vectors must be produced on a large scale, without compromising their quality, in order to produce therapeutic drug products containing a sufficient number of engineered cells. It is estimated that manufacturing of such viral vectors requires weeks. (Levine et al. (2017) Mol. Ther. Methods Clin. Dev., 4:92-101). Engineering cells with viral vectors is equally time- and labor-consuming. Continuous monitoring is necessary to ensure the safety of the viral vectors and cells engineered therewith. Combined with a limited number of manufacturing facilities, these characteristics of viral vector-based cell engineering make scaled production challenging and expensive. (Eyles et al. (2019) J. Chem. Technol. Biotechnol., 94:1008- 16).

[0006] Another drawback of viral-vector based methods of cell engineering is their limitation in the size of the cargo (e.g., nucleic acid) they can deliver. For example, retroviral vectors, frequently used for gene delivery and capable of integrating into a host genome, contain approximate 8 kilobases (kb) of capacity for insertion of a transgene. Adenoviruses are able to deliver larger DNA particles, such as up to about 36 or 38 kb, but cannot integrate into a host genome. Adeno-associated vectors (AAV) are capable of integrating into a host genome, but have a packaging capacity of only about 4.7 kb. Thus, commonly used viral vectors can suffer from either an inability to integrate into a host genome, an inability to incorporate large transgenes, or both. (Nayerossadat et al. (2012) Adv. Biomed. Res., 1:27). By contrast, LNPs are capable of delivering larger cargo, including by delivery of nucleic acid molecules by transposon and CRISPR-Cas-mediated systems.

[0007] Non-viral methods of gene delivery and engineering have been investigated, including the use of DNA guns, electroporation, and ultrasound. However, these methods tend to suffer from low efficiency. Id. To date, a number of cationic lipid polymers have been developed for gene delivery, but in vivo studies have revealed substantial toxicity and low transfection efficiency. Id.

[0008] In addition, many methods of introducing nucleic acid into a cell, such as for genetic engineering purposes, rely on the introduction of ribonucleic acid (RNA) into cells. For example, it has been shown that lipid nanoparticles can be used to introduce RNA into cells. In some aspects, RNA encoding machinery of the CRISPR-Cas9 system is introduced 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). In other aspects, RNA such as short interfering RNA (siRNA) or short hairpin RNA (shRNA) is introduced into cells, for the purpose of suppressing or disrupting a gene and / or its expression. (Cullis and Hope, Mol Ther (2017) 25(7): 1467-75). By contrast, delivery of DNA into T cells, particular primary T cells, remains challenging. Non-viral methods of gene delivery to primary T cells often suffer from low efficiency, toxicity, or both (Rahimmanesh et al., Res Pharm Sci (2020) 15(5):437-46).SUMMARY

[0009] Provided herein are lipid nanoparticles (LNPs) that deliver deoxyribonucleic acid (DNA) and / or ribonucleic acid (RNA) payloads to cells. Also provide herein are fused nanoparticles that deliver multiple payloads to cells, in particular, nucleic acid molecules.

[0010] In one aspect, provided herein is a lipid nanoparticle (LNP) comprising: (1) an ionizable lipid comprising a squaramide moiety, wherein the ionizable lipid is an ionizable amino lipid; and (2) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the ionizable lipid comprising the squaramide moiety is referred to as Lipid 29 (also referred to as L29) and has the structure depicted below.LIPID 29

[0011] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising: (1) an ionizable lipid comprising Lipid 319 (also referred to as L319 or 319), wherein the ionizable lipid is an ionizable amino lipid; and (2) a deoxyribonucleic acid (DNA) molecule. The structure of Lipid 319 is depicted below.LIPID 319

[0012] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising: (1) an ionizable lipid comprising Lipid A6 (also referred to as A6), wherein the ionizable lipid is an ionizable amino lipid; and (2) a deoxyribonucleic acid (DNA) molecule. The structure of Lipid A6 is depicted below.LIPID A6

[0013] In some embodiments, the ionizable lipid is Lipid 29 and the mass fraction of the ionizable lipid in the LNP is between about 30% and about 65%. In some embodiments, the ionizable lipid is Lipid 29 and the mass fraction of the ionizable lipid in the LNP is between about 40% and about 60%. In some embodiments, the ionizable lipid is Lipid 29 and the mass fraction of the ionizable lipid in the LNP is between about 30% and about 50%. In some embodiments, the ionizable lipid is Lipid 29 and the mass fraction of the ionizable lipid in the LNP is between about 40% and about 45%. In some embodiments, the ionizable lipid is Lipid 29 and the mass fraction of the ionizable lipid in the LNP is between about 35% and about 45%. In some embodiments, the ionizable lipid is Lipid 29 and the mass fraction of the ionizable lipid in the LNP is about 40%, about 45%, about 50%, about 55% or about 60%.

[0014] In some embodiments, the ionizable lipid is Lipid 319 and the mass fraction of the ionizable lipid in the LNP is between about 30% and about 65%. In some embodiments, the ionizable lipid is Lipid 319 and the mass fraction of the ionizable lipid in the LNP is between about 40% and about 60%. In some embodiments, the ionizable lipid is Lipid 319 and the mass fraction of the ionizable lipid in the LNP is between about 30% and about 50%. In some embodiments, the ionizable lipid is Lipid 319 and the mass fraction of the ionizable lipid in the LNP is between about 40% and about 45%. In some embodiments, the ionizable lipid is Lipid 319 and the mass fraction of the ionizable lipid in the LNP is between about 35% and about 45%. In some embodiments, the ionizable lipid is Lipid 319 and the mass fraction of the ionizable lipid in the LNP is about 40%, about 45%, about 50%, about 55% or about 60%.

[0015] In some embodiments, the ionizable lipid is Lipid A6 and the mass fraction of the ionizable lipid in the LNP is between about 30% and about 65%. In some embodiments, the ionizable lipid is Lipid A6 and the mass fraction of the ionizable lipid in the LNP is between about 40% and about 60%. In some embodiments, the ionizable lipid is Lipid A6 and the mass fraction of the ionizable lipid in the LNP is between about 30% and about 50%. In some embodiments, the ionizable lipid is Lipid A6 and the mass fraction of the ionizable lipid in the LNP is between about 40% and about 45%. In some embodiments, the ionizable lipid is Lipid A6 and the mass fraction of the ionizable lipid in the LNP is between about 35% and about 45%. In some embodiments, the ionizable lipid is Lipid and the mass fraction of the ionizable lipid in the LNP is about 40%, about 45%, about 50%, about 55% or about 60%.

[0016] In some embodiments, the LNP comprises a helper lipid. In some embodiments, the mass fraction of the helper lipid in the LNP is between about 18% and about 22%. In some embodiments, the mass fraction of the helper lipid in the LNP is about 19%. In some embodiments, the helper lipid is l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC). In some embodiments, the helper lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0017] In some embodiments, the LNP comprises a polyethylene glycol (PEG)-conjugated lipid. In some embodiments, the mass fraction of the PEG-conjugated lipid in the LNP is between about 2% and about 3%. In some embodiments, the mass fraction of the PEG-conjugated lipid in the LNP is about 2.5%. In some embodiments, the PEG-conjugated lipid is DMG-PEG2000.

[0018] In some embodiments, the LNP comprises cholesterol. In some embodiments, the mass fraction of the cholesterol in the LNP is between about 30% and about 40%. In some embodiments, the mass fraction of the cholesterol in the LNP is about 35%.

[0019] In some embodiments, the LNP comprises more than one cationic lipid. In some embodiments, the LNP comprises two cationic lipids. In some embodiments, the LNP comprises an ionizable cationic lipid and a non-ionizable cationic lipid. The non-ionizable lipid has a higher pKa than the ionizable lipid and would predominantly be charged at physiological pH, such as in the bloodstream. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.1% and about 40%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.2% and about 20%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.2% and about 20%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.5% and about 10%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 1% and about 7%. In some embodiments, the mass fraction of the non- ionizable cationic lipid in the LNP is between about 1% and about 6%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 2% and about 5%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.2% and about 1%.

[0020] In some embodiments, the mass fraction of the DNA molecule in the LNP is between about 2% and about 6%. In some embodiments, the mass fraction of the DNA molecule in the LNP is between about 3% and about 4%. In some embodiments, the mass fraction of the DNA molecule in the LNP is about 3.5%.

[0021] In some embodiments, the DNA molecule comprises a transgene. In some embodiments, the transgene encodes a recombinant protein. In some embodiments, the transgene encodes a recombinant receptor. In some embodiments, the DNA molecule is a closed end DNA (ceDNA) vector. In some embodiments, the transgene is positioned between protelomerase binding sequences.In some embodiments, the transgene is operably linked to a promoter and positioned between inverted terminal repeats (ITRs). In some embodiments, the transgene is between about 2 kilobases and about 10 kilobases. In some embodiments, the transgene is between about 4 kilobases and about 8 kilobases.

[0022] In some embodiments, the ceDNA vector is between about 2 kilobases and about 10 kilobases. In some embodiments, the ceDNA vector is between about 4 kilobases and about 8 kilobases.

[0023] In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the recombinant receptor is a TCR. In some embodiments, the recombinant receptor is a CAR. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular region. In some embodiments, the extracellular antigen-binding domain is an antibody or an antigen-binding fragment thereof that binds to an antigen that is associated with, or expressed on, a cell or tissue of a disease or condition. In some embodiments, the CAR is a single antigen directed CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the DNA (e.g., ceDNA) molecule encoding the bispecific CAR is at least 5.5 kilobases, at least 6 kilobases, at least 7 kilobases, or at least 8 kilobases. In some embodiments, the bispecific CAR is between about 5.5 kilobases and about 8 kilobases. In some embodiments, the bispecific CAR is between about 6 kilobases and about 8 kilobases. In some embodiments, the bispecific CAR is about 8 kilobases.

[0024] In some embodiments, the antigen is selected from the group consisting of avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight- melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (R0R1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms Tumor 1 (WT-1). In some embodiments, the antigen is BCMA. In some embodiments, the antigen is CD19. In some embodiments, the antigen is CD20. In some embodiments, the antigen is CD22. In some embodiments, the antigen is GPRC5D.

[0025] In some embodiments, the intracellular region comprises an intracellular signaling domain that is or comprises an intracellular signaling domain of a CD3 chain, or a signaling portion thereof. In some embodiments, the intracellular region comprises one or more costimulatory signaling domain(s) comprising an intracellular signaling domain selected from the group consisting of: a CD28, a 4-1BB, an ICOS, or a signaling portion thereof. In some embodiments, the intracellular region comprises one or more costimulatory signaling domain(s) comprising an intracellular signaling domain of 4- IBB.

[0026] In some embodiments, the DNA molecule comprises a single-stranded DNA oligonucleotide (ssODN) or a double-stranded DNA oligonucleotide (dsODN), the ssODN or the dsODN comprising a nucleotide sequence that is homologous to a target genomic locus. In some embodiments, the DNA molecule comprises a single-stranded DNA oligonucleotide (ssODN) comprising a nucleotide sequence that is homologous to a target genomic locus. In some embodiments, the DNA molecule comprises a double-stranded DNA oligonucleotide (dsODN) comprising a nucleotide sequence that is homologous to a target genomic locus.

[0027] Also provided herein is a co-formulated lipid nanoparticle (co-LNP) comprising a fusion of a first lipid nanoparticle (LNP) and a second lipid nanoparticle (LNP), wherein, prior to fusion: (1) the first LNP comprises: (i) a deoxyribonucleic acid (DNA) molecule; and (ii) a first ionizable lipid; and (2) the second LNP comprises: (i) at least one ribonucleic acid (RNA) molecule (e.g., one RNA molecule or two RNA molecules); and (ii) a second ionizable lipid. In some embodiments where the second LNP comprises only one RNA molecule, the molecule is an mRNA. In some embodiments where the second LNP comprises only one RNA molecule, the molecule is a guide RNA. In some embodiments where the second LNP comprises two RNA molecule, one RNA molecule is an mRNA and the other RNA is a guide RNA (gRNA).

[0028] In some embodiments, the LNP is a co-LNP comprising a fusion of a first precursor lipid nanoparticle (LNP) and a second precursor lipid nanoparticle (LNP), wherein, prior to fusion: (1) the first precursor LNP comprises: (i) a deoxyribonucleic acid (DNA) molecule; and (ii) a first ionizable lipid; and (2) the second precursor LNP comprises: (i) a first ribonucleic acid (RNA) molecule and optionally a second ribonucleic acid (RNA) molecule; and (ii) a second ionizable lipid, wherein the first and / or second ionizable lipids are selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6.

[0029] In some embodiments, the LNP is a co-LNP comprising: (1) a deoxyribonucleic acid (DNA) molecule, a first ribonucleic acid (RNA) molecule and optionally a second ribonucleic acid (RNA) molecule; and (2) a first ionizable lipid and a second ionizable lipid, wherein the first and / or second ionizable lipids are selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6. In some embodiments, the DNA molecule is associated with the first ionizable lipid; and the first RNA molecule is associated with the second ionizable lipid .

[0030] In some embodiments, the first LNP and second LNP, prior to fusion, are precursor LNPs that are not fully formed. As set forth herein, precursor LNPs are generated in an acidic environment (e.g., at a pH between about 4 and about 5). In some embodiments, the first ionizable lipid of the first LNP forms an ionic bond with the DNA molecule and the second ionizable lipid of the second LNP forms an ionic bond with the RNA molecule(s). In some embodiments, following fusion of the first and second precursor LNPs to form the fused co-LNP, the first ionizable lipid remains substantially associated (complexed) with the DNA molecule and the second ionizable lipid remains substantially associated (complexed) with the RNA molecule(s). For instance, in some embodiments, more than 75% of the first ionizable lipid remains associated with the DNA molecule and more than 75% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, more than 80% of the first ionizable lipid remains associated with the DNA molecule and more than 80% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, more than 85% of the first ionizable lipid remains associated with the DNA molecule and more than 85% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, more than 90% of the first ionizable lipid remains associated with the DNA molecule and more than 90% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, more than 95% of the first ionizable lipid remains associated with the DNA molecule and more than 95% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, more than 99% of the first ionizable lipid remains associated with the DNA molecule and more than 99% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, all of the first ionizable lipid remains associated with the DNA molecule and all of the second ionizable lipid remains associated with the RNA molecule(s) in thefused co-LNP. In some embodiments, between about 75% and about 90% of the first ionizable lipid remains associated with the DNA molecule and between about 75% and about 90% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, more than 75% of the first ionizable lipid remains associated with the DNA molecule and more than 75% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP. In some embodiments, between about 75% and about 99% of the first ionizable lipid remains associated with the DNA molecule and between about 75% and about 99% of the second ionizable lipid remains associated with the RNA molecule(s) in the fused co-LNP.

[0031] In some embodiments, the shell of the fused co-LNP comprises a mixture of lipids from each of the precursor LNPs. In some embodiments, the shell of the fused co-LNP is a hybrid of the lipids that comprise the two precursor LNPs.

[0032] In some embodiments, at least one of the first ionizable lipid or second ionizable lipid of the co-LNP is Lipid 29. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and at least one RNA molecule by methods described herein. In other such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 29 and at least one RNA molecule by methods described herein. In other embodiments, both the first ionizable lipid and second ionizable lipid of the co-LNP is Lipid 29. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising Lipid 29 and at least one RNA molecule. Following fusion of the precursor LNPs, the resultant co-LNP will comprise a single ionizable lipid (Lipid 29).

[0033] In some embodiments, at least one of the first ionizable lipid or second ionizable lipid of the co-LNP is Lipid 319. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and at least one RNA molecule by methods described herein. In other such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and at least one RNA molecule by methods described herein. In other embodiments, both the first ionizable lipid and second ionizable lipid of the co-LNP are Lipid 319. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising Lipid 319 and at least one RNA molecule. Following fusion of the precursor LNPs, the resultant co-LNP will comprise a single ionizable lipid (Lipid 319).

[0034] In some embodiments, at least one of the first ionizable lipid or second ionizable lipid of the co-LNP is Lipid A6. In some such embodiments, the co-LNP is formed by fusing a first precursorLNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and at least one RNA molecule by methods described herein. In other such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid A6 and at least one RNA molecule by methods described herein. In other embodiments, both the first ionizable lipid and second ionizable lipid of the co-LNP are Lipid A6. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising Lipid A6 and at least one RNA molecule. Following fusion of the precursor LNPs, the resultant co-LNP will comprise a single ionizable lipid (Lipid A6).

[0035] In some embodiments, the co-LNP formed by fusing two precursor LNPs comprises two different ionizable lipids. For instance, in some embodiments, the co-LNP comprises both Lipid 29 and Lipid 319. In some embodiments, the co-LNP comprises Lipid 29, Lipid 319, or Lipid A6 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the co-LNP comprises Lipid 29, Lipid 319, or Lipid A6 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the co-LNP comprises Lipid 29, Lipid 319 or Lipid A6 and the ionizable lipid OF-C4-Deg-Lin (also referred to herein as Lipid C4 or C4), or an analog thereof. In some embodiments, the co-LNP comprises Lipid 29, Lipid 319, or Lipid A6 and the ionizable lipid DLin- KC2-DMA, or an analog thereof.

[0036] In some embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 29, Lipid 319 or Lipid A6 with a second precursor LNP comprising an RNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail. In other embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail with a second precursor LNP comprising an RNA molecule and Lipid 29, Lipid 319, or Lipid A6.

[0037] In some embodiments, the co-LNP comprises a volumetric ratio of the first LNP to the second LNP that is between about 3:1 and about 1:3. In some embodiments, the co-LNP comprises a volumetric ratio of the first LNP to the second LNP that is between about 2:1 and about 1:2. In some embodiments, the co-LNP comprises a volumetric ratio of the first LNP to the second LNP of about 1:1. In some embodiments, the co-LNP comprises a volumetric ratio of the first LNP to the second LNP of about 1:2. In some embodiments, the co-LNP comprises a volumetric ratio of the first LNP to the second LNP of about 2:1.

[0038] In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) in the co-LNP is between about 3:1 and about 1:3. In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) in the co-LNP is between about 2: 1 and about 1 :2. In someembodiments, the mass ratio between the DNA molecule and RNA molecule(s) is about 1:1. In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) is about 2:1.

[0039] Also provided herein is a tri-formulated lipid nanoparticle (tri-LNP) comprising a fusion of a first LNP, a second LNP, and a third LNP, wherein, prior to fusion: (1) the first LNP comprises: (i) a deoxyribonucleic acid (DNA) molecule; and (ii) a first ionizable lipid; (2) the second LNP comprises: (i) a first ribonucleic acid (RNA) molecule; and (ii) a second ionizable lipid; and 3) the third LNP comprises: (i) a second ribonucleic acid (RNA) molecule; and (ii) a third ionizable lipid. In some embodiments, the first RNA molecule is an mRNA. In some embodiments, the second RNA molecule is a guide RNA (gRNA).

[0040] In some embodiments, the tri-LNP comprises a fusion of a first precursor lipid nanoparticle (LNP), a second precursor lipid nanoparticle (LNP), and a third precursor lipid nanoparticle (LNP) precursor lipid nanoparticle (LNP), wherein, prior to fusion: (1) the first precursor LNP comprises: (i) a deoxyribonucleic acid (DNA) molecule; and (ii) a first ionizable lipid; and (2) the second precursor LNP comprises: (i) a first ribonucleic acid (RNA) molecule; and (ii) a second ionizable lipid; (3) the third precursor LNP comprises: (i) a second ribonucleic acid (RNA) molecule; and (ii) a third ionizable lipid, wherein the first and / or second and / or third ionizable lipids are selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6.

[0041] In some embodiments, the tri-LNP comprises (1) a deoxyribonucleic acid (DNA) molecule, a first ribonucleic acid (RNA) molecule and a second ribonucleic acid (RNA) molecule; and (2) a first ionizable lipid, a second ionizable lipid, and a third ionizable lipid, wherein the first and / or second and / or third ionizable lipids are selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6. In some embodiments, the DNA molecule is associated with the first ionizable lipid; the first RNA molecule is associated with the second ionizable lipid; and the third RNA molecule is associated with the third ionizable lipid.

[0042] In some embodiments, the mass ratio between the two RNA molecules (i.e., first RNA molecule and second RNA molecule) and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is between about 0.5:1 and about 1:3. In some embodiments, the mass ratio between the two RNA molecules (i.e., first RNA molecule and second RNA molecule) and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is between about 1:1 and about 1:2. In some embodiments, the mass ratio between the two RNA molecules (i.e., first RNA molecule and second RNA molecule) and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is between about 1:1.2 and about 1:2.4. In some embodiments, the mass ratio between the two RNA molecules (i.e., first RNA molecule and second RNA molecule) and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is between about 0.8:1 and about 0.8:2. In some embodiments, the mass ratio between the two RNA molecules (i.e., first RNA molecule and secondRNA molecule) and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1. In some embodiments, the mass ratio between the two RNA molecules (i.e., first RNA molecule and second RNA molecule) and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1 :2.

[0043] In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:1. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:2. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:3. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:4.

[0044] In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is between about 1 : 1 :0.5 and about 1:1:6. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:1. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:2. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:3. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:4.

[0045] In some embodiments, one of the precursor RNA LNPs comprises a guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. In some embodiments, the other of the precursor RNA LNPs comprises mRNA encoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the DNA LNP comprises HDRt DNA. Thus, in some embodiments, a tri-LNP generated by the trifusion method comprises a gRNA, mRNA encoding a recombinant nuclease capable of inducing a DNA break, and HDRt DNA. In some embodiments, the molar ratio between the sgRNA, the mRNA encoding a recombinant nuclease capable of inducing a DNA break and the HDRt DNA in the tri- LNP is about 1:1:1. In some embodiments, the molar ratio between the sgRNA, the mRNA encoding a recombinant nuclease capable of inducing a DNA break and the HDRt DNA in the tri-LNP is about 1:1:2. In some embodiments, the molar ratio between the sgRNA, the mRNA encoding a recombinant nuclease capable of inducing a DNA break and the HDRt DNA in the tri-LNP is about 1:1:4.

[0046] In some embodiments, the mass fraction of the ionizable lipids in the tri-LNP is between about 40% and about 60%. In some embodiments, the mass fraction of the ionizable lipids in the tri- LNP is between about 40% and about 50%. In some embodiments, the mass fraction of the ionizable lipids in the tri-LNP is between about 45% and about 55%. In some embodiments, the mass fraction of the ionizable lipids in the tri-LNP is between about 45% and about 50%. In any of the foregoing embodiments, the mass fraction of the helper lipid is between about 15% and 25%, 15% and 23%, or 15% and 20% (e.g., 15%, 16%, 17%, 18%, 19% or 20%).

[0047] In some embodiments, the first LNP, second LNP and third LNP, prior to fusion, are precursor LNPs that are not fully formed. In some embodiments, the precursor LNPs are generated in an acidic environment (e.g., at a pH between about 4 and about 5). In some embodiments, the first ionizable lipid of the first LNP forms an ionic bond with the DNA molecule and the second ionizable lipid of the second LNP forms an ionic bond with the first RNA molecule and the third ionizable lipid of the third LNP forms an ionic bond with the second RNA molecule. In some embodiments, following fusion of the first, second and third precursor LNPs to form the fused tri-LNP, the first ionizable lipid remains substantially associated (complexed) with the DNA molecule, the second ionizable lipid remains substantially associated (complexed) with the first RNA molecule, and the third ionizable lipid remains substantially associated (complexed) with the second RNA molecule.For instance, in some embodiments, more than 75% of the first ionizable lipid remains associated with the DNA molecule, more than 75% of the second ionizable lipid remains associated with the first RNA molecule and more than 75% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP.

[0048] In some embodiments, the shell of the fused tri-LNP comprises a mixture of lipids from each of the three precursor LNPs. In some embodiments, the shell of the fused tri-LNP is a hybrid of the lipids that comprise the three precursor LNPs.

[0049] In some embodiments, at least one of the first ionizable lipid, second ionizable lipid or third ionizable lipid of the tri-LNP is Lipid 29. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 29 and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 29 and a first RNA molecule and a third precursor LNP comprising Lipid 29 and a second RNA molecule by methods described herein.

[0050] In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising Lipid 29 and a first RNA molecule and a third precursor LNP comprising Lipid 29 and a second RNA molecule by methods described herein. In these embodiments, the tri-LNP following fusion of the precursor LNPs include one ionizable lipid, wherein one of the ionizable lipids is Lipid 29.

[0051] In some embodiments, at least one of the first ionizable lipid, second ionizable lipid or third ionizable lipid of the tri-LNP is Lipid 319. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising Lipid 319 and a second RNA molecule by methods described herein.

[0052] In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising Lipid 319 and a second RNA molecule by methods described herein. In these embodiments, the tri-LNP following fusion of the precursor LNPs include one ionizable lipid, wherein one of the ionizable lipids is Lipid 319.

[0053] In some embodiments, at least one of the first ionizable lipid, second ionizable lipid or third ionizable lipid of the tri-LNP is Lipid A6. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid A6 and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising Lipid A6 and a second RNA molecule by methods described herein.

[0054] In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising Lipid A6 and a first RNA molecule and a third precursor LNP comprising Lipid A6 and a second RNA molecule by methods described herein. In these embodiments, the tri-LNP following fusion of the precursor LNPs include one ionizable lipid, wherein one of the ionizable lipids is Lipid A6.

[0055] In some embodiments, the tri-LNP formed by fusing three precursor LNPs comprises at least two different ionizable lipids. For instance, in some embodiments, the tri-LNP comprises Lipid 29 and Lipid 319. In some embodiments, the tri-LNP comprises Lipid 29 and Lipid A6. In some embodiments, the tri-LNP comprises Lipid 319 and Lipid A6. In some embodiments, the tri-LNP comprises Lipid 29, Lipid 319 and Lipid A6. In some embodiments, the tri-LNP comprises Lipid 29 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the co-LNP comprises Lipid 319 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the tri-LNP comprises Lipid A6 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the tri-LNP comprises Lipid 29 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the tri-LNP comprises Lipid 319 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the tri-LNP comprises Lipid A6 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the tri-LNP comprises Lipid 29, Lipid 319 or Lipid A6 and the ionizable lipid OF-C4-Deg-Lin, or an analog thereof. In some embodiments, the tri-LNP comprises Lipid 29, Lipid 319 or Lipid A6 and the ionizable lipid DLin-KC2-DMA, or an analog thereof.

[0056] In some embodiments, the tri-LNP formed by fusing three precursor LNPs comprises three different ionizable lipids. In some such embodiments, at least one of the ionizable lipids is Lipid 29. In other embodiments, at least one of the ionizable lipids is Lipid 319. In other embodiments, at least one of the ionizable lipids is Lipid A6.

[0057] In some embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 29, Lipid 319 or Lipid A6 with a second precursor LNP comprising a first RNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail and a third precursor LNP comprising a second RNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK- E12) or an unsaturated linoleil tail. In other embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail with a second precursor LNP comprising a first RNA molecule and Lipid 29 or Lipid 319 and a third precursor LNP comprising a second RNA molecule and Lipid 29, Lipid 319 or Lipid A6.

