WO2025219491A1 - Method and composition for hdr template dna delivery - Google Patents

Abstract

Provided are compositions and methods for transfecting a cell of hematopoietic lineage, using a lipid nanoparticle (LNP) that includes a lipid mix composition including an ionizable lipid, the lipid mix composition encapsulating an HDR template DNA comprising a gene of interest for insertion into a desired chromosomal locus.

Description

METHOD AND COMPOSITION FOR HDR TEMPLATE DNA DELIVERY

CROSS REFERENCE TO RELATED APPLICATION

[0001] This patent application claims the benefit of U.S. Provisional Patent Application No. 63/635,298, filed on April 17, 2024, which is incorporated by reference in its entirety herein.

FIELD

[0002] The disclosure generally relates to compositions and methods for delivering homology- directed repair (HDR) template DNA to cells of hematopoietic origin.

BACKGROUND

[0003] Lipid nucleic acid-based cell therapy reagents offer advantages over electroporation in traditional oncological treatments in terms of safety and efficacy. Primary human T cells are notoriously difficult to transfect without impairing their survival, even with lipid nanoparticles as carriers. While mRNA need only be present in the cytoplasm, DNA for chromosomal alteration has to cross the nuclear membrane to function. Homology-directed repair (HDR) is a mechanism in cells to repair double-strand DNA breaks and must occur in the nucleus. HDR approaches to genetic therapy using lipid nanoparticles (LNPs) are particularly challenging, especially in human primary cells, which are known to be sensitive to foreign DNA. In addition, native HDR pathways are inactive in mature primary cells.

[0004] In many instances, proteins such as DNases present in the cytoplasm actively degrade any foreign DNA. However, this degradation process doesn’t apply to mRNA since it is not perceived as mislocated in the context of cellular homeostasis. This difference in cellular response underscores the importance of considering the unique characteristics of mRNA and DNA when developing transfection strategies for various cell types using LNPs, in contrast to conventional approaches such as electroporation.

[0005] Cell therapy involves removing a sample of blood from a patient or a healthy volunteer, isolating or enriching desired target cells, transforming these target cells, and then returning the cells back into the body of the donor patient or another patient. One of the goals of cell therapy is to replace diseased or damaged genetics or augment natural immunity with additional expressed proteins. [0006] Currently, electroporation is considered the most feasible nonviral way to genetically modify T cells. This process physically disrupts the cell membrane to force genetic material(s) into cells, and results in some T cells being “irreversibly electroporated” or killed. This process has been considered necessary because of the resistance that T cells have toward “alien DNA”. There is also some risk to the genetic materials being damaged before or during transfer. In addition, electroporated cells can take a long time to proliferate and a recent study showed that the viability of T cells after electroporation was 37%.

[0007] Viral based T cell transfection is labor intensive, expensive and poses manufacturing and regulatory challenges. Also, virus manufacturing methods are expensive because they are highly regulated, need a lot of equipment, and are labor intensive (and may be one batch for each patient). Viral based transfection also poses the risk that viral genome may randomly insert into the human genome and requires that the patient leave the hospital to have T cells harvested and treated at a specialized viral manufacturing facility. Recently FDA has given out a guidance that cell therapy treated patients are to be monitored for several years following chimeric antigen receptor (CAR)T or TCR therapy due to possible risk of gene integration caused by the viral means of manipulation of cells.

[0008] Examples of cell products available commercially for immuno-oncology applications include Kymriah™ for B cell precursor acute lymphoblastic leukemia and Yescarta™ for use in B cell lymphoma. This ex vivo therapy is also called CAR-T therapy wherein modified T cells with CD19-targeted chimeric antigen receptor attacks the CD 19 presenting cancer cells of the patient. Leukemia is the leading cause of mortality in pediatric patients. Use of CAR-T therapy was transformative to the patient’s cancer free recovery.

[0009] Lipid nanoparticles (LNPs) generally consist of different lipids, each serving distinct functions. These LNPs can have a lipidic or aqueous core and may contain bilayer structures depending on the abundance or structure of each type of lipids used. HDR template DNA technology requires approaches that optimize the transport of not only HDR template DNA but the associated Cas9 and guide RNA into the nucleus. Furthermore, the HDR template DNA must be present at the site of repair in adequate quantities.

[0010] Recognized challenges to creating successful LNP cell therapy are safety, manufacturability, stability, and efficacy. A better cell therapy delivery agent with both high efficiency and high live cell yield is still required. Furthermore, there remains an urgent need for HDR in human primary T cells using gentle non-viral systems for wider genomic medicine access to contain costs and increase safety.

BRIEF SUMMARY

[0011] In accordance with embodiments, the invention provides compositions and methods to achieve HDR template delivery to cells of hematopoietic origin at higher efficiency and with better cell survival than electroporation or permanently cationic lipids.