[0058] In some embodiments, the co-LNPs and tri-LNPs formed by fusion methods described herein show a fluorescence energy transfer (FRET). In particular embodiments, FRET is demonstrated by attaching individual fluorescent dyes, a donor and acceptor, to each of the precursor LNPs prior to mixing (under acidic conditions) and neutralization. Fluorescence emission from the acceptor dye indicates the level of fusion. In some embodiments, the FRET emission signal of the fused co-ENP is greater that the fluorescence emission signal of a mixture of two individual ENPs that are not fused together. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.3. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.35. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.38. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.4. In some embodiments, the normalized FRET signal immediately following neutralization is between about 0.35 and 0.42 In some embodiments, the normalized FRET signal immediately following neutralization is between about 0.38 and 0.42 In any of the foregoing embodiments, the normalized FRET signal may be calculated by the method described in Example 4.

[0059] In some embodiments, the co-LNP or tri-LNP comprises a first helper lipid and a second helper lipid and optionally a third helper lipid. In some embodiments, the co-LNP comprises a polyethylene glycol (PEG)-conjugated lipid. In some embodiments, the co-LNP or tri-LNP comprises cholesterol.

[0060] In some embodiments, the co-LNP or tri-LNP has an average size of between about 50 nm and 150 nm, or between about 75 nm and about 125 nm, as measured by dynamic light scattering (DLS). In some embodiments, the co-LNP has an average size of between about 50 nm and 150 nm, as measured by dynamic light scattering (DLS). In some embodiments, the co-LNP or tri-LNP has an average size of between about 75 nm and about 125 nm, as measured by dynamic light scattering (DLS).

[0061] In some embodiments, the first helper lipid of the co-LNP or tri-LNP is l-stearoyl-2- oleoyl-sn-glycero-3-phosphocholine (SOPC). In some embodiments, the second helper lipid of the co- LNP or tri-LNP is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the third LNP of the tri-LNP comprises a third helper lipid. In some embodiments, the second helper lipid and the third helper lipid of the tri-LNP are the same.

[0062] In some embodiments, a co-LNP or tri-LNP comprises a fusion of two or more LNPs, any of which may comprise a non-ionizable cationic lipid. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP or ti-LNP is between about 0.5% and about 7%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP or tri-LNP is between about 0.5% and about 5%. In other embodiments, the mass fraction of the non-ionizablecationic lipid in the co-LNP or tri-LNP is between about 1% and about 6%. In other embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP or tri-LNP is between about 2% and about 5%.

[0063] In some embodiments, the DNA molecule of the co-LNP or tri-LNP comprises a transgene. In some embodiments, the transgene encodes a recombinant receptor. In some embodiments, the DNA molecule is a closed end DNA vector. In some embodiments, the transgene is positioned between protelomerase binding sequences. In some embodiments, the transgene is operably linked to a promoter and positioned between inverted terminal repeats (ITRs).

[0064] In some embodiments, the recombinant receptor encoded by the co-LNP or tri-LNP is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the recombinant receptor is a TCR. In some embodiments, the recombinant receptor is a CAR. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular region. In some embodiments, the extracellular antigen-binding domain is an antibody or an antigen-binding fragment thereof that binds to an antigen that is associated with, or expressed on, a cell or tissue of a disease or condition. In some embodiments, the CAR is a single antigen directed CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the DNA (e.g., ceDNA) molecule encoding the bispecific CAR is at least 6 kilobases, at least 7 kilobases, or at least 8 kilobases. In some embodiments, the bispecific CAR is between about 6 kilobases and about 8 kilobases. In some embodiments, the bispecific CAR is about 8 kilobases.

[0065] In some embodiments, the DNA molecule of the co-LNP or tri-LNP comprises a singlestranded DNA oligonucleotide (ssODN) or a double-stranded DNA oligonucleotide (dsODN), the ssODN or the dsODN comprising a nucleotide sequence that is homologous to a target genomic locus. In some embodiments, the DNA molecule comprises a single-stranded DNA oligonucleotide (ssODN) comprising a nucleotide sequence that is homologous to a target genomic locus. In some embodiments, the DNA molecule comprises double-stranded DNA oligonucleotide (dsODN) comprising a nucleotide sequence that is homologous to a target genomic locus.

[0066] In some embodiments, the RNA molecule of the co-LNP or tri-LNP is or comprises a guide RNA (gRNA). In some embodiments, the RNA molecule is a guide RNA (gRNA). In some embodiments, the RNA molecule comprises a guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the gRNA is complexed with a recombinant nuclease capable of inducing a DNA break. In some embodiments, the RNA molecule is or comprises a nucleotide sequence encoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the RNA molecule is: (1) a gRNA; and (2) a nucleotide sequence encoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the RNA molecule of the second LNP is a gRNA, and the RNA molecule of the third LNP is a nucleotide sequenceencoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas). In some embodiments, the recombinant nuclease is a Cas nuclease. In some embodiments, the Cas is Cas9 or Casl2a. In some embodiments, the Cas is Cas9. In some embodiments, the Cas is Casl2a.

[0067] Also provided herein is an LNP comprising: (1) a first ribonucleic acid (RNA) molecule and a second ribonucleic acid (RNA) molecule; and (2) a first ionizable lipid and a second ionizable lipid, wherein one of the first and second RNA molecules encodes a recombinant nuclease capable of inducing a DNA break; and the other of the first and second RNA molecules is a guide RNA (gRNA). In some embodiments, the LNP comprising the first RNA molecule and second RNA molecule is formed by mixing of the first RNA molecule and second RNA molecule with other components of the LNP (e.g., helper lipid and cholesterol). In some embodiments, the LNP comprising the first RNA molecule and second RNA molecule is formed by a fusion of two precursor LNPs by methods disclosed herein, thereby generating a fused co-LNP. Such co-LNPS comprising two RNA components are also referred to herein as RNA co-LNPs. In some embodiments the RNA co-LNP can be fused with a precursor LNP comprising a DNA molecule by methods disclosed herein, thereby generating a co-LNP or a tri-LNP.

[0068] Also provided herein is a co-formulated lipid nanoparticle (co-LNP) comprising a fusion of a first lipid nanoparticle (LNP) and a second lipid nanoparticle (LNP), wherein: (1) the first LNP comprises: (i) a ribonucleic acid (RNA) molecule; and (ii) a first ionizable lipid; and (2) the second LNP comprises: (i) a ribonucleic acid (RNA) molecule; and (ii) a second ionizable lipid, wherein one of the first and second RNA molecules encodes a recombinant nuclease capable of inducing a DNA break; and the other of the first and second RNA molecules is a guide RNA (gRNA). In some embodiments the LNP (e.g., co-LNP) can be fused with a precursor LNP comprising a DNA molecule by methods disclosed herein, thereby generating a co-LNP comprising a DNA molecule and two RNA molecules or a tri-LNP.

[0069] In some embodiments, the gRNA of the RNA co-LNP is a single guide RNA (sgRNA). In some embodiments, the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas). In some embodiments, the recombinant nuclease is a Cas nuclease. In some embodiments, the Cas is Cas9 or Casl2a. In some embodiments, the Cas is Cas9. In some embodiments, the Cas is Casl2a.

[0070] In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid 29. In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid 319. In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid A6. In some embodiments, the second ionizable lipid of the RNA co-LNP is Lipid 29. In some embodiments, the second ionizable lipid of the RNAco-LNP is Lipid 319. In some embodiments, the second ionizable lipid of the RNA co-LNP is Lipid A6. In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid 29 and the second ionizable lipid is Lipid 29. In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid 319 and the second ionizable lipid is Lipid 319. In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid A6 and the second ionizable lipid is Lipid A6. In some embodiments, the first ionizable lipid of the RNA co-LNP is Lipid 29 or Lipid 319 and the second ionizable lipid comprises a diketopiperazine ring core or an unsaturated lipid tail. In some embodiments, the first ionizable lipid of the RNA co-LNP comprises second a diketopiperazine ring core or an unsaturated lipid tailand the second ionizable lipid is Lipid 29, Lipid 319 or Lipid A6. In some embodiments, one of the ionizable lipids is (a) OF-C4-Deg-Lin (also referred to herein as Lipid C4 or C4), or an analog thereof; or (b) cKK-E12, or an analog thereof. In some embodiments, the first ionizable lipid of the RNA co-LNP is OF-C4-Deg-Lin, or an analog thereof. In some embodiments, the first ionizable lipid is OF-C4-Deg-Lin. In some embodiments, the first ionizable lipid of the RNA co-LNP is cKK-E12, or an analog thereof. In some embodiments, the first ionizable lipid of the RNA co-LNP is cKK-E12. In some embodiments, the first ionizable lipid is DLin-MC3-DMA, or an analog thereof. In some embodiments, the first ionizable lipid is DLin-MC3-DMA.

[0071] Also provided herein is a method of producing a lipid nanoparticle (LNP), the method comprising: (1) adding to an organic solvent comprising ethanol: (a) an ionizable lipid (e.g., Lipid 29, Lipid 319 or Lipid A6); (b) a helper 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 deoxyribonucleic acid (DNA) 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 an LNP.

[0072] In some embodiments, a flow rate of the aqueous phase in the device is between about 8 mL / min and about 10 mL / min. In some embodiments, a flow rate of the organic phase in the device is between about 2 mL / min and about 4 mL / min. In some embodiments, the flow rate of the aqueous phase is about 9 mL / min, and the flow rate of the organic phase is about 3 mL / min.

[0073] In some embodiments, the aqueous solvent is an acetate 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 4.0.

[0074] In some embodiments, the method comprises collecting the generated LNP from the device in the acetate buffer. In some embodiments, the method comprises washing the collected LNP with an isotonic buffer. In some embodiments, the method comprises filtering the LNP in the isotonic buffer with a filter to remove an LNP greater than 200 nM in diameter. In some embodiments, the isotonic buffer is PBS.

[0075] Also provided herein is a method of producing a co-LNP, comprising: (1) mixing, in an acidic buffer: (a) a first lipid nanoparticle (LNP) comprising a first ionizable lipid and a nucleic acid molecule; and (b) a second LNP comprising a second ionizable lipid and a ribonucleic acid (RNA) molecule, thereby generating a composition comprising the first LNP and the second LNP; and (2) neutralizing the composition comprising the first LNP and the second LNP, thereby generating a co- LNP, which is a fusion of the first LNP and the second LNP, wherein the nucleic acid molecule in (a) is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule. In some embodiments, the first LNP and second LNP, prior to fusion, are precursor LNPs that are not fully formed.

[0076] In some embodiments, the nucleic acid molecule in (a) is a DNA molecule. In some embodiments, the nucleic acid molecule in (a) is an RNA molecule.

[0077] In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is between about 3:1 and about 1:3. In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is between about 2:1 and about 1:2.

[0078] In some embodiments, the method comprises mixing, in the acidic buffer, (c) a third LNP comprising a third ionizable lipid and an RNA molecule, thereby generating a composition comprising the first, second, and third LNPs. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs in the composition is between about 3:1 and about 1:3. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs in the composition is between about 2: 1 and about 1 :2.

[0079] In some embodiments, the acidic buffer is an acetate buffer. In some embodiments, the acidic buffer has a pH of between about 3.0 and about 4.5. In some embodiments, the acidic buffer has a pH of between about 4.0 and about 5.0. In some embodiments, the acidic buffer has a pH of about 4.0. In some embodiments, the acidic buffer is neutralized to a pH of between about 6.0 and about 7.5, or between about 6.5 and about 7.0. In some embodiments, the acidic buffer is neutralized to a pH of between about 6.0 and about 7.5. In some embodiments, the acidic buffer is neutralized to a pH of between about 6.5 and about 7.0. In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding an isotonic buffer. In some embodiments, the isotonic buffer has a pH of about 7.4. In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding at least about 6 parts of the isotonic buffer to 1 part of the acidic buffer. In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding between about 6-7 parts of the isotonic buffer to 1 part of the acidic buffer. In some embodiments, the isotonic buffer is phosphate buffered saline (PBS).

[0080] Also provided herein is a co-LNP or tri-LNP produced by any of the methods provided herein.

[0081] Also provided herein is a method of genetically engineering an immune cell, the method comprising incubating an immune cell with a co-LNP or tri-LNP provided herein or a composition thereof. In some embodiments, the immune cell is a lymphocyte. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a primary T cell. In some embodiments, the T cell is a CD4+ T cell or a CD8+ T cell.

[0082] In some embodiments, at the time of incubating the immune cell with the LNP, the co- LNP (or tri-LNP), or the composition, the immune cell is activated. In some embodiments, at the time of incubating the immune cell with the LNP, the co-LNP (or tri-LNP), or the composition, the immune cell expresses CD25, CD26, CD27, CD28, CD30, CD71, CD154, CD40L, CD134, or a combination thereof. In some embodiments, the immune cell is incubated under stimulating conditions prior to incubating the immune cell with the LNP, the co-LNP (or tri-LNP), or the composition. In some embodiments, the immune cell is incubated under stimulating conditions for between about 24 hours and about 72 hours, or for about 48 hours. In some embodiments, the stimulating conditions comprise incubation with a stimulatory reagent capable of activating an intracellular signaling domain of a component of a TCR complex and an intracellular signaling domain of a costimulatory molecule. In some embodiments, the stimulatory reagent comprises a primary agent that binds to CD3 and a secondary agent that binds to a T cell costimulatory molecule. In some embodiments, the costimulatory molecule is selected from the group consisting of CD28, 4- 1BB, 0X40, and ICOS. In some embodiments, the primary agent is an anti-CD3 antibody or antigenbinding fragment, and the secondary agent is an anti-CD28 antibody or antigen-binding fragment. In some embodiments, the immune cell is incubated with apolipoprotein E (ApoE) prior to incubating the immune cell with the LNP, the co-LNP (or tri-LNP), or the composition. In some embodiments, the ApoE is ApoE4.

[0083] Also provided herein is an immune cell produced by any of the methods herein. Also provided herein is a composition comprising a plurality of immune cells produced by any of the methods herein.

[0084] Also provided herein is a method of treatment comprising administering the immune cell produced by any of the methods herein, or a composition thereof, to a subject having a disease or disorder. Also provided herein is use of the immune cell produced by any of the methods herein, or a composition thereof, for treating a disease or disorder. Also provided herein is use of the immune cell produced by any of the methods herein, or a composition thereof, in the manufacture of a medicament for treating a disease or disorder.BRIEF DESCRIPTION OF THE DRAWINGS

[0085] FIG. 1 shows that following incubation of T cells with various LNPs comprising different ionizable lipids, transient and stable integration of CAR is achieved.

[0086] FIG. 2 shows the percentage of CD3- (bars) and viable (dots) cells.

[0087] FIG. 3A depicts the generation of a co-formulated LNP containing RNA and DNA through fusion of a RNA-containing LNP and a DNA-containing LNP.

[0088] FIG. 3B depicts the generation of a co-formulated LNP containing RNA and DNA through double sequential fusion.

[0089] FIG. 3C depicts the generation of a co-formulated LNP containing RNA and DNA through fusion of two RNA-containing LNPs and one DNA-containing LNP.

[0090] FIG. 4A depicts the percentage of CD3- viable cells (dots) and cells exhibiting transient expression of a BCMA CAR (bars).

[0091] FIG. 4B depicts the percentage of CD3- viable cells (dots) and cells exhibiting integrated expression of a BCMA CAR (bars).

[0092] FIGS. 5A-B show the relationship between the percentage of CD3- cells and the percentage of cells exhibiting integrated BCMA CAR expression following CAR knock-in using a closed end DNA (ceDNA) construct or a DNA nanoplasmid (NP), respectively.

[0093] FIG. 6A and FIG. 6B show the percentage of fully edited cells 7 days and 14 days posttransfection, respectively, following transfection using electroporation, a hybrid approach, co-LNPs or tri-LNPs. Fully edited cells display TCR knockout (TCR-) and expression of CAR T.

[0094] FIG. 7A shows fully edited (TCR- CAR+) T cell populations enrich over time following transfection with tri-LNPs.

[0095] FIG. 7B shows fully edited (TCR- CAR+) T cell populations enrich over time following electroporation, but to a lesser extent than tri-LNP administration.

[0096] FIG. 8A shows CD4 / CD8 ratios at various timepoints following transfection of T cells with LNPs. Initially, the CD4 population is enhanced relative to the CD8 population.

[0097] FIG. 8B shows CD4 / CD8 ratios at various timepoints following transfection of T cells using electroporation.

[0098] FIG. 9 shows an experimental overview for FRET experiments used to characterize co- LNPs.

[0099] FIG. 10 shows a compilation of four independent experiments that demonstrate that significant lipid fusion occurs immediately when LNPs are mixed (0 hr. timepoint) at pH4 (Sample:co-LNP, pH4), and that maximal fusion occurs when LNPs are mixed at pH4 and then neutralized to pH 7 (Sample: co-LNP, pH7).

[0100] FIG. 11 shows normalized FRET signals calculated across the four independent signals, for non-fused LNPs (dual LNPs), acidic co-LNPs, and co-LNPs formed following neutralization of the acidic co-LNPs.

[0101] FIG. 12 shows that at 3 days post transfection, respectively, tri-LNPs comprising OF-C4- Deg-Lin (C4), Lipid 29 (L29), Lipid 319 (L319), or combinations thereof, effectively mediated TRAC knockout.

[0102] FIG. 13 shows that at 3 days post transfection, respectively, tri-LNPs comprising OF-C4- Deg-Lin (C4), Eipid 29 (L29), Eipid 319 (E319), or combinations thereof, effectively mediated TRAC knockout.

[0103] FIG. 14 shows the percent change between days 3 and 7 in TRAC knockout following tranf ections with the various ENPs comprising different ionizable lipids.

[0104] FIG. 15 shows that at 3 days post transfection, tri-ENPs comprising OF-C4-Deg-Ein (C4), Eipid 29 (L29), Eipid 319 (L319), or combinations thereof, resulted us CAR+ expression.

[0105] FIG. 16 shows that at 7 days post-transfection, tri-LNPs with various ionizable lipids provided comparable or higher fully edited cells (CAR+ expression and TRAC knockout) than the electroporation (EP) control.

[0106] FIG. 17 shows the yield of CAR T cells 7 days after transfections of the different tri- ENPs.

[0107] FIG. 18 shows CD4 / CD8 ratios at various timepoints following transfection of ENPs. Initially, the CD4 population is enhanced relative to the CD8 population.

[0108] FIG. 19A shows components of various precursor LNPs used in experiments designed to assess delivery of tandemGFP CAR (5.7 kb ceDNA) and TRAC knockout using tri-LNPs comprising different ionizable lipids including Lipid 29 (L29), Lipid 319 (L319), Lipid and A6 (A6).

[0109] FIG. 19B shows the estimated mass fractions of the tri-LNP formed from mixing the Cas9 mRNA (Formula Yl), TRAC sgRNA (Formula Y2), and ceDNA (F9) precursor ENPs at a ratio of 1:1:2. The total mass fraction of nucleic acid (NA) in the tri-ENP is approximately 4%. The ratio of total RNA (Cas9 RNA and TRAC sgRNA) to ceDNA in the tri-ENP is approximately 1:2.

[0110] FIG. 19C shows the estimated mass fractions of the tri-LNP formed from mixing the Cas9 mRNA (Formula Yl), TRAC sgRNA (Formula Y2), and ceDNA (F9) precursor ENPs at a ratio of 1:1:4. The total mass fraction of nucleic acid (NA) in the tri-ENP is approximately 4.3%. The ratio of total RNA (Cas9 RNA and TRAC sgRNA) to ceDNA in the tri-LNP is approximately 1:1.

[0111] FIG. 20 shows the tri-LNP components and various process parameters in experiments designed to assess delivery of a GFP tandem dual-targeted CAR (5.7 kb ceDNA) and TRAC knockout using tri-LNPs comprising different ionizable lipids including Lipid 29 (L29), Lipid 319 (L319), Lipid and A6 (A6).

[0112] FIG. 21A shows that at the third day post-transfection, tri-LNPs containing 5.7 kb tandemGFP CAR, with a 1:1:4 v / v / v precursor ratio of Cas9:TRAC:CAR DNA, generated CAR T cells at a similar rate of efficiency as the electroporation control with the same sequence (-8-9% CAR+). FIG. 21B shows the CD3- % knockout (KO) was comparable to the electroporation control for several of the LNPs tested with different ionizable lipids.

[0113] FIG. 22A shows tri-LNP conditions with each ionizable lipid mediated higher cell viability (live cell %) and growth over time compared to the EP control of RNP + tandemGFP CAR, demonstrating reduced toxicity to cells achieved with LNP-delivery of large DNA sequences over electroporation. FIG. 22B shows total number of live cells over time. FIG. 22C shows total number of cells (TNC) over time.

[0114] FIG. 23A shows that tri-LNPs containing a larger ratio of DNA to total RNA, namely the 1:1:4 Cas9 mRNA:TRAC sgRNA:DNA ratio, mediated the highest CAR KI compared to tri-LNPs with larger ratios of RNA (1:1:2 and 1:1:1) 3 days post-transfection, regardless of the ionizable lipid used in the formulation. Each tri-LNP ratio produced similar total numbers of cells (TNC) at day 7 (FIG. 23B). FIG. 23C shows that the CAR T yield that expanded through day 7 post-transfection, correlated with the % editing efficiency for each tri-LNP samples. FIG. 23D shows the total number of CAR+ cells at day 7 post-transfection.

[0115] FIG. 24A shows the capability of ionizable lipids to mediate TRAC / CD3 KO in RNA- only containing co-LNPs, created from fusing Cas9 mRNA and TRAC sgRNA precursors together in a 1:1 volume ratio (1:1 Co-RNA), correlated with their gene editing capabilities in tri-LNPs containing both the RNA and DNA. FIG. 24B shows ionizable lipids L29, L319, and A6 appear to mediate better RNA delivery and TRAC / CD3 KO (%CD3- cells) while in the context of tri-LNPs over the RNA-only co-LNPs, generating and maintain over 70% TRAC KO through day 7.

[0116] FIG. 25A shows that increasing the % mass fraction of tandemGFP CAR ceDNA up to 5% in F9B, compared to 3.5% in F9, does not appear to have a significant effect on resulting CAR+ expression. FIG. 25B shows that increasing the % mass fraction of tandemGFP CAR ceDNA up to 5% in F9B, compared to 3.5% in F9, does not appear to have a significant effect on cell growth.

[0117] FIG. 26A shows an overview of the CAR T cell production process use to develop functional CAR T cells to assess functionality of LNPs. FIG. 26B shows the %CAR+ T cells 7 days post-transfection of a Homology Directed Repair (HDR) using the various delivery methods (AAV,electroporation or tri-LNPs). FIG. 26C shows the CAR T cell yield 7 days post-transfection of a Homology Directed Repair (HDR). using the various delivery methods.

[0118] FIG. 27A shows a flowchart to assess functionality of LNP-produced CAR T cells with tumor killing capacity produced by different methods (AAV, electroporation or tri-LNPs) of delivering a Homology Directed Repair (HDR) template. FIG. 27B shows that tri-LNP2 samples exhibited CD4 / CD8 ratios at Day 0 similar to those of untreated mock samples. FIG. 27C shows electroporated (EP) samples experienced substantial loss in polyfunctionality post-chronic stimulation on day 10(dl0) relative to cells transfected with the tri-LNPs.

[0119] FIGs. 28A-D shows tumor killing capability of CAR T cells produced by various methods (AAV, electroporation or tri-LNPs) of delivering a Homology Directed Repair (HDR) template. FIG. 28A shows that the CAR T cells produced by the various methods are capable of lysing OPM-2 cells. FIG. 28B shows that the CAR T cells produced by the various methods, are capable of lysing RPMI8226 cells. FIG. 28C shows the % lysis of OPM-2 cells achieved at day 10 (dlO) using the various methods to deliver the Homology Directed Repair (HDR) template. FIG. 28D shows the % lysis of RPMI8226 cells achieved at day 10 (dlO) using the various methods to deliver the Homology Directed Repair (HDR) template.

[0120] FIG. 29A, FIG. 29B, and FIG. 29C, each demonstrating results of different dose groups, show that gene editing T cells via Tri-LNP suppressed tumor growth in the OPM2 xenograft mouse model.

[0121] FIG. 30A shows tumor control index (TCI) in the OPM2 xenograft mouse model as a function of donor and CAR T dose across three different donors and doses (1E6, 3.3E5, and 1.1E5). FIG. 30B shows tumor control index (TCI) of all three donors compiled at the 3.3E5 dose.

[0122] FIG. 31 shows CAR+ T cell counts compiled from all three donors at the 3.3E5 dose of T cells gene edited via Tri-LNP, electroporation, or mock edited.DETAILED DESCRIPTION

[0123] Provided herein are lipid nanoparticle (LNP) compositions for delivering nucleic acid molecules, including deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or both, into a lymphocyte, e.g., a T cell, and methods of producing and using the same, such as in connection with cell therapy. In any of the provided embodiments, the lipid nanoparticles contain an ionizable lipid, e.g., an ionizable amino lipid, a helper lipid, a polyethylene glycol (PEG) lipid, cholesterol, and a nucleic acid molecule (e.g., RNA or DNA). In some aspects, the nucleic acid molecule is DNA, such as a DNA molecule comprising a transgene encoding a recombinant receptor (e.g., a T cell receptor (TCR) or a chimeric antigen receptor (CAR)). In particular embodiments, T cells transduced using theLNPs are for use in cell therapy, such as adoptive cell therapy. In some aspects, the nucleic acid molecule is RNA, such as an RNA molecule encoding a recombinant nuclease capable of inducing a DNA break, a guide RNA (gRNA), or a combination, or an RNA molecule encoding a transposase. In some aspects, cells incubated with the LNPs provided herein are genetically edited, such as knocked out and / or knocked-in, at a genomic locus.

[0124] Results herein demonstrate that LNPs and compositions thereof are suitable for delivery of nucleic acids, including DNA, into T cells, such as for use in adoptive cell therapies and / or gene editing. In some aspects, the provided LNPs and compositions thereof deliver a DNA molecule comprising a transgene that encodes a recombinant receptor (e.g., a CAR) to genetically engineer T cells to express the receptor. In some aspects, the provided LNPs and compositions thereof deliver an RNA molecule, such as an mRNA encoding a transposase or nuclease, or a guide RNA (gRNA). In some embodiments, the LNP compositions methods are advantageous by virtue of being nonviral, in that the time, labor, and safety risks associated with viral vector-based cell engineering techniques are mitigated or avoided. Further, the LNP compositions provided herein exhibit high transfection and transduction efficiency, with no or limited cell toxicity observed.

[0125] In further aspects, LNPs can be co-formulated by the fusion of separate precursor LNPs to contain one or more nucleic acid molecules. A co- LNP can be generated from the fusion of a first precursor LNP containing a first nucleic acid molecule and a second precursor LNP containing a second nucleic acid molecule and optionally, a third nucleic acid molecule. For instance, a co-LNP can be generated to contain a DNA molecule and one or more types of RNA molecule (e.g., a transposon and transposase mRNA or repair template and gRNA) by fusing a DNA-containing precursor LNP with a RNA-containing precursor LNP. A co-LNP can also be generated to contain multiple types of RNA molecules by fusing a first precursor LNP containing a first RNA molecule with a second precursor LNP containing a second RNA molecule (e.g., mRNA encoding a nuclease (e.g., Cas) and a gRNA). The resultant RNA-containing LNP can be further fused with a DNA- containing precursor LNP to create a co-LNP containing e.g., two types of RNA molecules and a DNA molecule (e.g., mRNA encoding a nuclease, a gRNA, and repair template DNA). Alternatively, three separate precursor LNPs can be fused by techniques described herein, hence generating a tri- LNP. For instance, a first precursor RNA comprising a first RNA molecule (e.g., mRNA encoding a nuclease (e.g., Cas)), can be fused to a second precursor LNP comprising a gRNA and a third precursor LNP comprising a DNA (e.g., a repair template DNA). In some cases, transfection of cells with a co-LNP or tri-LNP generated from separate precursor LNPs may result in higher gene editing efficiency, as compared to the separate LNPs. In some cases, it is contemplated that the improved efficiency may be due to reduced competitive uptake, in that the cell is only required to uptake a single type of co- LNP or tri-LNP.

[0126] While lipid nanoparticles are already used or being investigated for delivery of ribonucleic acid (RNA), drugs, antioxidants, and contrast agents, they can be plagued by issues with dose-limiting toxicities and reproducibility. In addition, 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). By contrast, the compositions provided herein, and uses thereof, demonstrate that lipid nanoparticles can be formulated reproducibly, stably, and without targeting moieties to genetically engineer cells in a nontoxic manner. Further, observations herein demonstrate that a non-viral hybrid approach to genetic engineering, such as by delivering RNA into a cell by electroporation, coupled with delivery of DNA into the cell by incubation with DNA-containing LNPs, achieves high rates of transduction efficiency, while resulting in low or negligible cellular toxicity.

[0127] 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.

[0128] 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 FOR DELIVERING DNA

[0129] Provided herein are lipid nanoparticles (LNPs) and compositions containing the same, such as for delivering DNAinto a cell (e.g. a T cell). In some embodiments, the LNP comprises an ionizable lipid, such as Lipid 29. In some embodiments, the LNP comprises an ionizable lipid, such as Lipid 319. In some embodiments, the LNP comprises an ionizable lipid, such as Lipid A6. In some embodiments, the LNP further comprise a helper lipid, a polyethylene glycol (PEG)-conjugated lipid, and cholesterol. Also provided herein are methods for producing the LNPs and compositions and uses thereof, such as in connection with cell therapy. In some embodiments, the DNA LNPs described in this section are used to generate co-LNPs and tri-LNPs described in Section II below.A. DNA LNPs1. Components

[0130] Provided herein are LNPs comprising a deoxyribonucleic acid (DNA) molecule (also referred to as DNA LNPs or DNA-containing LNPs). In some embodiments, the DNA LNP comprises an ionizable lipid; a helper lipid; a polyethylene glycol (PEG)-conjugated lipid; cholesterol;and a DNA molecule. In other embodiments, the DNA LNP comprises a non-ionizable lipid cationic lipid; a helper lipid; a polyethylene glycol (PEG)-conjugated lipid; cholesterol; and a DNA molecule.

[0131] In one aspect, provided herein is a lipid nanoparticle (LNP) comprising: (1) an ionizable lipid comprising a squaramide moiety, wherein the ionizable lipid is an ionizable amino lipid; and (2) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the ionizable lipid comprising the squaramide moiety is referred to as Lipid 29 (also referred to as LIPID 29) and has the structure depicted below. The synthesis of Lipid 29 was described in Cornebise, E. Narayanan, Y. Xia, E. Acosta, L. Ci, H. Koch, J. Milton, S. Sabnis, T. Salerno and K. E. Benenato, Adv. Funct. Mater., 2022, 32, 2106727.LIPID 29

[0132] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising: (1) an ionizable lipid comprising Lipid 319 (also referred to as L319 or 319), wherein the ionizable lipid is an ionizable amino lipid; and (2) a deoxyribonucleic acid (DNA) molecule. The structure of Lipid 319 is depicted below. The synthesis of Lipid 319 was described in Maier, M. A., M., J., Matsuda, S., et al. Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics. Mol. Ther. 21(8), 1570-1578 (2013).LIPID 319

[0133] In another aspect, provided herein is a lipid nanoparticle (LNP) comprising: (1) an ionizable lipid comprising Lipid A6, wherein the ionizable lipid is an ionizable amino lipid; and (2) a deoxyribonucleic acid (DNA) molecule. The structure of Lipid A6 is depicted below.LIPID A6

[0134] In some embodiments, the mass fraction of the ionizable lipid in the LNP is between about 30% and about 65%. In some embodiments, the mass fraction of the ionizable lipid in the LNP is between about 32% and about 65%. In some embodiments, the mass fraction of the ionizable lipid in the LNP is between about 40% and about 60%. In some embodiments, the mass fraction of the ionizable lipid in the LNP is between about 35% and about 45%. In some embodiments, the mass fraction of the ionizable lipid in the LNP is between about 40% and about 45%. In some embodiments, the mass fraction of the ionizable lipid in the LNP is between about 50% and about 60%. In some embodiments, the mass fraction of the ionizable lipid is about 30%, about 32%, about 32.5%, about 33%, about 33.5%, about 34%, about 34.5%, about 35%, about 35.5%, about 36%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, or about 65%. In some embodiments, the mass fraction of the ionizable lipid is about 32%. In some embodiments, the mass fraction of the ionizable lipid is about 32.5%. In some embodiments, the mass fraction of the ionizable lipid is about 33%. In some embodiments, the mass fraction of the ionizable lipid is about 33.5%. In some embodiments, the mass fraction of the ionizable lipid is about 34%. In some embodiments, the mass fraction of the ionizable lipid is about 34.5%. In some embodiments, the mass fraction of the ionizable lipid is about 35%. In some embodiments, the mass fraction of the ionizable lipid is about 35.5%. In some embodiments, the mass fraction of the ionizable lipid is about 36%. In some embodiments, the mass fraction of the ionizable lipid is about 54%. In some embodiments, the mass fraction of the ionizable lipid is about 55%. In some embodiments, the mass fraction of the ionizable lipid is about 56%. In some embodiments, the mass fraction of the ionizable lipid is about 57%. In some embodiments, the mass fraction of the ionizable lipid is about 58%. In some embodiments, the mass fraction of the ionizable lipid is about 59%. In some embodiments, the mass fraction of the ionizable lipid is about 60%. In some embodiments, the mass fraction of the ionizable lipid is about 61%.

[0135] In some embodiments, the LNP comprises more than one cationic lipid. In some embodiments, the LNP comprises two cationic lipids. In some embodiments, the LNP comprises an ionizable cationic lipid and a non-ionizable cationic lipid. The non-ionizable lipid has a higher pKa than the ionizable lipid and would predominantly be charged in both the bloodstream. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non- ionizable cationic lipid in the LNP is between about 0.1% and about 40%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.2% and about 20%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.2% and about 20%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.5%and about 10%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 1% and about 7%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non- ionizable cationic lipid in the LNP is between about 1% and about 6%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 2% and about 5%. In some embodiments where the LNP comprises at least one ionizable lipid, the mass fraction of the non-ionizable cationic lipid in the LNP is between about 0.2% and about 1%.

[0136] In some embodiments, the non-ionizable cationic lipid has the following structure:which is referred to herein as Lipid Tl. In some embodiments, the mass fraction of Lipid T1 in the LNP is between about 0.1% and about 40%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 0.2% and about 20%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 0.2% and about 20%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 0.5% and about 10%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 1% and about 7%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 1% and about 6%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 2% and about 5%. In some embodiments, the mass fraction of Lipid Tl in the LNP is between about 0.2% and about 1%.

[0137] In some cases, the non-ionizable cationic lipid is DOTMA. In some cases, the ionizable lipid has the structure of

[0138] In some cases, the non-ionizable cationic lipid is DOTMA. In some cases, the ionizable lipid has the structure of

[0139] In some cases, the non-ionizable lipid is DODAP. In some cases, the ionizable lipid has the structure of

[0140] In some cases, the non-ionizable lipid is DOSPA. In some cases, the ionizable lipid has the structure of

[0141] In some embodiments, the helper lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1- Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and l,2-dioleoyl-sn-glycero-sn-3- phosphatidylcholine (DOPC). In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid has the structure of

[0142] In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid has the structure of

[0143] In some embodiments, the helper lipid is SOPC. In some embodiments, the helper lipid has the structure of

[0144] In some embodiments, the helper lipid is DOPC. In some embodiments, the helper lipid has the structure of

[0145] In some embodiments, the mass fraction of the helper lipid in the LNP is between about 15% and about 25%, or between about 18% and about 22%. In some embodiments, the mass fraction of the helper lipid is between about 12% and about 20%, or between about 16% and about 20%. In some embodiments, the mass fraction of the helper lipid is about 15%, about 15.5%, about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, about 20%, about 20.5%, 21%, about 21.5%, about 22%, about 22.5%, about 23%, about 23.5%, about 24%, about 24.5%, or about 25%. In some embodiments, the mass fraction of the helper lipid is about 15%. In some embodiments, the mass fraction of the helper lipid is about 15.5%. In some embodiments, the mass fraction of the helper lipid is about 16%. In some embodiments, the mass fraction of the helper lipid is about 16.5%. In some embodiments, the mass fraction of the helper lipid is about 17%. In some embodiments, the mass fraction of the helper lipid is about 17.5%. In some embodiments, the mass fraction of the helper lipid is about 18%. In some embodiments, the mass fraction of the helper lipid is about 18.5%. In some embodiments, the mass fraction of the helper lipid is about 19%. Insome embodiments, the mass fraction of the helper lipid is about 19.5%. In some embodiments, the mass fraction of the helper lipid is about 20%. In some embodiments, the mass fraction of the helper lipid is about 20.5%. In some embodiments, the mass fraction of the helper lipid is about 21%. In some embodiments, the mass fraction of the helper lipid is about 21.5%. In some embodiments, the mass fraction of the helper lipid is about 22%. In some embodiments, the mass fraction of the helper lipid is about 22.5%. In some embodiments, the mass fraction of the helper lipid is about 23%. In some embodiments, the mass fraction of the helper lipid is about 23.5%. In some embodiments, the mass fraction of the helper lipid is about 24%. In some embodiments, the mass fraction of the helper lipid is about 24.5%. In some embodiments, the mass fraction of the helper lipid is about 25%.

[0146] In some embodiments, the polyethylene glycol (PEG)-conjugated lipid is DMG- PEG2000. In some embodiments, the PEG-conjugated lipid has the structure of

[0147] In some embodiments, the mass fraction of the PEG-conjugated lipid in the LNP is between about 2% and about 10%, or between about 3% and about 7%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%. 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%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 3%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 3.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 4%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 4.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 5%. In some embodiments, the mass fraction of the PEG- conjugated lipid is about 5.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 6%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 6.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 7%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 7.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 8%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 8.5%.

[0148] In some embodiments, cholesterol has the structure of

[0149] In some embodiments, the mass fraction of cholesterol in the LNP is between about 30% and about 45% or between about 33% and 37%. 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 mass fraction of cholesterol is about 44%. In some embodiments, the mass fraction of cholesterol is about 45%.

[0150] In some embodiments, the DNA molecule comprises a transgene. In some embodiments, the transgene encodes a recombinant receptor. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the recombinant receptor is a CAR. In some embodiments, the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular region. In some embodiments, the extracellular antigen-binding domain is an antibody or an antigen-binding fragment thereof that binds to an antigen that is associated with, or expressed on a cell or tissue of a disease or condition. In some embodiments, the intracellular region comprises an intracellular signaling domain that is or comprises an intracellular signaling domain of a CD3 chain, or a signaling portion thereof. In some embodiments, the intracellular region comprises one or more costimulatory signaling domain(s) comprising an intracellular signaling domain selected from the group consisting of: a CD28, a 4-1BB, an ICOS, or a signaling portion thereof. In some embodiments, the CAR is a single antigen directed CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the DNA (e.g.,ceDNA) molecule encoding the bispecific CAR is at least 6 kilobases, at least 7 kilobases, or at least 8 kilobases. In some embodiments, the bispecific CAR is between about 6 kilobases and about 8 kilobases. In some embodiments, the bispecific CAR is about 8 kilobases.

[0151] In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is selected from among avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7- H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (LI -CAM), CE7 epitope of LI -CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomer ase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1). In some embodiments, the antigen is BCMA. In some embodiments, the antigen is CD19.

[0152] In some embodiments, the transgene is operably linked to a promoter and positioned between inverted terminal repeats (ITRs). In some embodiments, the DNA molecule is a closed end DNA vector. In some embodiments, the transgene is positioned between protelomerase binding sequences. In some embodiments, the DNA molecule is a doggybone DNA vector.

[0153] In some embodiments, the DNA molecule comprises a single-stranded DNA oligonucleotide (ssODN) or a double-stranded DNA oligonucleotide (dsODN). In some embodiments, the DNA molecule comprises a single-stranded DNA oligonucleotide (ssODN). In some embodiments, the ssODN comprises a nucleotide sequence that is homologous to a target genomic locus. In some embodiments, the DNA molecule comprises a double-stranded DNA oligonucleotide (dsODN). In some embodiments, the dsODN comprises a nucleotide sequence that is homologous to a target genomic locus.

[0154] In some embodiments, the mass fraction of the DNA in the LNP is between about 1% and about 10%, between about 2% and about 6%, or between about 3% and about 5%. In some embodiments, the mass fraction of the DNA is about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, or about 13%. In some embodiments, the mass fraction of the DNA is about 3%. In some embodiments, the mass fraction of the DNA is about 3.5%. In some embodiments, the mass fraction of the DNA is about 4%. In some embodiments, the mass fraction of the DNA is about 5%.

[0155] In some embodiments, the total DNA concentration in the LNP is about between about 0.5 ug / mL and about 10 ug / mL, between about 0.75 ug / mL and about 8 ug / mL, or between about 1.5 ug / mL and about 6 ug / mL. In some embodiments, the total DNA concentration is about 0.5 ug / mL. In some embodiments, the total DNA concentration is about 0.75 ug / mL. In some embodiments, the total DNA concentration is about 1 pg / mL. In some embodiments, the total DNA concentration is about 1.25 ug / mL. In some embodiments, the total DNA concentration is about 1.5 ug / mL. In some embodiments, the total DNA concentration is about 1.75 ug / mL. In some embodiments, the total DNA concentration is about 2 ug / mL. In some embodiments, the total DNA concentration is about2.25 ug / mL. In some embodiments, the total DNA concentration is about 2.5 ug / mL. In some embodiments, the total DNA concentration is about 2.75 ug / mL. In some embodiments, the total DNA concentration is about 3ug / mL. In some embodiments, the total DNA concentration is about3.25 ug / mL. In some embodiments, the total DNA concentration is about 3.5 ug / mL. In some embodiments, the total DNA concentration is about 3.75 ug / mL. In some embodiments, the total DNA concentration is about 4 ug / mL. In some embodiments, the total DNA concentration is about4.25 ug / mL. In some embodiments, the total DNA concentration is about 4.5 ug / mL. In some embodiments, the total DNA concentration is about 4.75 ug / mL. In some embodiments, the total DNA concentration is about 5 ug / mL. In some embodiments, the total DNA concentration is about5.25 ug / mL. In some embodiments, the total DNA concentration is about 5.5 ug / mL. In some embodiments, the total DNA concentration is about 5.75 ug / mL. In some embodiments, the total DNA concentration is about 6 ug / mL. In some embodiments, the total DNA concentration is about6.25 ug / mL. In some embodiments, the total DNA concentration is about 6.5 ug / mL. In some embodiments, the total DNA concentration is about 6.75 ug / mL. In some embodiments, the total DNA concentration is about 7 ug / mL. In some embodiments, the total DNA concentration is about7.25 ug / mL. In some embodiments, the total DNA concentration is about 7.5 ug / mL. In some embodiments, the total DNA concentration is about 7.75 ug / mL. In some embodiments, the total DNA concentration is about 8 ug / mL.2. Formulations

[0156] Provided herein are LNPs containing: (a) an ionizable lipid selected from the group consisting of Lipid 319, Lipid A6, and Lipid L29; (b) a helper lipid that is l-stearoyl-2-oleoyl-sn- glycero-3-phosphocholine (SOPC); (c) a polyethylene glycol (PEG)-conjugated lipid that is DMG- PEG2000; (d) cholesterol; and (e) a deoxyribonucleic acid (DNA) molecule.

[0157] In some embodiments, the ionizable lipid is Lipid 319. In some embodiments, the mass fraction of Lipid 319 is between about 35% and about 45%. In some embodiments, the mass fraction of Lipid 319 is between about 30% and about 40%. In some embodiments, the mass fraction of Lipid 319 is between about 35% and about 40%. In some embodiments, the mass fraction of Lipid 319 is between about 30% and about 35%. In some embodiments, the mass fraction of SOPC is between about 15% and about 25%. In some embodiments, the mass fraction of DMG-PEG2000 is between about 2% and about 3%. In some embodiments, the mass fraction of cholesterol is between about 30% and about 40%. In some embodiments, the mass fraction of the DNA molecule is between about 3% and about 4%.

[0158] In some embodiments, the ionizable lipid is Lipid 29. In some embodiments, the mass fraction of Lipid 29 is between about 35% and about 45%. In some embodiments, the mass fraction of Lipid 29 is between about 30% and about 40%. In some embodiments, the mass fraction of Lipid 29 is between about 35% and about 40%. In some embodiments, the mass fraction of Lipid 29 is between about 30% and about 35%. In some embodiments, the mass fraction of SOPC is between about 15% and about 25%. In some embodiments, the mass fraction of DMG-PEG2000 is between about 2% and about 3%. In some embodiments, the mass fraction of cholesterol is between about 30% and about 40%. In some embodiments, the mass fraction of the DNA molecule is between about 3% and about 4%.

[0159] In some embodiments, the ionizable lipid is Lipid A6. In some embodiments, the mass fraction of Lipid A6 is between about 35% and about 45%. In some embodiments, the mass fraction of Lipid A6 is between about 30% and about 40%. In some embodiments, the mass fraction of Lipid A6 is between about 35% and about 40%. In some embodiments, the mass fraction of Lipid A6 is between about 30% and about 35%. In some embodiments, the mass fraction of SOPC is between about 15% and about 25%. In some embodiments, the mass fraction of DMG-PEG2000 is betweenabout 2% and about 3%. In some embodiments, the mass fraction of cholesterol is between about 30% and about 40%. In some embodiments, the mass fraction of the DNA molecule is between about 3% and about 4%.

[0160] In some embodiments, the LNP contains: (a) Lipid 29 with a mass fraction of between about 35% and about 45%; (b) SOPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2000 with a mass fraction of between about 2% and about 3%; (d) cholesterol with a mass fraction of between about 30% and about 40%; and (e) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the LNP contains: (a) Lipid 29 with a mass fraction of between about 30% and about 40%; (b) SOPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2000 with a mass fraction of between about 2% and about 3%; (d) cholesterol with a mass fraction of between about 30% and about 40%; and (e) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the LNP contains: (a) Lipid 29 with a mass fraction of about 40%; (b) SOPC with a mass fraction of about 19%; (c) DMG-PEG2000 with a mass fraction of about 2.5%; (d) cholesterol with a mass fraction of about 35%; and (e) a deoxyribonucleic acid (DNA) molecule with a mass fraction of about 3.5%. In some embodiments, the LNP further comprise a non-ionizable cationic lipid. In some embodiments, the mass fraction of the non-ionizable cationic lipid is between about 1% and 6% or between about 2% and about 5%.

[0161] In some embodiments, the LNP contains: (a) Lipid 319 with a mass fraction of between about 35% and about 45%; (b) SOPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2000 with a mass fraction of between about 2% and about 3%; (d) cholesterol with a mass fraction of between about 30% and about 40%; and (e) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the LNP contains: (a) Lipid 319 with a mass fraction of between about 30% and about 40%; (b) SOPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2000 with a mass fraction of between about 2% and about 3%; (d) cholesterol with a mass fraction of between about 30% and about 40%; and (e) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the LNP contains: (a) Lipid 319 with a mass fraction of about 40%; (b) SOPC with a mass fraction of about 19%; (c) DMG-PEG2000 with a mass fraction of about 2.5%; (d) cholesterol with a mass fraction of about 35%; and (e) a deoxyribonucleic acid (DNA) molecule with a mass fraction of about 3.5%. In some embodiments, the LNP further comprise a non-ionizable cationic lipid. In some embodiments, the mass fraction of the non-ionizable cationic lipid is between about 1% and 6% or between about 2% and about 5%.

[0162] In some embodiments, the LNP contains: (a) Lipid A6 with a mass fraction of between about 35% and about 45%; (b) SOPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2000 with a mass fraction of between about 2% and about 3%; (d) cholesterol with a mass fraction of between about 30% and about 40%; and (e) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the LNP contains: (a) Lipid A6 with a mass fraction of between about 30% andabout 40%; (b) SOPC with a mass fraction of between about 15% and about 25%; (c) DMG-PEG2000 with a mass fraction of between about 2% and about 3%; (d) cholesterol with a mass fraction of between about 30% and about 40%; and (e) a deoxyribonucleic acid (DNA) molecule. In some embodiments, the LNP contains: (a) Lipid A6 with a mass fraction of about 40%; (b) SOPC with a mass fraction of about 19%; (c) DMG-PEG2000 with a mass fraction of about 2.5%; (d) cholesterol with a mass fraction of about 35%; and (e) a deoxyribonucleic acid (DNA) molecule with a mass fraction of about 3.5%. In some embodiments, the LNP further comprise a non-ionizable cationic lipid. In some embodiments, the mass fraction of the non-ionizable cationic lipid is between about 1% and 6% or between about 2% and about 5%.B. Method for Producing LNPs

[0163] Provided herein are methods for producing LNPs for delivering a nucleic acid molecule into a T cell and uses thereof, the lipid nanoparticles containing an ionizable lipid, a helper lipid, a polyethylene glycol (PEG) lipid, cholesterol, and a DNA molecule or an RNA molecule. In some embodiments, the RNA and DNA LNPs described herein may be produced by methods described herein.

[0164] In some embodiments, the method includes (1) adding to an organic solvent comprising ethanol (a) an ionizable lipid, wherein the lipid is selected from the group consisting of Lipid 29, Lipid 319, Lipid A6 and Lipid 215; (b) a helper 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 deoxyribonucleic acid (DNA) 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 DNA. In some embodiments, the ionizable lipid is Lipid 29. In some embodiments, the ionizable lipid is Lipid 319. In some embodiments, the ionizable lipid is Lipid A6. In some embodiments, the ionizable lipid is Lipid 215.

[0165] In some embodiments, the method includes (1) adding to an organic solvent comprising ethanol (a) an ionizable lipid, wherein the lipid is selected from the group consisting of Lipid 29, Lipid 319 and Lipid A6; (b) a helper 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 deoxyribonucleic acid (DNA) 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 DNA. In some embodiments, the ionizable lipid is Lipid 29. In some embodiments, the ionizable lipid is Lipid 319. In some embodiments, the ionizable lipid is Lipid A6.

[0166] In some embodiments, the method includes (1) adding to an organic solvent comprising ethanol (a) an ionizable lipid, wherein the lipid is selected from the group consisting of Lipid 29,Lipid 319, Lipid A6 and Lipid 215; (b) a helper 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. In some embodiments, the ionizable lipid is Lipid 29. In some embodiments, the ionizable lipid is Lipid 319. In some embodiments, the ionizable lipid is Lipid A6. In some embodiments, the ionizable lipid is Lipid 215.

[0167] In some embodiments, the method includes (1) adding to an organic solvent comprising ethanol (a) an ionizable lipid, wherein the lipid is selected from the group consisting of Lipid 29, Lipid 319 and Lipid A6; (b) a helper 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. In some embodiments, the ionizable lipid is Lipid 29. In some embodiments, the ionizable lipid is Lipid 319. In some embodiments, the ionizable lipid is Lipid A6.

[0168] In some embodiments, the flow rate of the aqueous phase in the device is between about 8 mL / min and about 10 mL / min. In some embodiments, the flow rate of the aqueous phase in the device is about 8 mL / min. In some embodiments, the flow rate of the aqueous phase in the device is about 9 mL / min. In some embodiments, the flow rate of the aqueous phase in the device is about 10 mL / min. In some embodiments, the flow rate of the organic phase in the device is between about 2 mL / min and about 4 mL / min. In some embodiments, the flow rate of the organic phase in the device is about 2 mL / min. In some embodiments, the flow rate of the organic phase in the device is about 3 mL / min. In some embodiments, the flow rate of the organic phase in the device is about 4 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 9 mL / min, and the flow rate of the organic phase is about 3 mL / min.

[0169] In some embodiments, the aqueous solvent is an acetate 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.

[0170] In some embodiments, the molarity of the acetate 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 mL / min and about 150 mM. Insome embodiments, the molarity of the acetate 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.

[0171] In some embodiments, the molarity of the acetate buffer is about 25 mM, about 112.5 mM, or about 200 mM. In some embodiments, the molarity of the acetate buffer is about 25 mM. In some embodiments, the molarity of the acetate buffer is about 112.5 mM. In some embodiments, the molarity of the acetate buffer is about 200 mM.

[0172] In some embodiments, the method comprises collecting the generated LNP from the device in the acetate buffer. In some embodiments, the method comprises washing the collected LNP 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.

[0173] 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.

[0174] In some embodiments, the method includes storing the generated LNP 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 LNP at about 4 degrees Celsius for at least about an hour. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for at least about 2 hours. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for at least about 4 hours. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for at least about 6 hours. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for at least about 8 hours. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for about 12 hours. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for about 18 hours. In some embodiments, the method includes storing the generated LNP at about 4 degrees Celsius for about 24 hours. In some embodiments, the method includes storing the generated LNP at about 12 degrees Celsius for between about 2 hours and about 12 hours. In some embodiments, the method includes storing the generated LNP at about 12 degrees Celsius for at least about 2 hours.

[0175] 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.C. Exemplary Features of LNPs

[0176] In some embodiments, the size of an LNP provided herein is measured by dynamic light scattering (DLS).

[0177] In some embodiments, the LNP comprises a DNA molecule. In some embodiments, the size of the LNP as measured by DLS (Z-ave) is between about 50 nm and about 200 nm, between about 75 nm and about 175 nm, or between about 100 nm and about 150 nm. In some embodiments, the size of the LNP (Z-ave) is between about 50 nm and about 200 nm. In some embodiments, the size of the LNP (Z-ave) is between about 75 nm and about 175 nm. In some embodiments, the size of the LNP (Z-ave) is between about 100 nm and about 150 nm. In some embodiments, the size of the LNP (Z-ave) is about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, or about 200 nm. In some embodiments, the size of the LNP (Z-ave) is about 50 nm. In some embodiments, the size of the LNP (Z-ave) is about 60 nm. In some embodiments, the size of the LNP (Z-ave) is about 70 nm. In some embodiments, the size of the LNP (Z-ave) is about 80 nm. In some embodiments, the size of the LNP (Z-ave) is about 90 nm. In some embodiments, the size of the LNP (Z-ave) is about 100 nm. In some embodiments, the size of the LNP (Z-ave) is about 110 nm. In some embodiments, the size of the LNP (Z-ave) is about 120 nm. In some embodiments, the size of the LNP (Z-ave) is about 130 nm. In some embodiments, the size of the LNP (Z-ave) is about 140 nm. In some embodiments, the size of the LNP (Z-ave) is about 150 nm. In some embodiments, the size of the LNP (Z-ave) is about 160 nm. In some embodiments, the size of the LNP (Z-ave) is about 170 nm. In some embodiments, the size of the LNP (Z-ave) is about 180 nm. In some embodiments, the size of the LNP (Z-ave) is about 190 nm. In some embodiments, the size of the LNP (Z-ave) is about 200 nm.