[0012] In embodiments, the LNP is formulated in a lipid composition for cell therapy. In embodiments, the LNP encapsulates a nucleic acid of interest, e.g., a gene of interest, encoding a protein of interest. In embodiments, the LNP encapsulates a gene editing element, such as a Homology Directed Repair (HDR) element, or guide and CRISPR elements. In embodiments, the LNP encapsulates an endonuclease. In embodiments, the LNP encapsulates an HDR element, a guide and CRISPR elements, and an endonuclease.

[0013] In embodiments, the invention comprises compositions comprising a first population of LNPs encapsulating an HDR element and a second population of LNPs encapsulating a gene editing element, and combinations thereof.

[0014] In embodiments, for ex vivo applications, the compositions are administered to biological samples that have been removed from the organism, then those samples treated, washed and restored to the organism. The organism may be a mammal, and may be human. This process is used for cell reprogramming, genetic restoration, or immunotherapy, for example. The drug product is the modified cell.

[0015] In embodiments, the present invention provides a method of modifying human T cells with chimeric antigen receptor (CAR) encoded mRNA to produce CAR-T cell product to be infused back into the patient, without any viral means of delivery of nucleic acid. Non-viral delivery can be a safer technology for modulating the T cell than a virus for programming the cells.

[0016] In embodiments, the present invention provides a method of modulating the T cell receptors to recognize and destroy neoantigens or tumor antigens present on the surface of the tumor cells of the patient, or to modulate T cell populations to treat cancer. T cells may also be modified in other embodiments to ameliorate autoimmune disorders such as celiac disease, Lupus, and diabetes. [0017] In one aspect, the present invention provides a lipid nanoparticle (LNP) for transfecting a cell of hematopoietic lineage, the lipid nanoparticle (LNP) comprising a lipid mix composition comprising an ionizable lipid, the lipid mix composition encapsulating an HDR template DNA comprising a gene of interest for insertion into a target chromosomal locus. [0018] In embodiments, the HDR template DNA is double stranded.

[0019] In embodiments, the HDR template DNA is single stranded.

[0020] In embodiments, the HDR template DNA comprises a chimeric antigen receptor.

[0021] In embodiments, the LNP further comprises an endonuclease or an mRNA encapsulated by the lipid mix composition, wherein the mRNA encodes the endonuclease.

[0022] In embodiments, the endonuclease is a CRISPR-associated endonuclease.

[0023] In embodiments, the LNP further comprises a single guide RNA.

[0024] In embodiments, the HDR template DNA is accompanied with a guide RNA in combination with an endonuclease or an mRNA encoding the endonuclease.

[0025] In embodiments, the lipid mix composition provides a higher knock-in efficiency in an LNP-mediated delivery of the HDR template DNA in primary T cells compared to a lipid mix composition comprising MC3 (4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12- octadecadien-l-yl-10,13-nonadecadien-l-yl ester) or SM-102 (9-Heptadecanyl 8- {(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} octanoate).

[0026] In embodiments, the ionizable lipid comprises a tetrahydrofuranyl or a cyclopentyl head group.

[0027] In embodiments, the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combination thereof.

[0028] In embodiments, the lipid mix composition further comprises a structural lipid, a sterol, a stabilizer, or any combinations thereof.

[0029] In embodiments, the lipid mix composition comprises about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.

[0030] In embodiments, the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijSlO), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof. [0031] In embodiments, the lipid mix composition comprises about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.

[0032] In embodiments, the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijSlO).

[0033] In embodiments, the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).

[0034] In another aspect, the present invention provides a lipid mix composition for encapsulating a HDR template DNA including a gene of interest for insertion into a desired chromosomal locus, the lipid mix composition comprising an ionizable lipid.

[0035] In embodiments, the lipid mix composition provides a higher knock-in efficiency in an LNP-mediated delivery of the HDR template DNA in primary T cells compared to a lipid mix composition comprising MC3 (4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12- octadecadien-l-yl-10,13-nonadecadien-l-yl ester) or SM-102 (9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} octanoate).

[0036] In embodiments, the ionizable lipid comprises a tetrahydrofuranyl or a cyclopentyl head group.

[0037] In embodiments, the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combinations thereof.

[0038] In embodiments, the lipid mix composition further comprises a structural lipid, a sterol, a stabilizer, or any combinations thereof.

[0039] In embodiments, the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijSlO), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof.

[0040] In embodiments, the lipid mix composition comprises about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%. [0041] In embodiments, the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijSlO).

[0042] In embodiments, the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).