[0178] In some embodiments, the LNPs and compositions thereof provided herein are stable at temperatures lower than room temperature. In some embodiments, the size of the LNPs provided herein are stable at temperatures lower than room temperature. In some embodiments, the size of the LNP is assessed by any technique known in the art, such as dynamic light scattering (DLS). In some embodiments, the amount of nucleic acid contained in the LNPs provided herein are stable at temperatures lower than room temperature.

[0179] In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about one day, at least about 3 days, at least about 5 days, or at least about 7 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about 3 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about 5 days. In some embodiments, the size of the LNPdoes not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about 7 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 3 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 5 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 7 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 10 days. In some embodiments, the size of the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 14 days.

[0180] In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for at least about one day, at least about 3 days, at least about 5 days, or at least about 7 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for at least about 3 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for at least about 5 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for at least about 7 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for about 3 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for about 5 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for about 7 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for about 10 days. In some embodiments, the size of the LNP does not change more than about 5% when the LNP is stored at about 4 degrees Celsius for about 14 days.

[0181] In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for at least about one day, at least about 3 days, at least about 5 days, or at least about 7 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for at least about 3 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for at least about 5 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for at least about 7 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for about 3 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for about 5 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for about 7 days. In some embodiments, the size of the LNP does notchange more than about 1% when the LNP is stored at about 4 degrees Celsius for about 10 days. In some embodiments, the size of the LNP does not change more than about 1% when the LNP is stored at about 4 degrees Celsius for about 14 days.

[0182] In some embodiments, the amount of DNA contained in the LNP does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about one day, at least about 3 days, at least about 5 days, or at least about 7 days. In some embodiments, the amount DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about one day. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about 3 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about 5 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for at least about 7 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about one day. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 3 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 5 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 7 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 10 days. In some embodiments, the amount of DNA does not change more than about 10% when the LNP is stored at about 4 degrees Celsius for about 14 days.II. CO-FORMULATED LIPID NANOPARTICLES (CO-LNPS)

[0183] Provided herein are co-formulated lipid nanoparticles (co-LNPs) and compositions containing the same, such as for delivering a nucleic acid molecule into a cell (e.g. a T cell). Coformulated LNPs of the disclosure are generated by fusion (e.g., pH neutralization-mediated fusion) of more than one precursor LNP into a single, co-formulated LNP. Co-formulated LNPs can be generated by fusing three precursor LNPs by methods described herein. Such co-formulated LNPs are also referred to as tri-LNPs. Also provided herein are methods for producing the co-formulated LNPs and compositions and uses thereof, such as in connection with cell therapy.A. DNA and RNA Co-LNPs and Tri-LNPs1. Components

[0184] Provided herein are co-formulated LNPs (co-LNPs) comprising a ribonucleic acid (RNA) molecule and a deoxyribonucleic acid (DNA) molecule. In some cases, the co-LNP can be generated to contain one or more types of RNA molecules and / or one or more types of DNA molecules. In some cases, a co-formulated LNP is generated from one or more RNA LNPs, including any of those asdescribed in Section II.B., and one or more DNA LNPs, including any of those as described in Section I. It is contemplated herein that the provision of a co-LNP containing RNA and DNA may result in higher gene editing efficiency, as compared to the provision of separate RNA LNPs and DNA LNPs. In some cases, it is contemplated that the improved efficiency may be due to reduced competitive uptake, in that the cell is only required to uptake a co-LNP, rather than multiple types of LNPs (e.g., RNA LNP and DNA LNP).

[0185] In some embodiments, the co-LNP comprises a DNA molecule, an RNA molecule, a first ionizable lipid, and a second ionizable lipid. In some embodiments, the co-LNP further comprises a third ionizable lipid. In some embodiments, the DNA molecule is associated with the first ionizable lipid, and the RNA molecule is associated with the second and / or third ionizable lipid.

[0186] Also provided herein are co-LNPs comprising a fusion of a first LNP and a second LNP, wherein: (1) the first LNP comprises a DNA molecule and a first ionizable lipid; and (2) the second LNP comprises an RNA molecule and a second ionizable lipid. In some embodiments, the fusion of the first LNP and the second LNP is performed by methods disclosed herein. In some embodiments, the volumetric ratio of the first LNP to the second LNP is between about 1:3 and about 3:1. In some embodiments, the volumetric ratio of the first LNP to the second LNP is about 1:3. In some embodiments, the volumetric ratio of the first LNP to the second LNP is about 1:2. In some embodiments, the volumetric ratio of the first LNP to the second LNP is about 1:1. In some embodiments, the volumetric ratio of the first LNP to the second LNP is about 2:1. In some embodiments, the volumetric ratio of the first LNP to the second LNP is about 3:1.

[0187] In some embodiments, the first LNP and second LNP, prior to fusion, are precursor LNPs (also referred to herein as acidic LNPs) that are not fully formed. As set forth herein, precursor LNPs are generated in an acidic environment (e.g., at a pH between about 4 and about 5). It will be understood that in an acidic environment, the individual lipids that ultimately comprise the shell of LNP are loosely associated (aggregated). Following pH neutralization, the extent of lipid association increases, hence generating the fully formed fused LNP. Nonetheless, in the precursor LNPs, the extent of association between the ionizable lipid and the nucleic acid molecule would be high owing to the acidic nature of the medium. Without being bound by theory, it is expected that after pH neutralization and fusion of the first and second LNP, the nucleic acid molecule from each of the precursor LNPs remains substantially associated with the ionizable lipid to which it was bound in the individual precursor LNPs. For instance, the DNA molecule would remain substantially associated with the first ionizable lipid and the RNA molecule would remain substantially associated with the second ionizable lipid. Accordingly, the fused co-LNPs would not demonstrate substantial exchange of the nucleic acids from one ionizable lipid to another upon mixing and neutralization.

[0188] In some embodiments, the first ionizable lipid of the first LNP forms an ionic bond with the DNA molecule and the second ionizable lipid of the second LNP forms an ionic bond with the RNA molecule. In some embodiments, following fusion of the first and second precursor LNPs to form the fused co-LNP, the first ionizable lipid remains substantially associated (complexed) with the DNA molecule and the second ionizable lipid remains substantially associated (complexed) with the RNA molecule.

[0189] In some embodiments, following fusion of the first and second precursor LNPs to form the fused co-LNP, the first ionizable lipid remains substantially associated (complexed) with the DNA molecule and the second ionizable lipid remains substantially associated (complexed) with the RNA molecule. For instance, in some embodiments, more than 75% of the first ionizable lipid remains associated with the DNA molecule and more than 75% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, more than 80% of the first ionizable lipid remains associated with the DNA molecule and more than 80% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, more than 85% of the first ionizable lipid remains associated with the DNA molecule and more than 85% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, more than 90% of the first ionizable lipid remains associated with the DNA molecule and more than 90% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, more than 95% of the first ionizable lipid remains associated with the DNA molecule and more than 95% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, more than 99% of the first ionizable lipid remains associated with the DNA molecule and more than 99% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, all of the first ionizable lipid remains associated with the DNA molecule and all of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, more than 75% of the first ionizable lipid remains associated with the DNA molecule and more than 75% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, between about 75% and about 90% of the first ionizable lipid remains associated with the DNA molecule and between about 75% and about 90% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP. In some embodiments, between about 75% and about 99% of the first ionizable lipid remains associated with the DNA molecule and between about 75% and about 99% of the second ionizable lipid remains associated with the RNA molecule in the fused co-LNP.

[0190] In some embodiments, the shell of the fused co-LNP comprises a mixture of lipids from each of the precursor LNPs. In some embodiments, the shell of the fused co-LNP is a hybrid of the lipids that comprise the two precursor LNPs. For instance, in some embodiments where the first LNP(prior to fusion) comprises a mass fraction of about 20% of a first helper lipid and the second LNP (prior to fusion) comprises a mass fraction of about 8% of a second helper lipid, the fused co-LNP comprises a mass fraction of greater than 8% and less than 20% total helper lipid, wherein the shell of the co-LNP comprises the first and the second helper lipid. In some such embodiments, the shell of the co-LNP comprises a mass fraction from about 14% to 16% helper lipid.

[0191] In some embodiments, the fusion further comprises (3) a third precursor LNP comprising an RNA molecule and a third ionizable lipid. Such co-LNPs are also referred to herein as tri-LNPs. In some embodiments, the tri-LNP comprises the DNA molecule, and two RNA molecules (i.e. a first RNA molecule and a second RNA molecule). In some embodiments, the tri-LNP comprises a DNA molecule, a first RNA molecule, a second RNA molecule, a first ionizable lipid, a second ionizable lipid, and a third ionizable lipid. In some embodiments, the DNA molecule is associated with the first ionizable lipid (e.g., as a DNA LNP), the first RNA molecule is associated with the second ionizable lipid (e.g, as a first RNA LNP), and the second RNA molecule is associated with the third ionizable lipid (e.g. as a second RNA LNP).

[0192] In some embodiments, the tri-LNPs comprise a fusion of a first LNP (e.g., DNA LNP), a second LNP (e.g., first RNA LNP), and a third LNP (e.g., second RNA LNP), wherein: (1) the first LNP comprises a DNA molecule and a first ionizable lipid; (2) the second LNP comprises a first RNA molecule and a second ionizable lipid; and (3) the third LNP comprises a second RNA molecule and a third ionizable lipid. In some embodiments, the fusion of the first LNP, the second LNP and the third LNP is performed by methods disclosed herein.

[0193] In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (i.e. RNA:RNA:DNA) in the tri-LNP (following fusion) is about 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:1:11, 1:1:12, 1:1:13, 1:1:14, 1:1:15, 1:1:16, 1:1:17, 1:1:18, 1: 1:19, 1:1:20, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 2:1:11, 2:1:12, 2:1:13, 2:1:14, 2:1:15, 2:1:16, 2:1:17, 2:1:18, 2:1:19, 2:1:20, 3:1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:1:11, 3:1:12, 3:1:13, 3:1:14, 3:1:15, 3:1:16, 3:1:17, 3:1:18, 3:1:19, and 3:1:20. In some embodiments, the first RNA molecule is a gRNA and the second RNA is an mRNA and the mass ratio of the gRNA: mRNA: DNA in the tri-LNP (following fusion) is about 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:1:11, 1:1:12, 1:1:13, 1:1:14, 1:1:15, 1:1:16, 1:1:17, 1:1:18, 1:1:19, 1:1:20, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 2:1:11, 2:1:12, 2:1:13, 2:1:14, 2:1:15, 2:1:16, 2:1:17, 2:1:18, 2:1:19, 2:1:20, 3:1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:1:11, 3:1:12, 3:1:13, 3:1:14, 3:1:15, 3:1:16, 3:1:17, 3:1:18, 3:1:19, and 3:1:20. In some embodiments, the first RNA molecule is a guide RNA (gRNA), such as a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. In some embodiments, the second RNA molecule is a mRNA encoding a recombinant nuclease capable of inducing a DNA break (e.g., a Cas, such as a Cas9 or Casl2a). In some embodiments, the DNA molecule is a ceDNAmolecule encoding a recombinant receptor (e.g., CAR). In some embodiments, the DNA molecule (e.g., ceDNA molecule) is a HDRt DNA template.

[0194] In some embodiments, the tri-LNP composition is composed of a first RNA LNP, a second RNA LNP and a DNA LNP. In some embodiments, the tri-LNP comprises a volumetric ratio of the first RNA LNP, the second RNA LNP and the DNA LNP (i.e. RNA:RNA:DNA) that is about 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:1:11, 1:1:12, 1:1:13, 1:1:14, 1:1:15, 1:1:16, 1:1:17, 1:1:18, 1:1:19, 1:1:20, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2:1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 2:1:11, 2:1:12, 2:1:13, 2:1:14, 2:1:15, 2:1:16, 2:1:17, 2:1:18, 2:1:19, 2:1:20, 3:1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:1:11, 3:1:12, 3:1:13, 3:1:14, 3:1:15, 3:1:16, 3:1:17, 3:1:18, 3:1:19, and 3:1 :20. In some embodiments, the first RNA LNP contains a gRNA and the second RNA LNP contains an mRNA and the tri-LNP comprise a volumetric ratio of the gRNA LNP, mRNA LNP and DNA LNP (i.e. gRNA: mRNA: DNA) that is about 1:1:5, 1:1:6, 1:1:7, 1:1:8, 1:1:9, 1:1:10, 1:1:11, 1:1:12, 1:1:13, 1:1:14, 1:1:15, 1:1:16, 1:1:17, 1:1:18, 1:1:19, 1:1:20, 2:1:1, 2:1:2, 2:1:3, 2:1:4, 2: 1:5, 2:1:6, 2:1:7, 2:1:8, 2:1:9, 2:1:10, 2:1:11, 2:1:12, 2:1:13, 2:1:14, 2:1:15, 2:1:16, 2:1:17, 2:1:18, 2: 1:19, 2:1:20, 3:1:1, 3:1:2, 3:1:3, 3:1:4, 3:1:5, 3:1:6, 3:1:7, 3:1:8, 3:1:9, 3:1:10, 3:1:11, 3:1:12, 3:1:13, 3:1:14, 3:1:15, 3:1:16, 3:1:17, 3:1:18, 3:1:19, and 3:1:20. In some embodiments, the first LNP contains a guide RNA (gRNA), such as a single guide RNA (sgRNA) comprising a crispr RNA (crRNA) and a tracrRNA. In some embodiments, the second RNA LNP contains a mRNA encoding a recombinant nuclease capable of inducing a DNA break (e.g., a Cas, such as a Cas9 or Casl2a). In some embodiments, the DNA LNP contains a DNA molecule that is a ceDNA molecule encoding a recombinant receptor (e.g., CAR). In some embodiments, the DNA (e.g., ceDNA molecule) of the DNA LNP is a HDRt DNA template.

[0195] In some embodiments, the volumetric ratio of the first LNP (e.g., DNA LNP) to the second and third LNPs (e.g., first and second RNA LNPs) is between about 1:3 and about 3:1. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs is about 1:3. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs is about 1:2. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs is about 1:1. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs is about 2:1. In some embodiments, the volumetric ratio of the first LNP to the second and third LNPs is about 3:1.

[0196] In some embodiments, the co-LNP comprises (i) a guide RNA (gRNA) and / or mRNA encoding a recombinant nuclease capable of inducing a DNA break; and (ii) HDR template (HDRt) DNA. In some embodiments, the co-LNP comprises (i) a guide RNA (gRNA) and mRNA encoding a recombinant nuclease capable of inducing a DNA break; and (ii) HDR template (HDRt) DNA. In some embodiments, the co-LNP comprises (i) a guide RNA (gRNA) complexed with a recombinant nuclease capable of inducing a DNA break; and (ii) HDR template (HDRt) DNA. In someembodiments, the gRNA is a single guide RNA (sgRNA) comprising a crispr RNA (crRNA) and a tracrRNA.

[0197] In some embodiments, the HDRt DNA encodes a recombinant receptor (e.g., a CAR). In some embodiments, the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas). In some embodiments, the recombinant nuclease is a zinc finger nuclease (ZFN). In some embodiments, the recombinant nuclease is a transcription activator-like effector nuclease (TALEN). In some embodiments, the recombinant nuclease is a CRISPR-associated nuclease (Cas). In some of any such embodiments, the Cas nuclease is selected from the group consisting of Cas3, Cas9, CaslO, Casl2, Casl2a, and Casl3. In some of any such embodiments, the Cas nuclease is Cas9. In some of any such embodiments, the Cas nuclease is Cas9 or a variant thereof. In some embodiments, the Cas nuclease is an enhanced specificity Cas9 (eSpCas9). In some embodiments, the Cas nuclease is a high fidelity Cas9 (HiFi Cas9). In some of any such embodiments, the Cas9 is from a bacteria selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitides, Campylobacter jejuni, and Streptococcus thermophilis. In some of any such embodiments, the Cas9 is from Streptococcus pyogenes. In some of any such embodiments, the Cas9 or a variant thereof is from Streptococcus pyogenes. In some embodiments, the Cas is Casl2a.

[0198] In some embodiments, the co-LNP comprises (i) mRNA encoding a transposase; and (ii) a transposon. In some embodiments, the transposase is selected from the group consisting of: Sleeping Beauty, piggyBac, TcBuster, Frog Prince, Tol2, Tcl / mariner, or a derivative thereof having transposase activity. In some embodiments, the transposase is Sleeping Beauty, PiggyBac, or TcBuster. In some embodiments, the transposase is Sleeping Beauty. In some embodiments, the transposase is PiggyBac. In some embodiments, the transposase is TcBuster. In some embodiments, the transposon comprises a transgene encoding for a recombinant receptor (e.g., a CAR).

[0199] In some embodiments, the co-ENP comprises a first and second ionizable lipid. In some embodiments, the co-ENP further comprises a third ionizable lipid. In some embodiments, the co- ENP is generated from two LNPs, such that the co-LNP comprises a first and a second ionizable lipid. In some embodiments, the co-LNP is generated from three LNPs, such that the co-LNP comprises a first, second, and third ionizable lipid.

[0200] In some embodiments, the first, second, and / or third ionizable lipid is positively charged (e.g. a cationic lipid). In some embodiments, the first, second, and / or third ionizable lipid is a cationic lipid, including but not limited to those described in US Patent No. 9,593,077; US Patent No. 9,365,610; US Patent No. 9,670,152; and US Patent No. 9,458,090. In some embodiments, the first, second and / or third ionizable lipid is a cationic lipid, including but not limited to those described in Published PCT application W02013149140. In some embodiments, the first, second, and / or thirdionizable lipid is a cationic lipid, including but not limited to those described in published US Patent application US2019084965; US2019106379. In some embodiments, the first, second, and / or third ionizable lipid is a cationic lipid, including but not limited to those described in Published EP application EP2830595.

[0201] In some embodiments, the first, second, and / or third ionizable lipid is Lipid 29. In some embodiments, the first, second, and / or third ionizable lipid is Lipid 319. In some embodiments, the first, second, and / or third ionizable lipid is Lipid A6.

[0202] In some embodiments, the first, second, and / or third ionizable lipid comprises a diketopiperazine ring core. In some embodiments, the ionizable lipid is any as described in Fenton et al., Angew. Chem. Int. Ed. (2018) 57:13582 -86. In some embodiments, the ionizable lipid has an unsaturated linoleic tail.

[0203] In some embodiments, the first, second, and / or third ionizable lipid is OF-C4-Deg-Lin (also referred to herein as Lipid C4 or C4), which has the structure depicted below:

[0204] In some embodiments, the first, second, and / or third ionizable lipid is cKK-E12, which has the structure depicted below:

[0205] In some embodiments, the first, second, and / or third ionizable lipid is DLin-MC3-DMA, which has the structure depicted below:

[0206] In some embodiments, the first, second, and / or third ionizable lipid is DLin-KC2-DMA (also referred to as Lipid KC2 or KC2), which has the structure depicted below:

[0207] In some embodiments, the first, second, and / or third ionizable lipid is DLin-MC3-DMA, which has the structure depicted below:

[0208] In some embodiments, the first, second, and / or third ionizable lipid is Lipid 215, which has the structure depicted below:LIPID 215

[0209] In some embodiments, the first ionizable lipid and the second ionizable lipid are the same. In some embodiments, the first ionizable lipid and the second ionizable lipid are different. In some embodiments of tri-LNPs, the second ionizable lipid and the third ionizable lipid are the same. In some embodiments of tri-LNPs, the second ionizable lipid and the third ionizable lipid are different.In some embodiments of tri-LNPs, the first, second, and third ionizable lipids are the same. In some embodiments of tri-LNPs, each of the first, second, and third ionizable lipids are different.

[0210] In some embodiments, the co-LNP is generated from the fusion of a precursor RNA LNP and a precursor DNA LNP, such that the co-LNP comprises a first and second ionizable lipid. In someembodiments, prior to fusion with the precursor DNA LNP, the precursor RNA LNP comprised Lipid 29. In some embodiments, prior to fusion with the precursor DNA LNP, the precursor RNA LNP comprised Lipid 319. In some embodiments, prior to fusion with the precursor RNA LNP, the precursor DNA precursor LNP comprised Lipid 29. In some embodiments, prior to fusion with the precursor RNA LNP, the precursor DNA comprised Lipid 319. In some embodiments, prior to fusion with the precursor RNA LNP, the precursor DNA comprised Lipid A6. In some embodiments, prior to fusion with the precursor RNA LNP, the precursor DNA precursor LNP comprised Lipid A6. In any of the foregoing embodiments, one of the precursor LNPs, prior to fusion, comprised an ionizable lipid comprising a diketopiperazine ring core such as OF-C4-Deg-Lin or cKK-E12.

[0211] In some embodiments, at least one of the first ionizable lipid or second ionizable lipid of the co-LNP is Lipid 29. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and at least one RNA molecule by methods described herein. In other such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 29 and at least one RNA molecule by methods described herein. In other embodiments, both the first ionizable lipid and second ionizable lipid of the co-LNP is Lipid 29. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising Lipid 29 and at least one RNA molecule. Following fusion of the precursor LNPs, the resultant co-LNP will comprise a single ionizable lipid (Lipid 29).

[0212] In some embodiments, at least one of the first ionizable lipid or second ionizable lipid of the co-LNP is Lipid 319. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and at least one RNA molecule by methods described herein. In other such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and at least one RNA molecule by methods described herein. In other embodiments, both the first ionizable lipid and second ionizable lipid of the co-LNP are Lipid 319. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising Lipid 319 and at least one RNA molecule. Following fusion of the precursor LNPs, the resultant co-LNP will comprise a single ionizable lipid (Lipid 319).

[0213] In some embodiments, at least one of the first ionizable lipid or second ionizable lipid of the co-LNP is Lipid A6. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and at least one RNA molecule by methods described herein. In other such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipidand a DNA molecule with a second precursor LNP comprising Lipid 319 and at least one RNA molecule by methods described herein. In other embodiments, both the first ionizable lipid and second ionizable lipid of the co-LNP are Lipid A6. In some such embodiments, the co-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising Lipid A6 and at least one RNA molecule. Following fusion of the precursor LNPs, the resultant co-LNP will comprise a single ionizable lipid (Lipid A6).

[0214] In some embodiments, the co-LNP formed by fusing two precursor LNPs comprises two different ionizable lipids. For instance, in some embodiments, the co-LNP comprises both Lipid 29 and Lipid 319. In some embodiments, the co-LNP comprises Lipid 29 or Lipid 319 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the co-LNP comprises Lipid 29 or Lipid 319 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the co-LNP comprises Lipid 29 or Lipid 319 and the ionizable lipid OF-C4-Deg-Lin, or an analog thereof. In some embodiments, the co-LNP comprises Lipid 29 or Lipid 319 and the ionizable lipid DLin-KC2-DMA, or an analog thereof.

[0215] In some embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 29, Lipid 319 or Lipid A6 with a second precursor LNP comprising an RNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail. In other embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail with a second precursor LNP comprising an RNA molecule and Lipid 29, Lipid 319 or Lipid A6.

[0216] In some embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 29 with a second precursor LNP comprising an RNA molecule and OF- C4-Deg-Lin.

[0217] In some embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 319 with a second precursor LNP comprising an RNA molecule and OF- C4-Deg-Lin.

[0218] In some embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid A6 with a second precursor LNP comprising an RNA molecule and OF- C4-Deg-Lin.

[0219] In some embodiments, the co-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and first ionizable lipid with a second precursor LNP comprising an RNA molecule and a second ionizable lipid, wherein:(a) the first ionizable lipid is Lipid 29 and the second ionizable lipid is Lipid 319;(b) the first ionizable lipid is Lipid 29 and the second ionizable lipid is Lipid A6;(c) the first ionizable lipid is Lipid 319 and the second ionizable lipid is Lipid 29;(d) the first ionizable lipid is Lipid 319 and the second ionizable lipid is Lipid A6;(e) the first ionizable lipid is Lipid A6 and the second ionizable lipid is Lipid L29; or(f) the first ionizable lipid is Lipid A6 and the second ionizable lipid is Lipid 319.

[0220] In some embodiments, the co-LNP is generated from the fusion of a DNA LNP and a RNA LNP, such that the co-LNP comprises a first helper lipid and a second helper lipid.

[0221] In some embodiments, each of the first and second helper lipids is independently selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and 1 ,2-dioleoyl- sn-glycero-sn-3-phosphatidylcholine (DOPC). In some embodiments, the first and / or second helper lipid is DOPE. In some embodiments, the first and / or second helper lipid has the structure of

[0222] In some embodiments, the first and / or second helper lipid is DSPC. In some embodiments, the first and / or second helper lipid has the structure of

[0223] In some embodiments, the first and / or second helper lipid is SOPC. In some embodiments, the first and / or second helper lipid has the structure of

[0224] In some embodiments, the first and / or second helper lipid is DOPC. In some embodiments, the first and / or second helper lipid has the structure of

[0225] In some embodiments, the co-LNP is generated from the fusion of two separate RNA LNPs and a DNA LNP, such that the co-LNP comprises a first, second, and third helper lipid.

[0226] In some embodiments, each of the first, second, and third helper lipids is independently selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1 ,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and l,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC). In some embodiments, the first and / or second helper lipid is DOPE. In some embodiments, the first and / or second helper lipid has the structure of

[0227] In some embodiments, the first, second, and / or third helper lipid is DSPC. In some embodiments, the first, second, and / or third helper lipid has the structure of

[0228] In some embodiments, the first, second, and / or third helper lipid is SOPC. In some embodiments, the first, second, and / or third helper lipid has the structure of

[0229] In some embodiments, the first, second, and / or third helper lipid is DOPC. In some embodiments, the first, second, and / or third helper lipid has the structure of

[0230] In some embodiments, the co-LNP is generated from the fusion of a RNA LNP and a DNA LNP, such that the co-LNP comprises a first and second helper lipid. In some embodiments, prior to fusion with the DNA LNP, the RNA LNP comprised DSPC. In some embodiments, prior to fusion with the RNA LNP, the DNA LNP comprised SOPC.

[0231] In some embodiments, the co-LNP is generated from the fusion of two separate RNA LNPs and a DNA LNP, such that the co-LNP comprises a first, second, and third ionizable lipid. In some embodiments, prior to fusion with the DNA LNP, one of the RNA LNPs comprised DSPC. In some embodiments, prior to fusion with the DNA LNP, the other of the RNA LNPs comprised DSPC. In some embodiments, prior to fusion with the RNA LNPs, the DNA LNP comprised SOPC.

[0232] In some embodiments, the co-LNP comprises a non-ionizable cationic lipid. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP (or tri-LNP) is between about 0.5% and about 7%. In some embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP (or tri-LNP) is between about 0.5% and about 5%. In other embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP (or tri-LNP) is between about 1% and about 6%. In other embodiments, the mass fraction of the non-ionizable cationic lipid in the co-LNP (or tri-LNP) is between about 2% and about 5%.

[0233] In some embodiments, the co-LNP comprises a PEG-conjugated lipid. In some embodiments, the polyethylene glycol (PEG)-conjugated lipid is DMG-PEG2000. In some embodiments, the PEG-conjugated lipid has the structure of

[0234] In some embodiments, the co-LNP comprises cholesterol. In some embodiments, cholesterol has the structure of

[0235] In some embodiments, the size of the co-LNP as measured by DLS (Z-ave) is between about 50 nm and about 150 nm, or between about 75 nm and about 125 nm. In some embodiments, the size of the co-LNP (Z-ave) is between about 50 nm and about 150 nm. In some embodiments, the size of the co-LNP (Z-ave) is between about 75 nm and about 125 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 50 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 50 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 55 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 60 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 65 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 70 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 75 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 80 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 85 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 90 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 95 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 100 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 105 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 110 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 115 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 120 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 125 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 130 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 135 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 140 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 145 nm. In some embodiments, the size of the co-LNP (Z-ave) is about 150 nm.

[0236] In some embodiments, the co-LNPs formed by fusion methods described herein show a fluorescence energy transfer (FRET). In particular embodiments, FRET is demonstrated by attaching individual fluorescent dyes, a donor and acceptor, to each of the precursor LNPs prior to mixing (under acidic conditions) and neutralization. Fluorescence emission from the acceptor dye indicates the level of fusion. In some embodiments, the FRET emission signal of the fused co-ENP is greater that the fluorescence emission signal of a mixture of two individual ENPs that are not fused together.

[0237] In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.3. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.35. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.38. In some embodiments, the normalized FRET signal immediately following neutralization is greater than 0.4. In some embodiments, the normalized FRET signal immediately following neutralization is between about 0.35 and 0.42. In other embodiments, the normalized FRET signal immediately following neutralization is between about 0.38 and 0.42. In any of the foregoing embodiments, the normalized FRET signal may be calculated by the method described in Example 16.

[0238] In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) in the co-LNP is between about 3:1 and about 1:3. In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) in the co-LNP is between about 2: 1 and about 1 :2. In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) is about 1:1. In some embodiments, the mass ratio between the DNA molecule and RNA molecule(s) is about 2:1.

[0239] In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:1. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:2. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:3. In some embodiments, the mass ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:4.2. Methods for Producing DNA and RNA Co-LNPs

[0240] Provided herein methods for producing a co-formulated LNP (co-LNP). In some embodiments, the co-LNP comprises an RNA molecule and a DNA molecule.

[0241] In some embodiments, the co-LNP is generated from the fusion of a first precursor LNP comprising an RNA molecule and a second precursor LNP comprising a DNA molecule. In someembodiments, the first RNA molecule comprises a guide RNA (gRNA). In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. In some embodiments, the gRNA is complexed with a recombinant nuclease capable of inducing a DNA break. In some embodiments, the co-LNP is generated from the fusion of a first LNP comprising an RNA molecule and a second LNP comprising a DNA molecule. In some embodiments, the RNA molecule comprises a gRNA and mRNA encoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the DNA molecule comprises homology-directed repair template (HDRt) DNA.

[0242] In some embodiments, the co-LNP is generated from the fusion of a first precursor LNP comprising an RNA molecule and a second precursor LNP comprising a DNA molecule. In some embodiments, the RNA molecule comprises mRNA encoding a transposase. In some embodiments, the DNA molecule comprises a transposon encoding a recombinant receptor (e.g., a CAR), n some embodiments, the CAR is a single antigen directed CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the DNA (e.g., ceDNA) molecule encoding the bispecific CAR is at least 5.5 kilobases, at least 6 kilobases, at least 7 kilobases, or at least 8 kilobases. In some embodiments, the bispecific CAR is between about 5.5 kilobases and about 8 kilobases. In some embodiments, the bispecific CAR is between about 6 kilobases and about 8 kilobases. In some embodiments, the bispecific CAR is about 8 kilobases.

[0243] In some embodiments, the co-LNP is generated from the fusion of a first precursor LNP comprising an RNA molecule, a second LNP comprising an RNA molecule, and a third LNP comprising a DNA molecule. In some embodiments, the one of the first and second RNA LNPs comprises mRNA encoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the other of the first and second RNA LNPs comprises gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. In some embodiments, the DNA LNP comprises HDRt DNA.

[0244] In some embodiments, a co-LNP is generated from the fusion of any of the precursor RNA LNPs described herein with any of the DNA LNPs described herein.

[0245] Also provided herein is a co-LNP produced by any of the methods provided herein. a. Co-formulation

[0246] Provided herein are methods of producing co-LNPs comprising fusing a precursor RNA LNP comprising an RNA molecule with a precursor DNA LNP comprising a DNA molecule to produce a co-LNP comprising RNA and DNA. In some embodiments, the method comprises mixing a composition comprising a precursor RNA LNP with a composition comprising a DNA LNP, wherein the RNA LNP composition and the DNA LNP composition are both acidic. In some embodiments, the pH of the precursor RNA LNP composition is about 4.0. In some embodiments, the pH of the DNALNP composition is about 4.0. In some embodiments, the RNA LNPs are mixed with the DNA LNPs at a volume ratio of about 1:3 (RNA LNPs:DNA LNPs). In some embodiments, following mixing of the precursor RNA and DNA LNPs, at least about six parts by volume of an isotonic buffer are added to the mixture of RNA and DNA LNPs. In some embodiments, about six parts by volume of an isotonic buffer are added to the mixture of precursor RNA and DNA LNPs. In some embodiments, about seven parts by volume of an isotonic buffer are added to the mixture of precursor RNA and DNA LNPs. In some embodiments, the isotonic buffer is about pH 7.4. In some embodiments, the isotonic buffer is phosphate buffered saline. Thus, in some embodiments, the method comprises: (1) mixing the precursor RNA LNP composition and DNA LNP composition at a volume ratio of about 1:3 (RNA LNPs:DNA LNPs); and (2) adding at least about six parts by volume of an isotonic buffer (e.g. PBS) to the mixture of precursor RNA and DNA LNPs, thereby generating a co-LNP comprising the RNA and DNA molecules. It is contemplated herein that neutralization of the mixed composition by the isotonic buffer mediates fusion of the RNA LNP and the DNA LNP.

[0247] In some embodiments, the precursor RNA LNP comprises a gRNA complexed with a recombinant nuclease capable of inducing a DNA break. In some embodiments, the precursor RNA LNP comprises mRNA encoding a recombinant nuclease capable of inducing a DNA break and a gRNA. In some embodiments, the precursor DNA LNP comprises HDRt DNA. In some embodiments, the precursor RNA LNP comprises a gRNA complexed with a recombinant nuclease capable of inducing a DNA break, and the DNA LNP comprises HDRt DNA. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. Thus, in some embodiments, the co-LNP comprises a gRNA complexed with a recombinant nuclease capable of inducing a DNA break and HDRt DNA. In some embodiments, the RNA LNP comprises mRNA encoding a recombinant nuclease capable of inducing a DNA break and a gRNA, and the DNA LNP comprises HDRt DNA. Thus, in some embodiments, the co-LNP comprises a gRNA, mRNA encoding for a recombinant nuclease capable of inducing a DNA break, and HDRt DNA.

[0248] In some embodiments, the RNA LNP comprises mRNA encoding a transposase. In some embodiments, the DNA LNP comprises a transposon encoding a recombinant receptor (e.g., a CAR). In some embodiments, the RNA LNP comprises mRNA encoding a transposase, and the DNA LNP comprises a transposon encoding a recombinant receptor (e.g., a CAR). Thus, in some embodiments, the co-LNP comprises mRNA encoding a transposase and a transposon, e.g. encoding a recombinant receptor. b. Double Sequential Fusion

[0249] Provided herein are methods of producing co-LNPs by a double sequential fusion. In some embodiments, the methods comprise fusing a first precursor RNA LNP comprising a first precursor RNA molecule with a second RNA LNP comprising a second RNA molecule to generate aRNA co-LNP, which is then fused with z precursor DNA LNP to produce a co-LNP comprising RNA and DNA. In some embodiments, the method comprises mixing a first composition comprising a first precursor RNA LNP with a second composition comprising a second precursor RNA LNP, wherein the first RNA LNP composition and the second RNA LNP composition are both acidic. In some embodiments, the RNA LNPs compositions are acidic by virtue of comprising an acetate buffer. In some embodiments, the pH of the first and second RNA LNP compositions is about 4.0. In some embodiments, the first and second precursor RNA LNP compositions are mixed together at a volume ratio of about 1:1 (first RNA LNPs:second RNA LNPs). In some embodiments, following mixing of the first and second precursor RNA LNPs, about one part by volume of an isotonic buffer is added to the mixture of the first and second RNA LNPs. In some embodiments, the isotonic buffer is about pH 7.4. In some embodiments, the isotonic buffer is phosphate buffered saline (PBS). In some embodiments, this process generates a RNA co-LNP comprising the first and second RNA molecules in a single RNA co-LNP. In some embodiments, the RNA co-LNP is subsequently subjected to buffer exchange, such that the composition comprising the RNA co-LNP is about pH 4.0. In some embodiments, an acetate buffer is exchanged for the isotonic buffer. In some embodiments, the RNA LNPs are mixed with a composition of DNA LNPs having a pH of about 4.0 at a volume ratio of about 1:3 (RNA LNPs:DNA LNPs). In some embodiments, the DNA LNP composition is acidic by virtue of comprising an acetate buffer. In some embodiments, following mixing of the RNA and DNA LNPs, at least about six parts by volume of an isotonic buffer (e.g. PBS) are added to the mixture of RNA and DNA LNPs. In some embodiments, about six parts by volume of an isotonic buffer are added to the mixture of RNA and DNA LNPs. In some embodiments, about seven parts by volume of an isotonic buffer are added to the mixture of RNA and DNA LNPs. In some embodiments, the isotonic buffer is about pH 7.4. Thus, in some embodiments, the method comprises: (1) mixing the first precursor RNA LNP composition and the second precursor RNA LNP composition at a volume ratio of about 1:1; (2) adding about one part by volume of an isotonic buffer (e.g. PBS), thereby generating a RNA co-LNP; (3) exchanging the buffer of the RNA co-LNP composition for a buffer of about pH 4.0; (4) mixing the RNA co-LNP composition with the DNA LNP composition at a volume ratio of about 1:3 (RNA LNPs:DNA LNPs); and (5) adding at least about six parts by volume of an isotonic buffer (e.g. PBS) to the mixture of RNA and DNA LNPs, thereby generating a co-LNP comprising the RNA and DNA molecules. It is contemplated herein that neutralization of the mixed composition by the isotonic buffer mediates fusion of the precursor RNA LNPs, or of the precursor RNA LNP and the precursor DNA LNP.

[0250] In some embodiments, one of the RNA LNPs comprises a gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. In some embodiments, the other of the RNA LNPs comprises mRNA encoding a recombinant nuclease capable of inducing a DNA break. In some embodiments, the DNA LNP comprises HDRt DNA.Thus, in some embodiments, the co-LNP generated by the double sequential fusion method comprises a gRNA, mRNA encoding a recombinant nuclease capable of inducing a DNA break, and HDRt DNA. c. Tri-fusion

[0251] Provided herein are methods of producing co-LNPs comprising a tri-fusion LNP or “tri- LNP”. In some embodiments, the methods comprise fusing a first precursor RNA LNP comprising a first RNA molecule with a second precursor RNA LNP comprising a second RNA molecule and a DNA LNP comprising a DNA molecule to generate a tri-LNP containing RNA and DNA molecules. In some embodiments, the method comprises mixing a first composition comprising a first precursor RNA LNP with a second composition comprising a second precursor RNA LNP and a third composition comprising a precursor DNA molecule, wherein each of the compositions is acidic. In some embodiments, each of the composition is acidic by virtue of comprising an acetate buffer. In some embodiments, the pH of each composition is about 4.0. In some embodiments, the first and second precursor RNA LNP compositions and the precursor DNA LNP composition are mixed together at a volume ratio of about 1:2 (RNA LNPs:DNA LNPs). In some embodiments, following mixing of the first and second precursor RNA LNPs and the precursor DNA LNPs, at least about six parts by volume of an isotonic buffer are added to the mixture of the first and second precursor RNA LNPs and DNA LNPs. In some embodiments, the isotonic buffer is about pH 7.4. In some embodiments, the isotonic buffer is phosphate buffered saline (PBS). In some embodiments, this process generates a tri-LNP comprising the first and second RNA molecules and the DNA molecule in a single tri-LNP. Thus, in some embodiments, the method comprises: (1) mixing the first RNA LNP composition, the second RNA LNP composition, and the DNA LNP composition at a volume ratio of about 1:2 (RNA LNPs:DNA LNPs); (2) adding between about six and seven parts by volume of an isotonic buffer (e.g. PBS), thereby generating a co-LNP comprising the RNA and DNA molecules. It is contemplated herein that neutralization of the mixed composition by the isotonic buffer mediates fusion of the RNA LNPs and the DNA LNP.

[0252] In some embodiments, the molar ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:1. In some embodiments, the molar ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:2. In some embodiments, the molar ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:3. In some embodiments, the molar ratio between the first RNA molecule, second RNA molecule and DNA molecule (e.g., ceDNA molecule) in the tri-LNP (following fusion) is about 1:1:4.

[0253] In some embodiments, the first LNP, second LNP and third LNP, prior to fusion, are precursor LNPs that are not fully formed. In some embodiments, the precursor LNPs are generated in an acidic environment (e.g., at a pH between about 4 and about 5). In some embodiments, the first ionizable lipid of the first LNP forms an ionic bond with the DNA molecule and the second ionizable lipid of the second LNP forms an ionic bond with the first RNA molecule and the third ionizable lipid of the third LNP forms an ionic bond with the second RNA molecule. In some embodiments, following fusion of the first, second and third precursor LNPs to form the fused tri-LNP, the first ionizable lipid remains substantially associated (complexed) with the DNA molecule, the second ionizable lipid remains substantially associated (complexed) with the first RNA molecule, and the third ionizable lipid remains substantially associated (complexed) with the second RNA molecule.For instance, in some embodiments, more than 75% of the first ionizable lipid remains associated with the DNA molecule, more than 75% of the second ionizable lipid remains associated with the first RNA molecule and more than 75% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, more than 80% of the first ionizable lipid remains associated with the DNA molecule, more than 80% of the second ionizable lipid remains associated with the first RNA molecule and more than 80% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, more than 85% of the first ionizable lipid remains associated with the DNA molecule, more than 85% of the second ionizable lipid remains associated with the first RNA molecule and more than 85% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, more than 90% of the first ionizable lipid remains associated with the DNA molecule, more than 90% of the second ionizable lipid remains associated with the first RNA molecule and more than 90% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, more than 95% of the first ionizable lipid remains associated with the DNA molecule, more than 95% of the second ionizable lipid remains associated with the first RNA molecule and more than 95% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, more than 99% of the first ionizable lipid remains associated with the DNA molecule, more than 99% of the second ionizable lipid remains associated with the first RNA molecule and more than 99% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, between about 75% and about 99% of the first ionizable lipid remains associated with the DNA molecule, between about 75% and about 99% of the second ionizable lipid remains associated with the first RNA molecule, and between about 75% and about 99% of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP. In some embodiments, all of the first ionizable lipid remains associated with the DNA molecule, all of the second ionizable lipid remains associated with the first RNA molecule and all of the third ionizable lipid remains associated with the second RNA molecule in the fused tri-LNP.

[0254] In some embodiments, the shell of the fused tri-LNP comprises a mixture of lipids from each of the three precursor LNPs. In some embodiments, the shell of the fused tri-LNP is a hybrid of the lipids that comprise the three precursor LNPs.

[0255] In some embodiments, at least one of the first ionizable lipid, second ionizable lipid or third ionizable lipid of the tri-LNP is Lipid 29. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 29 and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 29 and a first RNA molecule and a third precursor LNP comprising Lipid 29 and a second RNA molecule by methods described herein.

[0256] In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 29 and a DNA molecule with a second precursor LNP comprising Lipid 29 and a first RNA molecule and a third precursor LNP comprising Lipid 29 and a second RNA molecule by methods described herein. In these embodiments, the tri-LNP following fusion of the precursor LNPs include one ionizable lipid, wherein one of the ionizable lipids is Lipid 29.

[0257] In some embodiments, at least one of the first ionizable lipid, second ionizable lipid or third ionizable lipid of the tri-LNP is Lipid 319. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising Lipid 319 and a second RNA molecule by methods described herein.

[0258] In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid 319 and a DNA molecule with a second precursor LNP comprising Lipid 319 and a first RNA molecule and a third precursor LNP comprising Lipid 319 and a second RNA molecule bymethods described herein. In these embodiments, the tri-LNP following fusion of the precursor LNPs include one ionizable lipid, wherein one of the ionizable lipids is Lipid 319.

[0259] In some embodiments, at least one of the first ionizable lipid, second ionizable lipid or third ionizable lipid of the tri-LNP is Lipid A6. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising a second ionizable lipid and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid A6 and a first RNA molecule and a third precursor LNP comprising a third ionizable lipid and a second RNA molecule by methods described herein. In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a first ionizable lipid and a DNA molecule with a second precursor LNP comprising Lipid A6 and a first RNA molecule and a third precursor LNP comprising Lipid A6 and a second RNA molecule by methods described herein.

[0260] In some such embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising Lipid A6 and a DNA molecule with a second precursor LNP comprising Lipid A6 and a first RNA molecule and a third precursor LNP comprising Lipid A6 and a second RNA molecule by methods described herein. In these embodiments, the tri-LNP following fusion of the precursor LNPs include one ionizable lipid, wherein one of the ionizable lipids is Lipid A6.

[0261] In some embodiments, the tri-LNP formed by fusing three precursor LNPs comprises at least two different ionizable lipids. For instance, in some embodiments, the tri-LNP comprises both Lipid 29 and Lipid 319. In some embodiments, the co-LNP comprises Lipid 29 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the co-LNP comprises Lipid 319 and an ionizable lipid comprising a diketopiperazine ring core. In some embodiments, the tri-LNP comprises Lipid 29 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the tri-LNP comprises Lipid 319 and an ionizable lipid comprising an unsaturated linoleil tail. In some embodiments, the tri-LNP comprises Lipid 29 or Lipid 319 and the ionizable lipid OF-C4-Deg-Lin, or an analog thereof. In some embodiments, the tri-LNP comprises Lipid 29 or Lipid 319 and the ionizable lipid DLin-KC2-DMA, or an analog thereof.

[0262] In some embodiments, the tri-LNP formed by fusing three precursor LNPs comprises three different ionizable lipids. In some such embodiments, at least one of the ionizable lipids is Lipid 29. In other embodiments, at least one of the ionizable lipids is Lipid 319.

[0263] In some embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 29 or Lipid 319 with a second precursor LNP comprising a first RNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-El 2) or an unsaturated linoleil tail and a third precursor LNP comprising a second RNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4-Deg-Lin or cKK-E12) or an unsaturated linoleil tail. In other embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and an ionizable lipid comprising a diketopiperazine core (e.g., OF-C4- Deg-Lin or cKK-E12) or an unsaturated linoleil tail with a second precursor LNP comprising a first RNA molecule and Lipid 29 or Lipid 319 and a third precursor LNP comprising a second RNA molecule and Lipid 29 or Lipid 319.

[0264] In some embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 29 with a second precursor LNP comprising a first RNA molecule and OF-C4-Deg-Lin, and a third precursor LNP comprising a second RNA molecule and OF-C4-Deg-Lin.

[0265] In some embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid 319 with a second precursor LNP comprising a first RNA molecule and OF-C4-Deg-Lin, and a third precursor LNP comprising a second RNA molecule and OF-C4-Deg-Lin.

[0266] In some embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and Lipid A6 with a second precursor LNP comprising a first RNA molecule and OF-C4-Deg-Lin, and a third precursor LNP comprising a second RNA molecule and OF-C4-Deg-Lin.

[0267] In some embodiments, the tri-LNP is formed by fusing a first precursor LNP comprising a DNA molecule and a first ionizable lipid with a second precursor LNP comprising a first RNA molecule and a second ionizable lipid, and a third precursor LNP comprising a second RNA molecule and a third ionizable lipid, wherein:(a) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 319;(b) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid A6;(c) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 29;(d) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 319;(e) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid A6;(f) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 29;(g) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 319;(h) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid A6;(i) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 29;(j) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid A6;(k) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 29;(l) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 319;(m) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid A6;(n) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 29;(o) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 319;(p) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid A6;(q) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 29;(r) the first ionizable lipid is Lipid A6 , the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid A6;(s) the first ionizable lipid is Lipid A6 , the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 319;(t) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 29;(u) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 319;(v) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid A6;(w) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 29; or(x) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 319.

[0268] In some embodiments, the mass fraction of the ionizable lipids in the tri-LNP is between about 40% and about 60%. In some embodiments, the mass fraction of the ionizable lipids in the tri- LNP is between about 40% and about 50%. In some embodiments, the mass fraction of the ionizable lipids in the tri-LNP is between about 45% and about 55%. In some embodiments, the mass fraction of the ionizable lipids in the tri-LNP is between about 45% and about 50%. In any of the foregoing embodiments, the mass fraction of the helper lipid is between about 15% and 25%, 15% and 23%, or 15% and 20% (e.g., 15%, 16%, 17%, 18%, 19% or 20%).

[0269] In some embodiments, one of the precursor RNA LNPs comprises a gRNA. In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) and a tracrRNA. In some embodiments, the other of the precursor RNA LNPs comprises mRNA encoding a recombinant nuclease capable of inducing a DNA break, such as an mRNA encoding Cas9 or Casl2a. In some embodiments, the DNA LNP comprises HDRt DNA. Thus, in some embodiments, a tri-LNP generated by the trifusion method comprises a gRNA, mRNA encoding a recombinant nuclease capable of inducing a DNA break, and HDRt DNA. In some embodiments, the molar ratio between the sgRNA, the mRNA and the HDRt DNA is 1:1:1. In some embodiments, the molar ratio between the sgRNA, the mRNA and the HDRt DNA is 1:1:2. In some embodiments, the molar ratio between the sgRNA, the mRNA and the HDRt DNA is 1:1:4.B. RNA Co-LNPs1. Components

[0270] Provided herein are co-formulated LNPs (co-LNPs) comprising a ribonucleic acid (RNA) molecule (also referred to as RNA co-LNPs or RNA-containing co-LNPs). In some embodiments, the RNA LNP comprises an ionizable lipid; a helper lipid; a polyethylene glycol (PEG)-conjugated lipid; cholesterol; and an RNA molecule. In some embodiments, the RNA co-LNP comprises a first RNA molecule and a second RNA molecule. In some embodiments, the RNA co-LNP comprises a first ionizable lipid and a second ionizable lipid.

[0271] In some embodiments, the RNA co-LNP comprises (1) a first RNA molecule and a second RNA molecule; and (2) a first ionizable lipid and a second ionizable lipid. In some embodiments, one of the first and second RNA molecules encodes a recombinant nuclease capable of inducing a DNA break, and the other of the first and second RNA molecules is a guide RNA (gRNA).In some embodiments, the gRNA is a single guide RNA (sgRNA) comprising a CRISPRRNA (crRNA) and a tracrRNA.

[0272] In some embodiments, the RNA co-LNP comprises a fusion of a first precursor LNP and a second precursor LNP, wherein: (1) the first precursor LNP comprises: an RNA molecule and a first ionizable lipid; and (2) the second precursor LNP comprises an RNA molecule and a second ionizable lipid. In some embodiments, one of the first and second RNA molecules encodes a recombinant nuclease capable of inducing a DNA break, and the other of the first and second RNA molecules is a guide RNA (gRNA). In some embodiments, the RNA co-LNP comprises a volumetric ratio of the first LNP to the second LNP that is about 1:1.

[0273] In some embodiments, the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas). In some embodiments, the recombinant nuclease is a zinc finger nuclease (ZFN). In some embodiments, the recombinant nuclease is a transcription activator-like effector nuclease (TALEN). In some embodiments, the recombinant nuclease is a CRISPR-associated nuclease (Cas). In some of any such embodiments, the Cas nuclease is selected from the group consisting of Cas3, Cas9, CaslO, Casl2, Casl2a, and Casl3. In some of any such embodiments, the Cas nuclease is Cas9. In some of any such embodiments, the Cas nuclease is Cas9 or a variant thereof. In some embodiments, the Cas nuclease is an enhanced specificity Cas9 (eSpCas9). In some embodiments, the Cas nuclease is a high fidelity Cas9 (HiFi Cas9). In some of any such embodiments, the Cas9 is from a bacteria selected from the group consisting of Streptococcus pyogenes, Staphylococcus aureus, Neisseria meningitides, Campylobacter jejuni, and Streptococcus thermophilis. In some of any such embodiments, the Cas9 is from Streptococcus pyogenes. In some of any such embodiments, the Cas9 or a variant thereof is from Streptococcus pyogenes. In some embodiments, the Cas is Casl2a.

[0274] In some embodiments, the RNA co-LNP comprises a first and second ionizable lipid. In some embodiments, the first and / or second ionizable lipid is Lipid 29. In some embodiments, the first and / or second ionizable lipid is Lipid 319. In some embodiments, the first ionizable lipid is Lipid 29 and the second ionizable lipid is Lipid 319. In some embodiments, the first ionizable lipid is Lipid 319 and the second ionizable lipid is Lipid 29. In some embodiments, the first ionizable lipid is Lipid 319 and the second ionizable lipid is Lipid A6. In some embodiments, the first ionizable lipid is Lipid 29 and the second ionizable lipid is Lipid A6. In some embodiments, the first ionizable lipid is Lipid A6 and the second ionizable lipid is Lipid 29. In some embodiments, the first ionizable lipid is Lipid A6 and the second ionizable lipid is Lipid 319.

[0275] In some embodiments, the first and / or second ionizable lipid comprises a diketopiperazine ring core. In some embodiments, the first and / or second ionizable lipid is any as described in Fenton et al., Angew. Chem. Int. Ed. (2018) 57:13582 -86. In some embodiments, theionizable lipid has an unsaturated linoleic tail. In some embodiments, the first and / or second ionizable lipid is OF-C4-Deg-Lin. In some embodiments, the first and / or second ionizable lipid is cKK-E12. In some embodiments, the first and / or second ionizable lipid is DLin-MC3-DMA. In some embodiments, the first and / or second ionizable lipid is DLin-KC2-DMA,

[0276] In some embodiments, the first and / or second ionizable lipid is positively charged (e.g. a cationic lipid). In some embodiments, the first and / or second ionizable lipid is a cationic lipid, including but not limited to those described in US Patent No. 9,593,077; US Patent No. 9,365,610; US Patent No. 9,670,152; and US Patent No. 9,458,090. In some embodiments, the first and / or second ionizable lipid is a cationic lipid, including but not limited to those described in Published PCT application W02013149140. In some embodiments, the first and / or second ionizable lipid is a cationic lipid, including but not limited to those described in published US Patent application US2019084965; US2019106379. In some embodiments, the first and / or second ionizable lipid is a cationic lipid, including but not limited to those described in Published EP application EP2830595.

[0277] In some embodiments, the mass fraction of the ionizable lipid in the RNA co-LNP is between about 32% and about 65%. In some embodiments, the mass fraction of the ionizable lipid is about 32%, about 32.5%, about 33%, about 33.5%, about 34%, about 34.5%, about 35%, about 35.5%, about 36%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, or about 65%. In some embodiments, the mass fraction of the ionizable lipid is about 32%. In some embodiments, the mass fraction of the ionizable lipid is about 32.5%. In some embodiments, the mass fraction of the ionizable lipid is about 33%. In some embodiments, the mass fraction of the ionizable lipid is about 33.5%. In some embodiments, the mass fraction of the ionizable lipid is about 34%. In some embodiments, the mass fraction of the ionizable lipid is about 34.5%. In some embodiments, the mass fraction of the ionizable lipid is about 35%. In some embodiments, the mass fraction of the ionizable lipid is about 35.5%. In some embodiments, the mass fraction of the ionizable lipid is about 36%. In some embodiments, the mass fraction of the ionizable lipid is about 54%. In some embodiments, the mass fraction of the ionizable lipid is about 55%. In some embodiments, the mass fraction of the ionizable lipid is about 56%. In some embodiments, the mass fraction of the ionizable lipid is about 57%. In some embodiments, the mass fraction of the ionizable lipid is about 58%. In some embodiments, the mass fraction of the ionizable lipid is about 59%. In some embodiments, the mass fraction of the ionizable lipid is about 60%. In some embodiments, the mass fraction of the ionizable lipid is about 61%.