[0043] In yet another aspect, the present invention provides a method for transfecting a cell of hematopoietic lineage, the method comprising: contacting cells of hematopoietic lineage with a lipid nanoparticle (LNP) including a lipid mix composition comprising an ionizable lipid, the lipid mix composition encapsulating an HDR template DNA comprising a gene of interest for insertion into a desired chromosomal locus, thereby transfecting the cells; and culturing the cells in a cell culture media.

[0044] In embodiments, the method further comprises isolating the cells from the cell culture media.

[0045] In embodiments, the HDR template DNA is double stranded.

[0046] In embodiments, the HDR template DNA is single stranded.

[0047] In embodiments, the HDR template DNA comprises a chimeric antigen receptor.

[0048] In embodiments, the HDR template DNA is accompanied by an endonuclease or an mRNA encoding the endonuclease.

[0049] In embodiments, the endonuclease is a CRISPR-associated endonuclease.

[0050] In embodiments, the HDR template DNA is accompanied by a single guide RNA.

[0051] In embodiments, the cells are contacted with the LNP containing the HDR template DNA accompanied by a guide RNA in combination with an endonuclease or an mRNA encoding the endonuclease.

[0052] In embodiments, the cells are contacted with a plurality of LNPs, each LNP of the plurality of LNPs containing the HDR template DNA, a guide RNA and/or endonuclease.

[0053] In embodiments, the method further comprises contacting a homology directed repair enhancer with the cells, wherein the homology directed repair enhancer comprises an HDR enhancer, NHEJ inhibitor, DNA sensor inhibitor, or cell cycle syncing molecule.

[0054] In embodiments, a target cell density is between 0.1 to 1 million cells/mL. [0055] In embodiments, contacting cells of hematopoietic lineage includes transfecting primary T cells with the lipid nanoparticle (LNP), wherein the LNP provides a higher knock-in efficiency in delivery of the HDR template DNA in primary T cells compared to a lipid nanoparticle comprising MC3 (4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12- octadecadien-l-yl-10,13-nonadecadien-l-yl ester) or SM-102 (9-Heptadecanyl 8- {(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} octanoate).

[0056] In embodiments, the ionizable lipid comprises a tetrahydrofuranyl or a cyclopentyl head group.

[0057] In embodiments, the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combinations thereof.

[0058] In embodiments, the lipid mix composition of the LNP further comprises a structural lipid, a sterol, a stabilizer, or any combinations thereof.

[0059] In embodiments, the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijSlO), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof.

[0060] In embodiments, the lipid mix composition comprises about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.

[0061] In embodiments, the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijSlO).

[0062] In embodiments, the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).

[0063] Further and alternative aspects and features of the disclosed principles will be appreciated from the following detailed description. As will be appreciated, the compositions and methods disclosed herein are capable of being carried out and used in other and different embodiments, and capable of being modified in various respects. Accordingly, it is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and do not restrict the scope of the appended claims.. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figs. 1A-1B are a bar graph and cytology plots showing the effect of cell density on homology-directed repair (HDR) efficiency during lipid nanoparticle (LNP)-mediated gene insertion in primary T cells. Fig. 1A shows the percent of HA+/CD5+ live T cells normalized to wild-type untreated controls across varying cell densities (0.1, 0.25, 0.5, and 1.0 million cells/mL). Fig. IB shows representative 2D flow cytometry plots comparing the frequency of HA-tagged CD5+ cells at two contrasting cell densities of 1 and 0.1 million cells/mL.

[0065] Figs. 2A-2B show LNP Treatment Kinetics of HDR Efficiency in Primary T Cells with Temporal Separation of Cas9 mRNA + sgRNA and ssODN Delivery. Fig. 2A shows a schematic representation of the timeline of two experimental sets for days of delivery of CRISPR components. In a first experiment set illustrating simultaneous delivery, all components (Cas9 mRNA, sgRNA, and ssODN) were delivered on day 1 (DI), day 2 (D2), or day 3 (D3). In a second set, the delivery of Cas9 mRNA and sgRNA occurred first, followed by the staggered delivery of the ssODN, 24 hours later. Fig. 2B shows raw HA+CD5+ levels, with background levels indicated as untreated (UT), across various time points (DI, D2, D3) and conditions: simultaneous delivery of all components (Dx all, x = 1, 2, or 3), and staggered delivery of ssODN added 24 hours post Cas9/sgRNA treatment (Dx Cas9 + 24hr ssODN). Treatments are further broken down by the weight of Cas9 mRNA/sgRNA and ssODN added per million T cells (5 pg : 1 pg, 1 pg : 1 pg, 5 pg : 5 pg, 5 pg : 10 pg). Significantly increased HDR efficiency is observed with simultaneous delivery of all components compared to the staggered delivery of ssODN added 24 hours post Cas9/sgRNA treatment. With the simultaneous delivery, the highest HDR efficiency is observed at the largest tested dose (5 pg Cas9/sgRNA : 10 pg ssODN per million T cells), among various doses tested. Error bars represent standard deviation across technical replicates.