[0278] In some embodiments, the helper lipid is selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1- Stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and l,2-dioleoyl-sn-glycero-sn-3-phosphatidylcholine (DOPC). In some embodiments, the helper lipid is DOPE. In some embodiments, the helper lipid has the structure of

[0279] In some embodiments, the helper lipid is DSPC. In some embodiments, the helper lipid has the structure of

[0280] In some embodiments, the helper lipid is SOPC. In some embodiments, the helper lipid has the structure of

[0281] In some embodiments, the helper lipid is DOPC. In some embodiments, the helper lipid has the structure of

[0282] In some embodiments, the mass fraction of the helper lipid in the RNA co-LNP is between about 4% and about 20%. In some embodiments, the mass fraction of the helper lipid is about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, 10%, about 10.5%, about 11%, about 11.5%, about 12%,about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, about 16%, about 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, or about 20%. In some embodiments, the mass fraction of the helper lipid is about 4%. In some embodiments, the mass fraction of the helper lipid is about 4.5%. In some embodiments, the mass fraction of the helper lipid is about 5%. In some embodiments, the mass fraction of the helper lipid is about 5.5%. In some embodiments, the mass fraction of the helper lipid is about 6%. In some embodiments, the mass fraction of the helper lipid is about 6.5%. In some embodiments, the mass fraction of the helper lipid is about 7%. In some embodiments, the mass fraction of the helper lipid is about 7.5%. In some embodiments, the mass fraction of the helper lipid is about 8%. In some embodiments, the mass fraction of the helper lipid is about 12%. In some embodiments, the mass fraction of the helper lipid is about 12.5%. In some embodiments, the mass fraction of the helper lipid is about 13%. In some embodiments, the mass fraction of the helper lipid is about 13.5%. In some embodiments, the mass fraction of the helper lipid is about 14%. In some embodiments, the mass fraction of the helper lipid is about 14.5%. In some embodiments, the mass fraction of the helper lipid is about 15%. In some embodiments, the mass fraction of the helper lipid is about 15.5%. In some embodiments, the mass fraction of the helper lipid is about 16%. In some embodiments, the mass fraction of the helper lipid is about 16.5%. In some embodiments, the mass fraction of the helper lipid is about 17%. In some embodiments, the mass fraction of the helper lipid is about 17.5. In some embodiments, the mass fraction of the helper lipid is about 18%. In some embodiments, the mass fraction of the helper lipid is about 18.5%. In some embodiments, the mass fraction of the helper lipid is about 19%. In some embodiments, the mass fraction of the helper lipid is about 19.5%. In some embodiments, the mass fraction of the helper lipid is about 20%.

[0283] In some embodiments, the polyethylene glycol (PEG)-conjugated lipid is DMG- PEG2000. In some embodiments, the PEG-conjugated lipid has the structure of

[0284] In some embodiments, the mass fraction of the PEG-conjugated lipid in the RNA co-LNP is between about 2% and about 10%, or between about 3% and about 7%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5% or about 10%. 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 isabout 2.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 3%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 3.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 4%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 4.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 5%. In some embodiments, the mass fraction of the PEG- conjugated lipid is about 5.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 6%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 6.5%. some embodiments, the mass fraction of the PEG-conjugated lipid is about 7%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 7.5%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 8%. In some embodiments, the mass fraction of the PEG-conjugated lipid is about 8.5%.

[0285] In some embodiments, cholesterol has the structure of

[0286] In some embodiments, the mass fraction of cholesterol in the RNA co-LNP is between about 15% and about 45%. In some embodiments, the mass fraction of cholesterol is about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, 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 15%. In some embodiments, the mass fraction of cholesterol is about 16%. In some embodiments, the mass fraction of cholesterol is about 17%. In some embodiments, the mass fraction of cholesterol is about 18%. In some embodiments, the mass fraction of cholesterol is about 19%. In some embodiments, the mass fraction of cholesterol is about 20%. 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 mass fraction of cholesterol is about 44%. In some embodiments, the mass fraction of cholesterol is about 45%.

[0287] In some embodiments, the mass fraction of the RNA in the RNA co-LNP is between about 2% and about 12%, between about 3% and about 11%, or between about 3.5% and about 10%. In some embodiments, the mass fraction of the RNA is about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, or about 13%. In some embodiments, the mass fraction of the RNA is about 3.5%. In some embodiments, the mass fraction of the RNA is about 10%.

[0288] In some embodiments, the total RNA concentration in the RNA co-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 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.2. Formulations

[0289] Provided herein are co-LNPs containing: (a) Lipid 29 or Lipid 319; (b) a helper lipid that is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC); (c) a polyethylene glycol (PEG)-conjugated lipid that is DMG-PEG2000; (d) cholesterol; and (e) a first and second RNA molecule.

[0290] In some embodiments, the mass fraction Lipid 29 or Lipid 319 in the RNA co-LNP is between about 35% and about 40%. In some embodiments, the mass fraction of DSPC in the RNA co-LNP is between about 10% and about 20%. In some embodiments, the mass fraction of DMG- PEG2000 is between about 3% and about 5%. In some embodiments, the mass fraction of cholesterol in the RNA co-LNP is between about 35% and about 45%. In some embodiments, the mass fraction of the RNA molecule in the RNA co-LNP is between about 3% and about 4%.

[0291] In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of between about 35% and about 40%; (b) DSPC with a mass fraction of between about 10% and about 20%; (c) DMG-PEG2000 with a mass fraction of between about 3% and about 5%; (d) cholesterol with a mass fraction of between about 35% and about 45%; and (e) a ribonucleic acid (RNA) molecule. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 37.8%; (b) DSPC with a mass fraction of about 15.2%; (c) DMG-PEG2000 with a mass fraction of about 3.5%; (d) cholesterol with a mass fraction of about 40%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 3.5%.

[0292] In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of between about 32% and about 36%; (b) DSPC with a mass fraction of between about 15% and about 20%; (c) DMG-PEG2000 with a mass fraction of between about 3.5% and about 5.5%; (d) cholesterol with a mass fraction of between about 35% and about 45%; and (e) a ribonucleic acid (RNA) molecule. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 34.4%; (b) DSPC with a mass fraction of about 17.6%; (c) DMG-PEG2000 with a mass fraction of about 4.5%; (d) cholesterol with a mass fraction of about 40%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 3.5%.

[0293] In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of between about 50% and about 65%; (b) DSPC with a mass fraction of between about 12% and about 24%; (c) DMG-PEG2000 with a mass fraction of between about 6% and about 7%; (d) cholesterol with a mass fraction of between about 17% and about 20%; and (e) a ribonucleic acid (RNA) molecule. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 60.21%; (b) DSPC with a mass fraction of about 13.64%; (c) DMG-PEG2000 with a mass fraction of about 6.77%; (d) cholesterol with a mass fraction of about 19.39%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 3.5%. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 60.21%; (b) DSPC with a mass fraction of about 13.64%; (c) DMG-PEG2000 with a mass fraction of about 6.77%; (d) cholesterol with a mass fraction of about 19.39%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 5%. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 60.21%; (b) DSPC with a mass fraction of about 13.64%; (c) DMG- PEG2000 with a mass fraction of about 6.77%; (d) cholesterol with a mass fraction of about 19.39%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 10%. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 54.19%; (b) DSPCwith a mass fraction of about 12.28%; (c) DMG-PEG2000 with a mass fraction of about 6.08%; (d) cholesterol with a mass fraction of about 17.45%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 3.5%. In some embodiments, the RNA co-LNP contains: (a) Lipid 29 or Lipid 319 with a mass fraction of about 54.19%; (b) DSPC with a mass fraction of about 12.28%; (c) DMG- PEG2000 with a mass fraction of about 6.08%; (d) cholesterol with a mass fraction of about 17.45%; and (e) a ribonucleic acid (RNA) molecule with a mass fraction of about 10%.3. Methods for Producing RNA Co-LNPs

[0294] Provided herein are methods for producing RNA co-LNPs, including any as described in the preceding section.

[0295] In some embodiments, the method comprises mixing, in an acidic buffer: (a) a first RNA LNP comprising a first ionizable lipid and a first RNA molecule; and (b) a second RNA LNP comprising a second ionizable lipid and a second RNA molecule. In some embodiments, the mixing generates a composition comprising the first LNP and the second LNP. In some embodiments, the method comprises neutralizing the composition comprising the first LNP and the second LNP. In some embodiments, the neutralizing generates a co-LNP, which is a fusion of the first LNP and the second LNP. In some embodiments, the method comprises (1) mixing, in an acidic buffer (a) a first RNA LNP comprising a first ionizable lipid and a first RNA molecule and (b) a second RNA LNP comprising a second ionizable lipid and a second RNA molecule; thereby generating a composition comprising the first LNP and the second LNP; and (2) neutralizing the composition comprising the first LNP and the second LNP, thereby generating a co-LNP, which is a fusion of the first LNP and the second LNP.

[0296] In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is between about 3:1 or about 1:3. In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is about 3:1. In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is about 2:1. In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is about 1:1. In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is about 1:2. In some embodiments, the volumetric ratio of the first LNP to the second LNP in the composition is about 1:3.

[0297] In some embodiments, the acidic buffer is an acetate buffer. In some embodiments, the acidic buffer has a pH of between about 3.0 and about 4.5, or of 4.0. In some embodiments, the acidic buffer has a pH of about 3.0. In some embodiments, the acidic buffer has a pH of about 3.5. In some embodiments, the acidic buffer has a pH of about 4.0. In some embodiments, the acidic buffer has a pH of about 4.5. In some embodiments, the acidic buffer is neutralized to a pH of between about 6.0 and about 7.5, or between about 6.5 and about 7.0. In some embodiments, the acidic buffer isneutralized to a pH of about 6.0. In some embodiments, the acidic buffer is neutralized to a pH of about 6.5. In some embodiments, the acidic buffer is neutralized to a pH of about 7.0. In some embodiments, the acidic buffer is neutralized to a pH of about 7.5.

[0298] In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding an isotonic buffer. In some embodiments, the isotonic buffer has a pH of about 7.4. In some embodiments, the isotonic buffer is phosphate buffered saline (PBS). In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding at least about 6 parts of the isotonic buffer to 1 part of the acidic buffer. In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding between about 6 parts and about 7 parts of the isotonic buffer to 1 part of the acidic buffer. In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding about 6 parts of the isotonic buffer to 1 part of the acidic buffer. In some embodiments, neutralizing the composition comprising the first LNP and the second LNP comprises adding about 7 parts of the isotonic buffer to 1 part of the acidic buffer.

[0299] Also provided herein is a RNA co-LNP produced by any of the methods provided herein.

[0300] Provided herein are methods of producing co-LNPs comprising a tri-fusion LNP or “tri-LNP” of three RNA LNPs. In some embodiments, the methods comprise fusing a first RNA LNP comprising a first RNA molecule with a second RNA LNP comprising a second RNA molecule and a third RNA LNP comprising a third RNA molecule to generate a co-LNP containing three RNA molecules. In some embodiments, the method comprises mixing a first composition comprising a first RNA LNP with a second composition comprising a second RNA LNP and a third composition comprising a third RNA LNP, wherein each of the compositions is acidic. In some embodiments, each of the composition is acidic by virtue of comprising an acetate buffer. In some embodiments, the pH of each composition is about 4.0. In some embodiments, following mixing of the three RNA LNPs, at least about six parts by volume of an isotonic buffer are added to the mixture. In some embodiments, the isotonic buffer is about pH 7.4. In some embodiments, the isotonic buffer is phosphate buffered saline (PBS). In some embodiments, this process generates a co-LNP comprising the three RNA molecules in a single co-LNP. It is contemplated herein that neutralization of the mixed composition by the isotonic buffer mediates fusion of the RNA LNPs.C. DNA co-LNPs

[0301] Provided herein are co-formulated LNPs (co-LNPs) comprising a deoxyribonucleic acid (DNA) molecule (also referred to as DNA co-LNPs or DNA-containing co-LNPs). In some embodiments, the DNA LNP comprises an ionizable lipid; a helper lipid; a polyethylene glycol (PEG)-conjugated lipid; cholesterol; and a DNA molecule. In some embodiments, the DNA co-LNP comprises a first DNA molecule and a second DNA molecule. In some embodiments, the DNA co-LNP comprises a first ionizable lipid and a second ionizable lipid. In some embodiments, the co-LNP is generated from the fusion of a first DNA LNP comprising a first DNA molecule and a second DNA LNP comprising a second DNA molecule.

[0302] In some embodiments, the co-LNP is generated from the fusion of a first DNA LNP comprising a first DNA molecule, a second DNA LNP comprising a second DNA molecule, and a third DNA LNP comprising a third DNA molecule. In certain embodiments, such a DNA tri-LNP can be generated by a double sequential fusion or tri-fusion described herein. In some embodiments, the method comprises mixing a first composition comprising a first DNA LNP with a second composition comprising a second DNA LNP and a third composition comprising a third DNA LNP, wherein each of the compositions is acidic. In some embodiments, each of the composition is acidic by virtue of comprising an acetate buffer. In some embodiments, the pH of each composition is about 4.0. In some embodiments, following mixing of the three DNA LNPs, at least about six parts by volume of an isotonic buffer are added to the mixture. In some embodiments, the isotonic buffer is about pH 7.4. In some embodiments, the isotonic buffer is phosphate buffered saline (PBS). In some embodiments, this process generates a co-LNP comprising the three DNA molecules in a single co-LNP. It is contemplated herein that neutralization of the mixed composition by the isotonic buffer mediates fusion of the DNA LNPs.

[0303] Any of the DNA LNPs described herein can be used to generate a DNA co-LNP comprising two or three DNA molecules.III. METHODS FOR INTRODUCING DNA INTO CELLS

[0304] In some embodiments, the LNPs or compositions containing the same, as produced by the methods described herein, are used to deliver a nucleic acid molecule containing a transgene into a cell such as an immune cell (e.g., a T cell), such as a transgene encoding a recombinant protein. In some embodiments, delivering a nucleic acid molecule containing a transgene into a cell using a LNP described herein includes contacting a cell with a LNP under conditions in which the LNP is introduced into the cell, wherein the LNPs contain the nucleic acid molecule. In some embodiments, the LNPs are fused co-LNPs or tri-LNPs, as described in Section II.

[0305] In some embodiments, the LNPs or compositions containing the same, as produced by the methods described herein, are used to deliver a nucleic acid molecule containing a transgene into a T cell, such as a transgene encoding a recombinant protein. In some embodiments, delivering a nucleic acid molecule containing a transgene 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 nucleic acid molecule. In some embodiments, the LNPs are fused co-LNPs or tri-LNPs, as described in Section II.

[0306] In some embodiments, incubating the T cell composition under stimulating conditions includes exposing the T cells to anti-CD3 / anti-CD28 antibodies or fragments thereof.

[0307] In some embodiments, the method further includes exposing the T cell composition to Apolipoprotein E (ApoE) before, during, or after incubation with the LNPs. In some embodiments, the T cell composition is exposed to ApoE before incubation with the LNPs. In some embodiments, the T cell composition is exposed to ApoE during at least a portion of incubation with the LNPs. In some embodiments, the ApoE is ApoE2. In some embodiments, the ApoE is ApoE3. In some embodiments, the ApoE is ApoE4. In some embodiments, the T cell composition is exposed to between about 0.1 pg / mL and about 10 pg / mL ApoE, between about 0.5 pg / mL and about 5 pg / mL ApoE, or between about 0.75 pg / mL and about 1.5 pg / mL ApoE. In some embodiments, the T cell composition is exposed to about 1 pg / mL ApoE. In some embodiments, the T cell composition is exposed to about 1 pg / mL ApoE4.

[0308] In some embodiments, the LNPs or compositions containing the same, as produced by the methods described herein, are administered directly to a patient (z.e., in vivo administration). In some embodiments, the LNPs are used to deliver a nucleic acid molecule to a patient in need thereof. In some embodiments, the LNPs or compositions containing the same are administered directly to a patient transduce T cells of the patient and deliver a payload to the T cells of the patient. In some embodiments, the payload is a DNA and / or RNA molecule, as described herein. In particular embodiments, co-LNPs or tri-LNPs are administered directly to a patient. The LNP or LNP compositions can be administered by any suitable means, for example, by bolus infusion, by injection, e.g., intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection, subscleral injection, intrachoroidal injection, intracameral injection, subconjectval injection, subconjuntival injection, sub-Tenon’s injection, retrobulbar injection, peribulbar injection, or posterior juxtascleral delivery. In some embodiments, they are administered by parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration.

[0309] In some embodiments, the nucleic acid is DNA or RNA. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0310] In some embodiments, the nucleic acid containing the transgene is a DNA molecule. In some embodiments, an LNP is used to deliver a DNA nucleic acid molecule containing a transgene into a cell (e.g., a T cell). In some embodiments, the nucleic acid molecule containing the transgene encodes a recombinant protein, such as a recombinant receptor. In some embodiments, the DNA (e.g., ceDNA) nucleic acid molecule containing the transgene is from about 1.5 kilobases (kb) to about 10 kb, such as from about 1.5 kb to about 7.5 kb, from about 1.5 kb to about 6 kb, from about 1.5 kb toabout 5.5 kb, from about 1.5 kb to about 5 kb, from about 1.5 kb to about 4 kb, from about 1.5 kb to about 3 kb, from about 1.5 kb to about 2 kb, from about 2 kb to about 10 kb, from about 2 kb to about7.5 kb, from about 2 kb to about 5.5 kb, from about 2 kb to about 5 kb, from about 2 kb to about 4 kb, from about 2 kb to about 3 kb, from about 3 kb to about 10 kb, from about 3 kb to about 7.5 kb, from about 3 kb to about 6 kb, from about 3 kb to about 5.5 kb, from about 3 kb to about 5 kb, from about 3 kb to about 4 kb, from about 4 kb to about 10 kb, from about 4 kb to about 7.5 kb, from about 4 kb to about 6 kb, from about 4 kb to about 5.5 kb, from about 4 kb to about 5 kb, from about 5 kb to about 10 kb, from about 5 kb to about 7.5 kb, from about 5 kb to about 6 kb, from about 5 kb to about 5.5 kb, from about 5.5 kb to about 10 kb, from about 5.5 kb to about 7.5 kb, from about 5.5 kb to about 6 kb, from about 6 kb to about 10 kb, from about 6 kb to about 7.5 kb, or from about 7.5 kb to about 10 kb. In some embodiments, the DNA (e.g., ceDNA) nucleic acid molecule containing the transgene is at least 5.5 kilobases, 6 kilobases, at least 7 kilobases, or at least 8 kilobases. In some embodiments, the DNA nucleic acid molecule containing the transgene is from 5.5 kilobases to 8 kilobases in size.

[0311] In some embodiments, the nucleic acid encodes a recombinant receptor. In particular embodiments, the nucleic acid encoding the recombinant receptor is DNA. In some embodiments, the recombinant receptor is a T cell receptor (TCR). In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR). In some embodiments, the CAR includes an extracellular antigenrecognition domain that specifically binds to the antigen and an intracellular signaling domain comprising an IT AM. In some embodiments, the CAR is a single antigen directed CAR, i.e. a CAR with an antigen binding domain that targets one antigen. In some embodiments, the CAR is a bispecific CAR in which the antigen binding domain is dual targeting for targeting two different antigens . In some embodiments, the DNA (e.g., ceDNA) molecule encoding the CAR is from about1.5 kilobases (kb) to about 10 kb, such as from about 1.5 kb to about 7.5 kb, from about 1.5 kb to about 6 kb, from about 1.5 kb to about 5.5 kb, from about 1.5 kb to about 5 kb, from about 1.5 kb to about 4 kb, from about 1.5 kb to about 3 kb, from about 1.5 kb to about 2 kb, from about 2 kb to about 10 kb, from about 2 kb to about 7.5 kb, from about 2 kb to about 5.5 kb, from about 2 kb to about 5 kb, from about 2 kb to about 4 kb, from about 2 kb to about 3 kb, from about 3 kb to about 10 kb, from about 3 kb to about 7.5 kb, from about 3 kb to about 6 kb, from about 3 kb to about 5.5 kb, from about 3 kb to about 5 kb, from about 3 kb to about 4 kb, from about 4 kb to about 10 kb, from about 4 kb to about 7.5 kb, from about 4 kb to about 6 kb, from about 4 kb to about 5.5 kb, from about 4 kb to about 5 kb, from about 5 kb to about 10 kb, from about 5 kb to about 7.5 kb, from about 5 kb to about 6 kb, from about 5 kb to about 5.5 kb, from about 5.5 kb to about 10 kb, from about 5.5 kb to about7.5 kb, from about 5.5 kb to about 6 kb, from about 6 kb to about 10 kb, from about 6 kb to about 7.5 kb, or from about 7.5 kb to about 10 kb. In some embodiments, the DNA (e.g., ceDNA) molecule encoding the CAR (e.g., bispecific CAR) is at least 5.5 kilobases, 6 kilobases, at least 7 kilobases, orat least 8 kilobases. In some embodiments, the DNA molecule (e.g, ceDNA) encoding the CAR (e.g., bispecific CAR) is from 5.5 kilobases to 8 kilobases in size.

[0312] In some embodiments, the antigen is a tumor antigen. In some embodiments, the antigen is selected from among avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7- H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (OGD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL-13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (LI -CAM), CE7 epitope of LI -CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomer ase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1). In some embodiments, the antigen is BCMA. In some embodiments, the antigen is CD19.

[0313] In some embodiments, the intracellular signaling domain includes an intracellular domain of a CD3-zeta (CD3Q chain. In some embodiments, the intracellular signaling region further includes a costimulatory signaling region. In some embodiments, the costimulatory signaling region includes a signaling domain of CD28, such as a human CD28. In some embodiments, the costimulatorysignaling region includes a signaling domain of 4-1BB, such as a human 4-1BB. In some embodiments, the co-stimulatory domain is or includes a signaling domain of CD28.

[0314] In some embodiments, the cells produced by the methods described herein (e.g. cells having the nucleic acid) are used for the treatment of cancer. In some embodiments, the cells produced by the methods described herein (e.g. cells having the nucleic acid) are used for treating a subject having a cancer, such as for use in adoptive cell therapy. In some embodiments, the cells produced by the methods described herein (e.g. cells having the nucleic acid) are for use as a medicament for treatment of a cancer. In some embodiments, the cells produced by the methods described herein (e.g. cells having the nucleic acid) are used in the manufacture of a medicament for treatment of a cancer.A. Cells and Preparation of Cells for Genetic Engineering

[0315] In some embodiments, provided are engineered cells, e.g., genetically engineered or modified cells, and methods of engineering cells (e.g., T cells). In some embodiments, one or more polynucleotides, e.g., encoding a recombinant receptor and / or additional polypeptide(s), such as any described herein, are introduced into a cell (e.g. a T cell) for engineering. 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 nucleic acid sequences are not naturally occurring, such as a nucleic acid sequences not found in nature or is modified from a nucleic acid sequence found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0316] The cells generally are eukaryotic cells, such as mammalian cells, and typically are human cells. 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. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells typically are primary cells, such as those isolated directly from a subject and / or isolated from a subject and frozen. 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. With reference to the subject to be treated, the cells may be allogeneic and / or autologous. Among the methods include off-the-shelf methods. Insome 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, processing, culturing, and / or engineering them, and re-introducing them into the same subject, before or after cryopreservation.

[0317] Among the sub-types and subpopulations of T cells and / or of CD4+ and / or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH 17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0318] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0319] In some embodiments, the cells include one or more nucleic acids introduced via genetic engineering, and thereby express recombinant or genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids 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 nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one comprising chimeric combinations of nucleic acids encoding various domains from multiple different cell types.

[0320] In some embodiments, preparation of the engineered cells includes one or more culture and / or preparation steps. The cells for introduction of the nucleic acid encoding the transgenic receptor such as the CAR, may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and / or engineered.

[0321] Accordingly, the cells in some embodiments are primary cells, e.g., primary human T cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and / or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biologicalsamples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0322] In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and / or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

[0323] In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate, and pig.

[0324] In some embodiments, isolation of the cells includes one or more preparation and / or nonaffinity based cell separation steps. In some examples, cells are washed, centrifuged, and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[0325] In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and / or platelets, and in some aspects contains cells other than red blood cells and platelets.

[0326] In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is accomplished a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer’s instructions. In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer’s instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca++ / Mg++free PBS. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media.

[0327] In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

[0328] In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells’ expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

[0329] Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and / or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population.

[0330] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

[0331] In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

[0332] For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+,CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+T cells, are isolated by positive or negative selection techniques.

[0333] For example, CD3+, CD28+T cells can be positively selected using anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0334] In some embodiments, isolation is carried out by enrichment for a particular cell population by positive selection, or depletion of a particular cell population, by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agent that specifically bind to one or more surface markers expressed or expressed (marker+) at a relatively higher level (markerhlgh) on the positively or negatively selected cells, respectively.

[0335] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some aspects, a CD4+or CD8+selection step is used to separate CD4+helper and CD8+cytotoxic T cells. Such CD4+and CD8+populations can be further sorted into sub-populations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and / or effector T cell subpopulations.

[0336] In some embodiments, CD8+cells are further enriched for or depleted of naive, central memory, effector memory, and / or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve long-term survival, expansion, and / or engraftment following administration, which in some aspects is particularly robust in such sub-populations. See Terakura et al. (2012) Blood.1:72-82; Wang et al. (2012) J Immunother. 35(9):689-701. In some embodiments, combining TcM-enriched CD8+T cells and CD4+T cells further enhances efficacy.

[0337] In embodiments, memory T cells are present in both CD62L+and CD62L subsets of CD8+peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L CD8+and / or CD62L+CD8+fractions, such as using anti-CD8 and anti-CD62L antibodies.

[0338] In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, CD27, and / or CD127. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD62L, CCR7, CD28, and / or CD27. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CCR7, CD28, and / orCD27. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD28 and CD27. In some aspects, isolation of a CD8+population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In some aspects, isolation of a CD8+population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD27 and CD28. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+cell population or subpopulation, also is used to generate the CD4+cell population or sub-population, such that both the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

[0339] In a particular example, a sample of PBMCs or other white blood cell sample is subjected to selection of CD4+cells, where both the negative and positive fractions are retained. The negative fraction then is subjected to negative selection based on expression of CD14 and CD45RA or CD19, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the positive and negative selections are carried out in either order.

[0340] CD4+T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+T lymphocytes are CD45RO , CD45RA+, CD62L+, CD4+T cells. In some embodiments, central memory CD4+cells are CD62L+and CD45RO+. In some embodiments, effector CD4+cells are CD62L and CD45RO .

[0341] In one example, to enrich for CD4+cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD 14, CD20, CD 11b, CD 16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and / or negative selection. For example, in some embodiments, the cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Protocols, Vol. 2: Cell Behavior In Vitro and In Vivo, p 17-25 Edited by: S. A. Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[0342] In some aspects, the sample or population of cells to be separated is incubated with small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads or MACS beads). Themagnetically responsive material, e.g., particle, generally is directly or indirectly attached to a binding partner, e.g., an antibody, that specifically binds to a molecule, e.g., surface marker, present on the cell, cells, or population of cells that it is desired to separate, e.g., that it is desired to negatively or positively select.

[0343] In some embodiments, the magnetic particle or bead comprises a magnetically responsive material bound to a specific binding member, such as an antibody or other binding partner. There are many well-known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Molday, U.S. Pat. No. 4,452,773, and in European Patent Specification EP 452342 B, which are hereby incorporated by reference. Colloidal sized particles, such as those described in Owen U.S. Pat. No. 4,795,698, and Liberti et al., U.S. Pat. No. 5,200,084 are other examples.

[0344] The incubation generally is carried out under conditions whereby the antibodies or binding partners, or molecules, such as secondary antibodies or other reagents, which specifically bind to such antibodies or binding partners, which are attached to the magnetic particle or bead, specifically bind to cell surface molecules if present on cells within the sample.

[0345] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from the unlabeled cells. For positive selection, cells that are attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are retained and further processed or subject to further separation steps.

[0346] In certain embodiments, the magnetically responsive particles are coated in primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to cells via a coating of primary antibodies specific for one or more markers. In certain embodiments, the cells, rather than the beads, are labeled with a primary antibody or binding partner, and then cell-type specific secondary antibody- or other binding partner (e.g., streptavidin)-coated magnetic particles, are added. In certain embodiments, streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[0347] In some embodiments, the magnetically responsive particles are left attached to the cells that are to be subsequently incubated, cultured and / or engineered; in some aspects, the particles are left attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, e.g., the use of competing non-labeled antibodies, andmagnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0348] In some embodiments, the affinity-based selection is via magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). Magnetic Activated Cell Sorting (MACS) systems are capable of high-purity selection of cells having magnetized particles attached thereto. In certain embodiments, MACS operates in a mode wherein the non-target and target species are sequentially eluted after the application of the external magnetic field. That is, the cells attached to magnetized particles are held in place while the unattached species are eluted. Then, after this first elution step is completed, the species that were trapped in the magnetic field and were prevented from being eluted are freed in some manner such that they can be eluted and recovered. In certain embodiments, the non-target cells are labelled and depleted from the heterogeneous population of cells.

[0349] In certain embodiments, the isolation or separation is carried out using a system, device, or apparatus that carries out one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the methods. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment, for example, to minimize error, user handling and / or contamination. In one example, the system is a system as described in International Pat. App. Pub. No. W02009 / 072003 or US 20110003380.

[0350] In some embodiments, the system or apparatus carries out one or more, e.g., all, of the isolation, processing, engineering, and formulation steps in an integrated or self-contained system, and / or in an automated or programmable fashion. In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, assess the outcome of, and / or adjust various aspects of the processing, isolation, engineering, and formulation steps.

[0351] In some aspects, the separation and / or other steps is carried out using CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells on a clinical-scale level in a closed and sterile system. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump, and various pinch valves. The integrated computer in some aspects controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit in some aspects includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate throughout the tubing set and, together with the pinch valves, ensures the controlled flow of buffer through the system and continual suspension of cells.

[0352] The CliniMACS system in some aspects uses antibody-coupled magnetizable particles that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labelling of cells with magnetic particles the cells are washed to remove excess particles. A cell preparation bag is thenconnected to the tubing set, which in turn is connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing, including a pre-column and a separation column, and are for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. Labelled cells are retained within the column, while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell populations for use with the methods described herein are unlabeled and are not retained in the column. In some embodiments, the cell populations for use with the methods described herein are labeled and are retained in the column. In some embodiments, the cell populations for use with the methods described herein are eluted from the column after removal of the magnetic field, and are collected within the cell collection bag.

[0353] In certain embodiments, separation and / or other steps are carried out using the CliniMACS Prodigy system (Miltenyi Biotec). The CliniMACS Prodigy system in some aspects is equipped with a cell processing unity that permits automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the optimal cell fractionation endpoint by discerning the macroscopic layers of the source cell product. For example, peripheral blood is automatically separated into erythrocytes, white blood cells and plasma layers. The CliniMACS Prodigy system can also include an integrated cell cultivation chamber which accomplishes cell culture protocols such as, e.g., cell differentiation and expansion, antigen loading, and long-term cell culture. Input ports can allow for the sterile removal and replenishment of media and cells can be monitored using an integrated microscope. See, e.g., Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and Wang et al. (2012) J Immunother. 35(9):689-701.

[0354] In some embodiments, a cell population described herein is collected and enriched (or depleted) via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluidic stream. In some embodiments, a cell population described herein is collected and enriched (or depleted) via preparative scale (FACS)-sorting. In certain embodiments, a cell population described herein is collected and enriched (or depleted) by use of microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. l(5):355-376. In both cases, cells can be labeled with multiple markers, allowing for the isolation of well-defined T cell subsets at high purity.

[0355] In some embodiments, the antibodies or binding partners are labeled with one or more detectable marker, to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to fluorescently labeled antibodies. In some examples, separation of cells based on binding of antibodies or other binding partners specific for one or more cell surface markers are carried in a fluidic stream, such as by fluorescence-activated cell sorting (FACS),including preparative scale (FACS) and / or microelectromechanical systems (MEMS) chips, e.g., in combination with a flow-cytometric detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[0356] In some embodiments, the preparation methods include steps for freezing, e.g., cryopreserving, the cells, either before or after isolation, incubation, and / or engineering. In some embodiments, the freeze and subsequent thaw step removes granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, e.g., following a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters in some aspects may be used. One example involves using PBS containing 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing media. This is then diluted 1:1 with media so that the final concentration of DMSO and HSA are 10% and 4%, respectively. The cells are generally then frozen to -80° C. at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[0357] In some embodiments, the cells are incubated and / or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. The incubation and / or engineering may be carried out in a culture vessel, such as a unit, chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, or other container for culture or cultivating cells. In some embodiments, the populations or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and / or survival of cells in the population, to mimic antigen exposure, and / or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor.

[0358] The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells.

[0359] In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR / CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR, e.g. anti-CD3. In some embodiments, the stimulating conditions include one or more agent, e.g. ligand, which is capable of stimulating a costimulatory receptor, e.g., anti-CD28. In some embodiments, such agents and / or ligands may be, bound to solid support such as a bead, and / or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, thestimulating agents include IL-2, IL-15 and / or IL-7. In some aspects, the IL-2 concentration is at least about 10 units / mL.

[0360] In some aspects, incubation is carried out in accordance with techniques such as those described in US Patent No. 6,040,177, Klebanoff et al. (2012) J Immunother. 35(9): 651-660, Terakura et al. (2012) Blood.1:72-82, and / or Wang et al. (2012) J Immunother. 35(9):689-701.

[0361] In some embodiments, the T cells are expanded by adding to a culture-initiating population feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g., such that the resulting population of cells contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (e.g. for a time sufficient to expand the numbers of T cells). In some aspects, the non-dividing feeder cells can comprise gamma-irradiated PBMC feeder cells. In some embodiments, the PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, the feeder cells are added to culture medium prior to the addition of the populations of T cells.

[0362] In some embodiments, the stimulating conditions include temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees Celsius, and generally at or about 37 degrees Celsius. Optionally, the incubation may further comprise adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. The LCL feeder cells in some aspects is provided in any suitable amount, such as a ratio of LCL feeder cells to initial T lymphocytes of at least about 10:1.

[0363] In embodiments, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating naive or antigen specific T lymphocytes with antigen. For example, antigen-specific T cell lines or clones can be generated to cytomegalovirus antigens by isolating T cells from infected subjects and stimulating the cells in vitro with the same antigen.B. Transgene Nucleic Acid Encoding Recombinant Proteins

[0364] In some embodiments, the provided methods are or include introducing a transgene nucleic acid or polynucleotide (also called a heterologous nucleic acid or polynucleotide) encoding a recombinant protein into cells of a population. In some embodiments, the transgene nucleic acid is a heterologous polynucleotide that encodes a recombinant protein. Such recombinant proteins may include recombinant receptors, such as any described herein. Introduction of the polynucleotides, e.g., heterologous or recombinant polynucleotides, encoding the recombinant protein into the cell may be carried out using the LNPs and compositions and uses thereof described herein. Exemplary methods of using the LNP compositions include those for non-viral introduction of heterologous polynucleotides encoding the receptors into the cells of a population, e.g., transfection. In some embodiments, a population of cells (e.g., T cells) is genetically engineered, such as to introduce aheterologous or recombinant polynucleotide encoding a recombinant receptor, thereby generating a population of transformed cells (also referred to herein as a transformed population of cells).

[0365] In certain embodiments, a transgene nucleic acid encoding the recombinant protein, e.g. a recombinant receptor, is introduced to the cells. In certain embodiments, the polynucleotide or nucleic acid molecule is heterologous to the cells. In particular embodiments, the heterologous polynucleotide is not native to the cells. In certain embodiments, the heterologous nucleic acid molecule or heterologous polynucleotide encodes a protein, e.g., a recombinant protein that is not natively expressed by the cell. In particular embodiments, the heterologous nucleic acid molecule or polynucleotide is or contains a nucleic acid sequence that is not found in the cell prior to the contact or introduction.

[0366] In particular embodiments, the heterologous polynucleotide encodes a recombinant protein. In certain embodiments, the recombinant protein is a recombinant receptor. In some embodiments, the recombinant protein is a recombinant antigen receptor, such as a recombinant TCR or a chimeric antigen receptor (CAR).

[0367] In some embodiments, the transgene nucleic acid is a DNA molecule. In some embodiments, the transgene nucleic acid molecule is a naked DNA molecule. In some embodiments, the nucleic acid molecule, e.g., naked DNA molecule, is a non-viral, capsid-free DNA molecule. In some embodiments, the naked DNA molecule is provided for non-viral gene transfer, such as a capsid-free DNA vector (also called plasmid) with covalently-closed ends.

[0368] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a modified DNA molecule. In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is modified to enhance its stability.

[0369] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a non-viral, capsid-free DNA molecule with covalently-closed ends (also referred to herein as a “closed-ended DNA” or a “ceDNA” molecule). In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a ceDNA molecule. In some embodiments, the transgene nucleic acid molecule is a naked ceDNA molecule.

[0370] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is selected from the group consisting of a closed-ended linear duplex (CELiD) DNA molecule, a minicircle DNA molecule, a minimalistic immunological-defined gene expression (MIDGE) DNA molecule, a ministring DNA molecule, a dumbbell-shaped linear duplex closed-ended DNA molecule, or a doggybone™ DNA molecule. In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a CELiD DNA molecule. In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a MIDGE DNA molecule. In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a ministring DNA. In someembodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a dumbbell-shaped linear duplex closed-ended DNA molecule. In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a doggybone™ DNA molecule.

[0371] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a doggybone™ DNA molecule. In some embodiments, the doggybone™ DNA (dbDNA™) molecule is a synthetic closed linear double-stranded DNA molecule. In some embodiments, the closed linear DNA molecule is double-stranded DNA that is covalently closed at each end. The double stranded section of the closed linear DNA molecule can be complementary. When denatured, closed linear DNA may form a single stranded circle. The DNA may be closed at each end by any suitable structure, including a cruciform, a hairpin, or a hairpin loop, depending on preference. The end of the closed linear DNA may be composed of a non-complementary sequence. In some embodiments, the non-complementary sequence forces the DNA into a single stranded configuration at the cruciform, hairpin, or hairpin loop.

[0372] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a closed-ended linear duplex (CELiD) DNA molecule. In some embodiments, the CELiD DNA molecule is a linear duplex molecule. In some embodiments, the CELiD DNA molecule is doublestranded DNA that is covalently closed at each end. In some embodiments, the CELiD DNA molecule may comprise heterologous DNA flanked by ITRs. In some embodiments, the heterologous DNA may encode a protein. In some embodiments, because the ends are covalently closed, the CELiD DNA molecule is exonuclease resistant.

[0373] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a minicircle DNA molecule. In some embodiments, the minicircle DNA molecule is a circular DNA molecule. In some embodiments, the circular DNA molecule contains at least one gene encoding a recombinant protein. In some embodiments, the minicircle DNA molecule is circular DNA having an attR site and genetically engineered gene expression cassette, and said gene expression cassette comprises a promoter, base sequence encoding immunoglobulin K chain signal peptide, base sequence encoding Flag tag, the gene encoding a recombinant protein, base sequence encoding His6 tag, stop codon, and polyA tailing signal linked sequentially.

[0374] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a minimalistic immunological-defined gene expression (MIDGE) DNA molecule. In some embodiments, the MIDGE DNA molecule is a circular DNA molecule. In some embodiments, the MIDGE DNA molecule is a circular DNA molecule that is double-stranded. In some embodiments, the MIDGE DNA molecule comprises an expression cassette containing a promoter, a gene of interest, and an RNA-stabilizing sequence, e.g., a poly A sequence. The complementary sense and antisense strands encoding the transgene can be connected at both the 5’ and 3’ ends of the doublestranded MIDGE DNA molecule by a single-stranded hairpin DNA having non-complementarysequence loop structures, so that the MIDGE DNA molecule has a “dumbbell” shape. The MIDGE DNA molecule can be resistant to enzymatic digestion and relatively stable in cells and serum.

[0375] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a ministring DNA molecule. In some embodiments, the ministring DNA molecule is an enhanced linear covalently closed (LCC) minivector. In some embodiments, the ministring DNA molecule is double-stranded. In some embodiments, the ministring DNA molecule comprises LCC ends, minimal transgene expression cassette elements, and DNA targeting sequences (DTS) at both ends.

[0376] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is a dumbbell-shaped linear duplex closed-ended DNA molecule. In some embodiments, the dumbbell-shaped linear duplex closed-ended DNA molecule is a linear, duplex molecule. In some embodiments, the dumbbell-shaped linear duplex closed-ended DNA molecule is covalently closed at each end.

[0377] In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, has covalently closed ends. In some embodiments, the transgene nucleic acid molecule, e.g., naked DNA molecule, is resistant to exonuclease digestion (e.g., exonuclease I or exonuclease III), e.g., for over an hour at 37°C.1. Recombinant Receptors

[0378] In some embodiments, provided herein are LNPs and compositions thereof for use in introducing a nucleic acid e.g., a DNA plasmid) encoding one or more recombinant receptor(s) into cells (e.g. T cells) of a population, thereby generating engineered cells that express or are engineered to express the one or more recombinant receptor(s). Among the receptors are antigen receptors and receptors containing one or more components thereof. The recombinant receptors may include chimeric receptors, such as those containing ligand-binding domains or binding fragments thereof and intracellular signaling domains or regions, functional non-TCR antigen receptors, chimeric antigen receptors (CARs), T cell receptors (TCRs), such as recombinant or transgenic TCRs, chimeric autoantibody receptor (CAAR) and components of any of the foregoing. The recombinant receptor, such as a CAR, generally includes the extracellular antigen (or ligand) binding domain linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). In some embodiments, the engineered cells express two or more receptors that contain different components, domains or regions. In some aspects, two or more receptors allows spatial or temporal regulation or control of specificity, activity, antigen (or ligand) binding, function and / or expression of the recombinant receptors. a. Chimeric Antigen Receptors ( CARs)

[0379] In some embodiments of the provided compositions and uses thereof, the chimeric receptors, such as a chimeric antigen receptors, contain one or more domains that combine a ligandbinding domain (e.g. antibody or antibody fragment) that provides specificity for a desired antigen(e.g., tumor antigen) with intracellular signaling domains. In some embodiments, the intracellular signaling domain is a stimulating or an activating intracellular domain portion, such as a T cell stimulating or activating domain, providing a primary activation signal or a primary signal. In some embodiments, the intracellular signaling domain contains or additionally contains a costimulatory signaling domain to facilitate effector functions. In some embodiments, chimeric receptors when genetically engineered into immune cells can modulate T cell activity, and, in some cases, can modulate T cell differentiation or homeostasis, thereby resulting in genetically engineered cells with improved longevity, survival and / or persistence in vivo, such as for use in adoptive cell therapy methods.

[0380] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, W02013 / 123061 U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995,7.446.190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762,7.446.191, 8,324,353, and 8,479,118, and European patent application number EP2537416, and / or those described by Sadelain et al., Cancer Discov. 2013 April; 3(4): 388-398; Davila et al. (2013) PLoS ONE 8(4): e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5): 633-39; Wu et al., Cancer, 2012 March 18(2): 160-75. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No.: 7,446,190, and those described in International Patent Application Publication No.: WO / 2014055668 Al. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No.: 7,446,190, US Patent No.: 8,389,282, Kochenderfer et al., 2013, Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al. (2012) J. Immunother. 35(9): 689-701; and Brentjens et al., Sci Transl Med. 2013 5(177). See also WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No.: 7,446,190, and US Patent No.: 8,389,282.

[0381] The chimeric receptors, such as CARs, generally include an extracellular antigen binding domain, such as a portion of an antibody molecule, generally a variable heavy (VH) chain region and / or variable light (VL) chain region of the antibody, e.g., an scFv antibody fragment.In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0382] In some embodiments, the antigen targeted by the receptor is or includes avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, alsoknown as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (0GD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight- melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE- A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

[0383] In some embodiments, the antigen is or includes a pathogen-specific or pathogen- expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0384] In some embodiments, the CAR is a bispecific CAR (also called a “dual targeting CAR”) in which the extracellular antigen binding domain of the CAR targets a first antigen and a secondantigen. In some embodiments, the bispecific CAR is a CAR that contains a first antigen binding domain that binds a first antigen and a second antigen binding domain that binds a second antigen, in which the antigen binding domains are in tandem in a single binding domain (also called a “tandem CAR”). In some embodiments, each antigen binding domain includes a heavy chain variable (VH) region and / or light chain variable (VL) region of an antibody that targets the antigen. In some embodiments, each antigen binding domain includes a heavy chain variable (VH) region and a light chain variable (VL) region of an antibody that targets the antigen. In some embodiments, the extracellular antigen binding domain is in a linear format in which the extracellular antigen binding domain includes, such as in order from N-terminus to C-terminus, the VH region and the VL region of the first antigen binding domain, and the VH region and the VL region of the second antigen binding domain. In some embodiments, the extracellular antigen binding domain is in a loop format in which the extracellular antigen binding domain includes, such as in order from N-terminus to C-terminus, one of the VH region and the VL region of the first antigen binding domain, one of the VH region and the VL region of the second antigen binding domain, the other of the VH region and the VL region of the first antigen binding domain, and the other of the VH region and the VL region of the second antigen binding domain. In some embodiments, the first and second antigen targeted by the first and second antigen binding domain, respectively, of a bispecific CAR are different. In some embodiments, the first and second antigens can be any as described above.

[0385] In some embodiments, an antigen targeted by the CAR is CD19, e.g., human CD19, and the antibody or antigen-binding domain includes an anti-CD19 antigen binding domain. In some embodiments, the antigen binding domain contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the anti-CD19 CAR includes an antigenbinding domain that is a single chain antibody derived from a fully human antibody. In some embodiments, the single chain antibody is an scFv. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD 19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, the anti-CD19 CAR includes an antigen-binding domain described in PCT Pub. No. WO2015187528. In some embodiments, the anti-CD19 CAR is a CAR described in PCT Pub. No. WO2015187528. In some embodiments, the antibody or antibody fragment is a human antibody, e.g., as described in U.S. Patent Publication No. US 2016 / 0152723. Exemplary fully human anti-CD19 antibodies are described in PCT Pub. No. W02016033570, PCT Pub. No.WO2020233589, U.S. Pub. No. US2010 / 0104509 and U.S. Pub. No. US20220220200.

[0386] Exemplary CD19-directed antigen receptors, e.g., CARs, also include the CARs of FDA- approved products BREYANZI® (lisocabtagene maraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), and YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagenemaraleucel), TECARTUS™ (brexucabtagene autoleucel), KYMRIAH™ (tisagenlecleucel), YESCARTA™ (axicabtagene ciloleucel). In some of any of the provided embodiments, the CAR is the CAR of BREYANZI® (lisocabtagene maraleucel, see Sehgal et al., 2020, Journal of Clinical Oncology 38:15_suppl, 8040; Teoh et al., 2019, Blood 134(Supplement_l):593; and Abramson et al., 2020, The Lancet 396(10254): 839-852). In some of any of the provided embodiments, the CAR is the CAR of TECARTUS™ (brexucabtagene autoleucel, see Mian and Hill, 2021, Expert Opin Biol Ther; 21(4):435-441; and Wang et al., 2021, Blood 138(Supplement 1):744). In some of any of the provided embodiments, the CAR is the CAR of KYMRIAH™ (tisagenlecleucel, see Bishop et al., 2022, N Engl J Med 386:629:639; Schuster et al., 2019, N Engl J Med 380:45-56; Halford et al., 2021, Ann Pharmacother 55(4):466-479; Mueller et al., 2021, Blood Adv. 5(23):4980-4991; and Fowler et al., 2022, Nature Medicine 28:325-332). In some of any of the provided embodiments, the CAR is the CAR of YESCARTA™ (axicabtagene ciloleucel, see Neelapu et al., 2017, N Engl J Med 377(26):2531-2544; Jacobson et al., 2021, The Lancet 23(l):P91-103; and Locke et al., 2022, N Engl J Med 386:640-654).

[0387] In some embodiments, an antigen targeted by the CAR is GPRC5D, e.g., human GPRC5D, and the antibody or antigen-binding domain includes an anti-GPRC5D antigen binding domain. In some embodiments, the antigen binding domain contains a VH and a VL derived from an antibody or an antibody fragment specific to GPRC5D. In some embodiments, the anti-GPRC5D CAR includes an antigen-binding domain that is a single chain antibody derived from a fully human antibody. In some embodiments, the single chain antibody is an scFv. In some embodiments, the antibody or antigen-binding domain can be any anti-GPRC5D antibody described or derived from any anti-GPRC5D antibody described (see, e.g., WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, and WO2018147245). Any of such anti-GPRC5D antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the CAR contains a variable heavy (VH) and / or a variable light (VL) region derived from an antibody described in WO 2016 / 090312, WO 2016 / 090329, WO 2018 / 017786, WO2020148677, WO2019154890, WO2021018859, WO2021018925, or WO2018147245.

[0388] In some embodiments, an antigen targeted by the CAR is BCMA, e.g., human BCMA, and the antibody or antigen-binding domain includes an anti-BCMA antigen binding domain. In some embodiments, the antigen binding domain contains a VH and a VL derived from an antibody or an antibody fragment specific to BCMA. In some embodiments, the anti-BCMA CAR includes an antigen-binding domain that is a single chain antibody derived from a fully human antibody. In some embodiments, the single chain antibody is an scFv. Chimeric antigen receptors containing anti-BCMA antibodies, including mouse anti-human BCMA antibodies and human anti-human antibodies, and cells expressing such chimeric receptors have been previously described. See Carpenter et al., Clin Cancer Res., 2013, 19(8):2048-2060, WO 2016 / 090320, W02016090327, W02010104949A2 andWO2017173256. In some embodiments, the antibody or antigen-binding domain can be any anti- BCMA antibody described or derived from any anti-BCMA antibody described (see, e.g., WO 2016 / 090320, WO 2016 / 090327, W02010104949A2 and WO2017173256). Any of such anti-BCMA antibodies or antigen-binding fragments can be used in the provided CARs. In some embodiments, the CAR contains a variable heavy (VH) and / or a variable light (VL) region derived from an antibody described in WO 2016 / 090320, WO 2016 / 090327, W02010104949A2 and WO2017173256).

[0389] Further exemplary antigen receptors, e.g., CARs, such as anti-BCMA CARs, include the CARs of idecabtagene vicleucel, ABECMA®, BCMA02, JCARH125, JNJ-68284528 (LCAR-B38M; ciltacabtagene autoleucel; CARVYKTI™) (Janssen / Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allol (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), ARI-002 (Hospital Clinic Barcelona, IDIBAPS), and CTX120 (CRISPR Therapeutics). In a particular embodiment, the CAR is the CAR of idecabtagene vicleucel cells. In a particular embodiment, the CAR is the CAR of ABECMA® cells (cells used in ABECMA® immunotherapy). In a particular embodiment, the CAR is the CAR of ciltacabtagene autoleucel cells. In a particular embodiment, the CAR is the CAR of CARVYKTI™ cells (cells used in CARVYKTI™ immunotherapy ).

[0390] In some embodiments, the antigen receptor comprises an intracellular domain linked directly or indirectly to the extracellular domain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain linking the extracellular domain and the intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises an ITAM. For example, in some aspects, the antigen recognition domain (e.g. extracellular domain) generally is linked to one or more intracellular signaling components, such as signaling components that mimic activation through an antigen receptor complex, such as a TCR complex, in the case of a CAR, and / or signal via another cell surface receptor. In some embodiments, the chimeric receptor comprises a transmembrane domain linked or fused between the extracellular domain (e.g. scFv) and intracellular signaling domain. Thus, in some embodiments, the antigen-binding component (e.g., antibody) is linked to one or more transmembrane and intracellular signaling domains.

[0391] In one embodiment, a transmembrane domain that naturally is associated with one of the domains in the receptor, e.g., CAR, is used. In some instances, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0392] The transmembrane domain in some embodiments is derived either from a natural or from a synthetic source. Where the source is natural, the domain in some aspects is derived from any membrane-bound or transmembrane protein. Transmembrane regions include those derived from (i.e.comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively the transmembrane domain in some embodiments is synthetic. In some aspects, the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine. In some aspects, a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. In some embodiments, the linkage is by linkers, spacers, and / or transmembrane domain(s). In some aspects, the transmembrane domain contains a transmembrane portion of CD28.

[0393] In some embodiments, the extracellular domain and transmembrane domain can be linked directly or indirectly. In some embodiments, the extracellular domain and transmembrane are linked by a spacer, such as any described herein. In some embodiments, the receptor contains extracellular portion of the molecule from which the transmembrane domain is derived, such as a CD28 extracellular portion.

[0394] Among the intracellular signaling domains are those that mimic or approximate a signal through a natural antigen receptor, a signal through such a receptor in combination with a costimulatory receptor, and / or a signal through a costimulatory receptor alone. In some embodiments, a short oligo- or polypeptide linker, for example, a linker of between 2 and 10 amino acids in length, such as one containing glycines and serines, e.g., glycine-serine doublet, is present and forms a linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0395] T cell activation is in some aspects described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). In some aspects, the CAR includes one or both of such signaling components.

[0396] The receptor, e.g., the CAR, generally includes at least one intracellular signaling component or components. In some aspects, the CAR includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs. Examples of IT AM containing primary cytoplasmic signaling sequences include those derived from CD3 zeta chain, FcR gamma, CD3 gamma, CD3 delta and CD3 epsilon. In some embodiments, cytoplasmic signaling molecule(s) in the CAR contain(s) a cytoplasmic signaling domain, portion thereof, or sequence derived from CD3 zeta.

[0397] In some embodiments, the receptor includes an intracellular component of a TCR complex, such as a TCR CD3 chain that mediates T-cell activation and cytotoxicity, e.g., CD3 zeta chain. Thus, in some aspects, the antigen-binding portion is linked to one or more cell signalingmodules. In some embodiments, cell signaling modules include CD3 transmembrane domain, CD3 intracellular signaling domains, and / or other CD transmembrane domains. In some embodiments, the receptor, e.g., CAR, further includes a portion of one or more additional molecules such as Fc receptor y, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor includes a chimeric molecule between CD3-zeta (CD3-Q or Fc receptor y and CD8, CD4, CD25 or CD 16.

[0398] In some embodiments, upon ligation of the CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune cell, e.g., T cell engineered to express the CAR. For example, in some contexts, the CAR induces a function of a T cell such as cytolytic activity or T- helper activity, such as secretion of cytokines or other factors. In some embodiments, a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal. In some embodiments, the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of coreceptors that in the natural context act in concert with such receptors to initiate signal transduction following antigen receptor engagement.

[0399] In the context of a natural TCR, full activation generally requires not only signaling through the TCR, but also a costimulatory signal. Thus, in some embodiments, to promote full activation, a component for generating secondary or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a costimulatory signal. In some aspects, an additional CAR is expressed in the same cell and provides the component for generating the secondary or costimulatory signal.

[0400] In some embodiments, the chimeric antigen receptor contains an intracellular domain of a T cell costimulatory molecule. In some embodiments, the CAR includes a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4- IBB, 0X40, DAP 10, and ICOS. In some aspects, the same CAR includes both the activating and costimulatory components. In some embodiments, the chimeric antigen receptor contains an intracellular domain derived from a T cell costimulatory molecule or a functional variant thereof, such as between the transmembrane domain and intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB.

[0401] In some embodiments, the activating domain is included within one CAR, whereas the costimulatory component is provided by another CAR recognizing another antigen. In some embodiments, the CARs include activating or stimulatory CARs, costimulatory CARs, both expressed on the same cell (see WO2014 / 055668). In some aspects, the cells include one or more stimulatory oractivating CAR and / or a costimulatory CAR. In some embodiments, the cells further include inhibitory CARs (iCARs, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December, 2013), such as a CAR recognizing an antigen other than the one associated with and / or specific for the disease or condition whereby an activating signal delivered through the disease-targeting CAR is diminished or inhibited by binding of the inhibitory CAR to its ligand, e.g., to reduce off-target effects.

[0402] In certain embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and CD137 (4-1BB, TNFRSF9) co-stimulatory domains, linked to a CD3 zeta intracellular domain.

[0403] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and an activation domain, e.g., primary activation domain, in the cytoplasmic portion. Exemplary CARs include intracellular components of CD3-zeta, CD28, and 4-1BB.

[0404] In some embodiments, the antigen receptor further includes a marker and / or cells expressing the CAR or other antigen receptor further includes a surrogate marker, such as a cell surface marker, which may be used to confirm transduction or engineering of the cell to express the receptor. In some aspects, the marker includes all or part (e.g., truncated form) of CD34, a NGFR, or epidermal growth factor receptor, such as truncated version of such a cell surface receptor (e.g., tEGFR). In some embodiments, the nucleic acid encoding the marker is operably linked to a polynucleotide encoding for a linker sequence, such as a cleavable linker sequence, e.g., T2A. For example, a marker, and optionally a linker sequence, can be any as disclosed in published patent application No. WO2014031687. For example, the marker can be a truncated EGFR (tEGFR) that is, optionally, linked to a linker sequence, such as a T2A cleavable linker sequence.

[0405] In some embodiments, the marker is a molecule, e.g., cell surface protein, not naturally found on T cells or not naturally found on the surface of T cells, or a portion thereof. In some embodiments, the molecule is a non-self molecule, e.g., non-self protein, i.e., one that is not recognized as “self’ by the immune system of the host into which the cells will be adoptively transferred.

[0406] In some embodiments, the marker serves no therapeutic function and / or produces no effect other than to be used as a marker for genetic engineering, e.g., for selecting cells successfully engineered. In other embodiments, the marker may be a therapeutic molecule or molecule otherwise exerting some desired effect, such as a ligand for a cell to be encountered in vivo, such as a costimulatory or immune checkpoint molecule to enhance and / or dampen responses of the cells upon adoptive transfer and encounter with ligand.

[0407] In some cases, CARs are referred to as first, second, and / or third generation CARs. In some aspects, a first generation CAR is one that solely provides a CD3 -chain induced signal uponantigen binding; in some aspects, a second-generation CARs is one that provides such a signal and costimulatory signal, such as one including an intracellular signaling domain from a costimulatory receptor such as CD28 or CD137; in some aspects, a third generation CAR is one that includes multiple costimulatory domains of different costimulatory receptors.

[0408] For example, in some embodiments, the CAR contains an antibody, e.g., an antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of CD28 or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some embodiments, the CAR contains an antibody, e.g., antibody fragment, a transmembrane domain that is or contains a transmembrane portion of CD28 or a functional variant thereof, and an intracellular signaling domain containing a signaling portion of a 4- IBB or functional variant thereof and a signaling portion of CD3 zeta or functional variant thereof. In some such embodiments, the receptor further includes a spacer containing a portion of an Ig molecule, such as a human Ig molecule, such as an Ig hinge, e.g. an IgG4 hinge, such as a hinge-only spacer.

[0409] In some aspects, the spacer contains only a hinge region of an IgG, such as only a hinge of IgG4 or IgGl. In other embodiments, the spacer is or contains an Ig hinge, e.g., an IgG4-derived hinge, optionally linked to a CH2 and / or CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge, linked to CH2 and CH3 domains. In some embodiments, the spacer is an Ig hinge, e.g., an IgG4 hinge...

Claims

CLAIMS1. A co-formulated lipid nanoparticle (co-LNP) comprising a fusion of a first precursor lipid nanoparticle (LNP) and a second precursor lipid nanoparticle (LNP), wherein, prior to fusion:(1) the first precursor LNP comprises:(i) a deoxyribonucleic acid (DNA) molecule; and(ii) a first ionizable lipid; and(2) the second precursor LNP comprises:(i) a first ribonucleic acid (RNA) molecule and optionally a second ribonucleic acid(RNA) molecule; and(ii) a second ionizable lipid, wherein the first and / or second ionizable lipids are selected from the group consisting of OF-C4-Deg- Lin, Lipid 29, Lipid 319, and Lipid A6.

2. A co-formulated lipid nanoparticle (co-LNP) comprising:(1) a deoxyribonucleic acid (DNA) molecule, a first ribonucleic acid (RNA) molecule and optionally a second ribonucleic acid (RNA) molecule; and(2) a first ionizable lipid and a second ionizable lipid, wherein the first and / or second ionizable lipids are selected from the group consisting of OF- C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6.

3. The co-LNP of claim 2, wherein (i) the DNA molecule is associated with the first ionizable lipid; and (ii) the first RNA molecule is associated with the second ionizable lipid.

4. The co-LNP of any one of claims 1-3, wherein the first ionizable lipid and / or the second ionizable lipid is Lipid 319.

5. The co-LNP of any one of claims 1-3, wherein the first ionizable lipid and / or the second ionizable lipid is Lipid 29.

6. The co-LNP of any one of claims 1-3, wherein the first ionizable lipid and / or the second ionizable lipid is Lipid A6.

7. The co-LNP of any one of claims 1 and 4-6, comprising a volumetric ratio of the first LNP to the second LNP that is between about 3:1 and about 1:3.

8. The co-LNP of any of claims 1-7, comprising a first helper lipid and a second helper lipid.

9. The co-LNP of any of claims 1-8, which has an average size of between about 50 nm and 150 nm, or between about 75 nm and about 125 nm, as measured by dynamic light scattering (DLS).

10. The co-LNP of any of claims 1-9, wherein the first ionizable lipid is Lipid 29 and the second ionizable lipid is OF-C4-Deg-Lin.

11. The co-LNP of any of claims 1-9, wherein the first ionizable lipid is Lipid 319 and the second ionizable lipid is OF-C4-Deg-Lin.

12. The co-LNP of any of claims 1-9, wherein the first ionizable lipid is Lipid A6 and the second ionizable lipid is OF-C4-Deg-Lin.

13. The co-LNP of any of claims 1-3, wherein(a) the first ionizable lipid is Lipid 29 and the second ionizable lipid is Lipid 319;(b) the first ionizable lipid is Lipid 29 and the second ionizable lipid is Lipid A6;(c) the first ionizable lipid is Lipid 319 and the second ionizable lipid is Lipid 29;(d) the first ionizable lipid is Lipid 319 and the second ionizable lipid is Lipid A6;(e) the first ionizable lipid is Lipid A6 and the second ionizable lipid is Lipid L29; or(f) the first ionizable lipid is Lipid A6 and the second ionizable lipid is Lipid 319.

14. The co-LNP of any of claims 8-13, wherein the first helper lipid is l-stearoyl-2- oleoyl-sn-glycero-3-phosphocholine (SOPC).

15. The co-LNP of any of claims 8-14, wherein the second helper lipid is 1 ,2-distearoyl- sn-glycero-3-phosphocholine (DSPC).

16. The co-LNP of any of claims 1-15, wherein the DNA molecule comprises a transgene.

17. The co-LNP of claim 16, wherein the transgene encodes a recombinant protein.

18. The co-LNP of claim 17, wherein the recombinant protein is a recombinant receptor.

19. The co-LNP of any one of claims 15-18, wherein the DNA molecule is a naked DNA molecule.

20. The co-LNP of any one of claims 15-19, wherein the DNA molecule is a closed-end DNA (ceDNA) molecule.

21. The co-LNP of any one of claims 15-19, wherein the DNA molecule is a nanoplasmid.

22. The co-LNP of any one of claims 16-18, wherein the transgene is positioned between protelomerase binding sequences.

23. The co-LNP of any one of claims 16-18 and 22, wherein the transgene is operably linked to a promoter and positioned between inverted terminal repeats (ITRs).

24. The co-LNP of any one of claims 16-23, wherein the transgene is from about 2 kilobases to about 10 kilobases.

25. The co-LNP of any one of claims 16-24, wherein the transgene is from about 4 kilobases to about 8 kilobases.

26. The co-LNP of any of claims 18-25, wherein the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

27. The co-LNP of any of claim 26, wherein the recombinant receptor is a CAR.

28. The co-LNP of claim 27, wherein the CAR is a bispecific CAR.

29. The co-LNP of claim 28, wherein the bispecific CAR is between about 5.5 kilobases and 8 kilobases or wherein the bispecific CAR is about 8 kilobases.

30. The co-LNP of any one of claims 27-29, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular region.

31. The co-LNP of claim 30, wherein the extracellular antigen-binding domain is an antibody or an antigen-binding fragment thereof that binds to an antigen that is associated with, or expressed on a cell or tissue of a disease or condition.

32. The co-LNP of claim 31 , wherein the antigen is selected from the group consisting of avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (0GD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight- melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE- A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms Tumor 1 (WT-1).

33. The co-LNP of any one of claims 1-32, wherein the first RNA molecule is or comprises a guide RNA (gRNA).

34. The co-LNP of claim 33, wherein the gRNA is a single guide RNA (sgRNA).

35. The co-LNP of claim 33 or claim 34, wherein the gRNA is complexed with a recombinant nuclease capable of inducing a DNA break.

36. The co-LNP of any of claims 1-35, wherein the co-LNP comprises the first RNA molecule and the second RNA molecule.

37. The co-LNP of any of claims 1-36, wherein the co-LNP further comprises a third ionizable lipid.

38. The co-LNP of claim 37, wherein the second RNA molecule is associated with the third ionizable lipid.

39. The co-LNP of claim 38, wherein the third ionizable lipid is selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A640. The co-LNP of any one of claims 1 and 4-39, wherein the second precursor LNP further comprises the second ribonucleic acid (RNA) molecule.

41. The co-LNP of any one of claims 1-40, wherein the second nucleotide sequence encodes a recombinant nuclease capable of inducing a DNA break.

42. The co-LNP of claim 41, wherein the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas).

43. The co-LNP of claim 42, wherein the Cas is Cas9 or Cas 12a.

44. The co-LNP of claim 42, wherein the nucleotide sequence encodes a transposase.

45. The co-LNP of claim 44, wherein the transposase is a piggyBac transposase or aSleeping Beauty transposase.

46. A tri-formulated lipid nanoparticle (tri-LNP) comprising a fusion of a first precursor lipid nanoparticle (LNP), a second precursor lipid nanoparticle (LNP), and a third precursor lipid nanoparticle (LNP) precursor lipid nanoparticle (LNP), wherein, prior to fusion:(1) the first precursor LNP comprises:(i) a deoxyribonucleic acid (DNA) molecule; and(ii) a first ionizable lipid; and(2) the second precursor LNP comprises:(i) a first ribonucleic acid (RNA) molecule; and(ii) a second ionizable lipid,(3) the third precursor LNP comprises:(i) a second ribonucleic acid (RNA) molecule; and(ii) a third ionizable lipid, wherein the first and / or second and / or third ionizable lipids are selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6.

47. A tri-formulated lipid nanoparticle (tri-LNP) comprising:(1) a deoxyribonucleic acid (DNA) molecule, a first ribonucleic acid (RNA) molecule and a second ribonucleic acid (RNA) molecule; and(2) a first ionizable lipid, a second ionizable lipid, and a third ionizable lipid. wherein the first and / or second and / or third ionizable lipids are selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6.

48. The tri-LNP of claim 47, wherein (i) the DNA molecule is associated with the first ionizable lipid; (ii) the first RNA molecule is associated with the second ionizable lipid; and (iii) the third RNA molecule is associated with the third ionizable lipid.

49. The co-LNP or the tri-LNP of any one of claims 37-48, wherein the first ionizable lipid and / or the second ionizable and / or the third ionizable lipid is Lipid 29.

50. The co-LNP or the tri-LNP of any one of claims 37-48, wherein the first ionizable lipid, the second ionizable and the third ionizable lipid are Lipid 29.

51. The co-LNP or the tri-LNP of any one of claims 37-48, wherein the first ionizable lipid and / or the second ionizable and / or the third ionizable lipid is Lipid 319.

52. The co-LNP or the tri-LNP of any one of claims 37-48, wherein the first ionizable lipid, the second ionizable and the third ionizable lipid are Lipid 319.

53. The co-LNP or the tri-LNP of any one of claims 37-48, wherein the first ionizable lipid and / or the second ionizable and / or the third ionizable lipid is Lipid A6.

54. The co-LNP or the tri-LNP of any one of claims 37-48, wherein the first ionizable lipid, the second ionizable and the third ionizable lipid are Lipid A6.

55. The tri-LNP of any one of claims 46 and 49-54, comprising a volumetric ratio of the first precursor LNP, the second precursor LNP and the third precursor LNP is about 2:1:1.

56. The tri-LNP of any one of claims 46 and 49-54, comprising a volumetric ratio of the first precursor LNP, the second precursor LNP and the third precursor LNP is about 4:1:1.

57. The co-LNP or the tri-LNP of any of claims 37-56, comprising a first helper lipid and a second helper lipid.

58. The co-LNP or the tri-LNP of any of claims 37-57, which has an average size of between about 50 nm and 150 nm, or between about 75 nm and about 125 nm, as measured by dynamic light scattering (DLS).

59. The co-LNP or the tri-LNP of any of claims 37-58, wherein the first ionizable lipid is Lipid 29, the second ionizable lipid is OF-C4-Deg-Lin, and the third ionizable lipid is OF-C4-Deg- Lin.

60. The co-LNP or the tri-LNP of any of claims 37-59, wherein the first ionizable lipid is Lipid 319, the second ionizable lipid is OF-C4-Deg-Lin, and the third ionizable lipid is OF-C4-Deg- Lin.

61. The co-LNP or the tri-LNP of any of claims 37-59, wherein the first ionizable lipid is Lipid A6, the second ionizable lipid is OF-C4-Deg-Lin, and the third ionizable lipid is OF-C4-Deg- Lin.

62. The co-LNP or the tri-LNP of any of claims 37-61, wherein:(a) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 319;(b) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid A6;(c) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 29;(d) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 319;(e) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid A6;(f) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 29;(g) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 319;(h) the first ionizable lipid is Lipid 29, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid A6;(i) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 29;(j) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid A6;(k) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 29;(l) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 319;(m) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid A6;(n) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 29;(o) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 319;(p) the first ionizable lipid is Lipid 319, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid A6;(q) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 29;(r) the first ionizable lipid is Lipid A6 , the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid A6;(s) the first ionizable lipid is Lipid A6 , the second ionizable lipid is Lipid 319, and the third ionizable lipid is Lipid 319;(t) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 29;(u) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid 319;(v) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid 29, and the third ionizable lipid is Lipid A6;(w) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 29; or(x) the first ionizable lipid is Lipid A6, the second ionizable lipid is Lipid A6, and the third ionizable lipid is Lipid 319.

63. The co-LNP or the tri-LNP of any of claims 57-62, wherein the first helper lipid is 1- stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC).

64. The co-LNP or the tri-LNP of any of claims 57-63, wherein the second helper lipid is 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC).

65. The tri-LNP of any of claims 46-64, wherein the DNA molecule comprises a transgene.

66. The tri-LNP of claim 65, wherein the transgene encodes a recombinant protein.

67. The tri-LNP of claim 66, wherein the recombinant protein is a recombinant receptor.

68. The tri-LNP of any one of claims 46-67, wherein the DNA molecule is a naked DNA molecule.

69. The tri-LNP of any one of claims 46-68, wherein the DNA molecule is a closed-end DNA (ceDNA) molecule.

70. The tri-LNP of any one of claims 46-68, wherein the DNA molecule is a nanoplasmid.

71. The tri-LNP of any one of claims 65-67, wherein the transgene is positioned between protelomerase binding sequences.

72. The tri-LNP of any one of claims 65-67 and 71, wherein the transgene is operably linked to a promoter and positioned between inverted terminal repeats (ITRs).

73. The tri-LNP of any one of claims 65-72, wherein the transgene is from about 2 kilobases to about 10 kilobases.

74. The tri-LNP of any one of claims 65-73, wherein the transgene is from about 4 kilobases to about 8 kilobases.

75. The tri-LNP of any of claims 67-74, wherein the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

76. The tri-LNP of any of claim 75, wherein the recombinant receptor is a CAR.

77. The tri-LNP of claim 76, wherein the CAR is a bispecific CAR.

78. The tri-LNP of claim 77, wherein the bispecific CAR is between about 5.5 kilobases and 8 kilobases or wherein the bispecific CAR is about 8 kilobases.

79. The tri-LNP of any one of claims 76-78, wherein the CAR comprises an extracellular antigen-binding domain, a transmembrane domain, and an intracellular region.

80. The tri-LNP of claim 79, wherein the extracellular antigen-binding domain is an antibody or an antigen-binding fragment thereof that binds to an antigen that is associated with, or expressed on a cell or tissue of a disease or condition.

81. The tri-LNP of claim 80, wherein the antigen is selected from the group consisting of avP6 integrin (avb6 integrin), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), epidermal growth factor protein (EGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrin receptor A2 (EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (0GD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPRC5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight- melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HLA-A1), Human leukocyte antigen A2 (HLA-A2), IL-22 receptor alpha(IL-22Ra), IL- 13 receptor alpha 2 (IL-13Ra2), kinase insert domain receptor (kdr), kappa light chain, LI cell adhesion molecule (Ll-CAM), CE7 epitope of Ll-CAM, Leucine Rich Repeat Containing 8 Family Member A (LRRC8A), Lewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE- A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesionmolecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (R0R1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta-isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), and Wilms Tumor 1 (WT-1).

82. The tri-LNP of any one of claims 46-81, wherein the first RNA molecule is or comprises a guide RNA (gRNA).

83. The tri-LNP of claim 82, wherein the gRNA is a single guide RNA (sgRNA).

84. The tri-LNP of claim 82 or claim 83, wherein the second ribonucleic acid (RNA) molecule encodes a recombinant nuclease capable of inducing a DNA break.

85. The tri-LNP of claim 84, wherein the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas).

86. The tri-LNP of claim 85, wherein the Cas is Cas9 or Casl2a.

87. The tri-LNP of claim 84, wherein the nucleotide sequence encodes a transposase.

88. The tri-LNP of claim 87, wherein the transposase is a piggyBac transposase or aSleeping Beauty transposase.

89. The co-LNP of any one of claims 36-45 or the tri-LNP of any one of claims 46-88, wherein the mass ratio between the two RNA molecules (the first RNA molecule and second RNA molecule) and DNA molecule in the tri-LNP is between about 0.5:1 and about 1:3.

90. The co-LNP of any one of claims 37-45 or the tri-LNP of any one of claims 46-88, wherein the mass ratio between the two RNA molecules (the first RNA molecule and second RNA molecule) and DNA molecule in the tri-LNP is between about 1 : 1 and about 1 :2.

91. The co-LNP of any one of claims 1 and 4-45 or tri-LNP of any one of claims 46 and 49-90, wherein the precursor LNPs are prepared in an acidic environment.

92. The co-LNP or tri-LNP of claim 91, wherein the pH of the acidic environment is between about 4 and about 5.

93. The co-LNP of any one of claims 1-45 or tri-LNP of any one of claims 46-92, wherein the co-LNP or tri-LNP shows a fluorescence energy transfer (FRET).

94. The co-LNP or tri-LNP of claim 93, wherein the normalized FRET signal is greater than 0.3.

95. The co-LNP or tri-LNP of claim 94, wherein the normalized FRET signal is greater than 0.35.

96. The co-LNP or tri-LNP of claim 94, wherein the normalized FRET signal is greater than 0.4.

97. A method of producing a co-formulated lipid nanoparticle (co-LNP), comprising:(1) mixing, in an acidic buffer:(a) a first lipid nanoparticle (LNP) comprising a first ionizable lipid and a nucleic acid molecule; and(b) a second LNP comprising a second ionizable lipid and a ribonucleic acid (RNA) molecule, thereby generating a composition comprising the first LNP and the second LNP; and(2) neutralizing the composition comprising the first LNP and the second LNP, thereby generating a co-LNP, which is a fusion of the first LNP and the second LNP, wherein the nucleic acid molecule in (a) is a deoxyribonucleic acid (DNA) molecule or a ribonucleic acid (RNA) molecule, wherein the first and / or second ionizable lipid are selected from the group consisting of OF-C4-Deg- Lin, Lipid 29, Lipid 319, and Lipid A6.

98. The method of claim 97, wherein the nucleic acid molecule in (a) is a DNA molecule.

99. The method of claim 97, wherein the nucleic acid molecule in (a) is an RNA molecule.

100. The method of any of claims 97-99, wherein the volumetric ratio of the first LNP to the second LNP in the composition is between about 3:1 and about 1:3.

101. The method of any of claims 97-100, further comprising mixing, in the acidic buffer, (a) a third LNP comprising a third ionizable lipid and an RNA molecule, wherein the third ionizable lipid is selected from the group consisting of OF-C4-Deg-Lin, Lipid 29, Lipid 319, and Lipid A6, thereby generating a composition comprising the first, second, and third LNPs, optionally wherein the RNA molecule in (b) is a first RNA molecule and the RNA molecule in (c) is a second RNA molecule.

102. The method of claim 101, wherein the method comprises neutralizing the composition comprising the first LNP, the second LNP, and the third LNP, thereby generating a tri- LNP, which is a fusion of the first LNP, the second LNP and the third LNP.

103. The method of claim 101 or claim 102, wherein the volumetric ratio of the first LNP to the second and third LNPs in the composition is between about 3:1 and about 1:3.

104. The method of any of claims 97-103, wherein the acidic buffer is an acetate buffer.

105. The method of any of claims 97-104, wherein the acidic buffer has a pH of between about 3.0 and about 4.5, or of 4.0.

106. The method of any of claims 97-105, wherein the acidic buffer is neutralized to a pH of between about 6.0 and about 7.5, or between about 6.5 and about 7.0.

107. The method of any of claims 97-106, wherein neutralizing the composition comprising the first LNP and the second LNP comprises adding an isotonic buffer.

108. The method of claim 107, wherein the isotonic buffer has a pH of about 7.4.

109. The method of claim 107 or claim 108, wherein neutralizing the composition comprising the first LNP and the second LNP comprises adding at least about 6 parts of the isotonic buffer to 1 part of the acidic buffer.

110. The method of any of claims 107-109, wherein neutralizing the composition comprising the first LNP and the second LNP comprises adding between about 6-7 parts of the isotonic buffer to 1 part of the acidic buffer.

111. The method of any of claims 107-109, wherein the isotonic buffer is phosphate buffered saline (PBS).

112. The method of any of claims 97-111, wherein the DNA molecule comprises a transgene.

113. The method of claim 112, wherein the transgene encodes a recombinant protein.

114. The method of claim 113, wherein the recombinant protein is a recombinant receptor.

115. The method of any of claims 97-114, wherein the DNA molecule is a naked DNA molecule.

116. The method of any of claims 97-114, wherein the DNA molecule is a closed-end DNA (ceDNA) molecule.

117. The method of any of claims 97-114, wherein the DNA molecule is a nanoplasmid.

118. The method of any of claims 114-117, wherein the recombinant receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

119. The method of any of claims 114-118, wherein the recombinant receptor is a CAR.

120. The method of claim 119, wherein the CAR is a bispecific CAR.

121. The method of any of claims 97-120, wherein the RNA molecule in (a) is a guide RNA (gRNA).

122. The method of claim 121, wherein the gRNA is a single guide RNA (sgRNA).

123. The method of any of claims 101-122, wherein the second ribonucleic acid (RNA) molecule in (c) encodes a recombinant nuclease capable of inducing a DNA break.

124. The method of claim 123, wherein the recombinant nuclease is a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN), or a CRISPR-associated nuclease (Cas).

125. The method of claim 124, wherein the Cas is Cas9 or Casl2a.

126. The method of claim 123, wherein the nucleotide sequence encodes a transposase.

127. The method of claim 126, wherein the transposase is a piggyBac transposase or aSleeping Beauty transposase.

128. A co-LNP or tri-LNP produced by the method of any of claims 97-127.

129. A method of genetically engineering an immune cell, the method comprising:(1) introducing a ribonucleic acid (RNA) molecule into an immune cell by electroporation; and(2) incubating the immune cell with a co-LNP of any of claims 1-45 or a tri-LNP of any one of claims 46-96.

130. A method of genetically engineering an immune cell, the method comprising incubating the immune cell with a co-LNP of any of claims 1-45 or a tri-LNP of any one of claims 46-96.

131. A method of genetically engineering an immune cell, the method comprising incubating an immune cell with the co-LNP of any of claims 1-45 or a tri-LNP of any one of claims 46-96.

132. The method of any of claims 129-131, wherein the immune cell is a lymphocyte.

133. The method of any of claims 129-132, wherein the immune cell is a T cell.

134. The method of claim 133, wherein the T cell is a primary T cell.

135. The method of claim 134, wherein the primary T cell is from a human subject.

136. The method of any one of claims 133-135, wherein the T cell is a CD4+ T cell or a CD8+ T cell.

137. The method of any of claims 129-136, wherein, at the time of incubating the immune cell with the LNP, the co-LNP, or the composition, the immune cell is activated.

138. The method of any of claims 129-136, wherein prior to the incubating, the method comprises activating the immune cells under conditions to activate the immune cells.

139. The method of claim 137 or claim 138, wherein the immune cell is a T cell and the T cell is activated with a reagent comprising an anti-CD3 antibody or antigen binding fragment and an anti-CD28 antibody or antigen binding fragment.

140. The method of any of claims 129-139, wherein, at the time of incubating the immune cell with the LNP, the co-LNP, or the composition, the immune cell expresses CD25, CD26, CD27, CD28, CD30, CD71, CD 154, CD40L, CD 134, or a combination thereof.

141. An immune cell produced by the method of any of claims 129-140.

142. A composition comprising a plurality of the immune cell of claim 141.

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