[0066] Figs. 3A-3C show the comparison of HDR efficiency between different LNP configurations. Fig. 3A shows a diagram depicting LNP encapsulation strategies: either (i) Cas9 mRNA and sgRNA in one LNP with ssODN in a separate LNP (“separate”) or (ii) all three components in the same LNP (“all-in-one”). Fig. 3B is a bar graph showing the HDR efficiency measured as the frequency of HA+ CD5+ primary T cells after treatment with LNPs encapsulating CRISPR/Cas9 components either in a “separate or as “all in one” manner. Fig. 3C shows the cell viability of HA+ CD5+ primary T cells after treatment with LNPs encapsulating CRISPR/Cas9 components either in a “separate or as “all in one” manner. [0067] Figs. 4A-4F are bar graphs and cytology plots showing evaluation of HDR efficiency and cell viability with different concentrations of Alt-R™ HDR Enhancer V2 (IDT, Coralville, IA). Figs. 4A-4C show LNPs produced on the NanoAssmblr® Spark™ nanoparticle formulation system (Cytiva, Marlborough, MA), without further purification. Figs. 4D-4F show LNPs produced on the NanoAssmblr® Ignite™ nanoparticle formulation system (Cytiva, Marlborough, MA), with complete buffer exchange and cleanup. Fig. 4A shows the HDR efficiency of LNPs produced on the NanoAssmblr® Spark™ containing Cas9 mRNA, sgRNA and ssODN, with varying concentrations of HDR Enhancer V2, as indicated. Fig. 4B shows corresponding cell viability normalized to untreated controls, at the indicated HDR Enhancer concentrations. Fig. 4C shows representative 2D flow cytometry plots showing the frequency of HA+CD5+ T cells at 0 pM and 4 pM HDR Enhancer V2. Fig. 4D shows HDR efficiency of LNPs produced on the NanoAssemblr Ignite containing Cas9 mRNA, sgRNA and ssODN, with varying concentrations of HDR Enhancer V2, as indicated. Fig. 4E shows corresponding cell viability normalized to untreated controls, at the indicated HDR Enhancer concentrations. Fig. 4F shows representative 2D flow cytometry plots showing the frequency of HA+/CD5+ T cells at 0 pM and 4 pM HDR Enhancer V2. Error bars represent technical replicates.

[0068] Figs. 5A-5B show the impact of small molecules and nucleic modulators on HDR efficiency and cell viability with PNI 550-LNP_l LNPs. Fig. 5A is a bar graph showing the knock-in efficiency of (HA+/CD5+) following treatment with PNI 550-LNP_l LNPs at various modulator doses (0-20 pM). Modulators tested include RU521 alone, Alt-R HDR Enhancer V2 alone, a combination of RU521 with V2, BX795, XL413, and A151. Fig. 5B shows the corresponding cell viability determined through flow cytometry after treatment with the indicated modulators. Data shows differential toxicity profiles among the modulators, with BX795 maintaining high cell viability at all the doses tested, whereas Alt-R HDR enhancer V2 significantly reduces cell viability at higher concentrations. XL413, Al 51, and RU521 alone show negligible effects on both knock in and viability.

[0069] Figs. 6A-6B show the comparison of proprietary PNI ionizable lipids to benchmark lipids for LNP-mediated HDR in primary human T cells. Fig. 6A is a bar graph showing HA+CD5+ knock-in efficiency normalized to untreated controls, comparing PNI 550 formulated in LNP l to benchmark lipid compositions (SM-102, MC3, BOCHD-DMA) formulated in LNP l and SM-102 formulated in Spikevax formulation. Fig. 6B is a bar graph showing HDR efficiency as GFP+TCR- cell frequency, demonstrating superior gene insertion with PNI lipids relative to MC3 (Onpattro®) and SM-102 (Spikevax®), with error bars reflecting both technical and donor variability. Experiment utilizes a large DNA donor (~2.5 kb, GFP expression) at the TRAC locus. [0070] Figs. 7A-7D are bar graphs and cytology plots showing HDR editing efficiency in primary human T cells using LNPs formulated with different PNI ionizable lipids and compositions. Fig 7A shows an expanded screen of ionizable lipids (PNI-550, 659, 762, 768, and 769) in the LNP l formulation. Fig 7B shows representative flow cytometry 2D plots for PNI 550, PNI 762 and PNI 659 showing the distribution of CD5 and HA antibody stained cells. Fig. 7C is a bar graph showing HA+CD5+ knock-in efficiency for two LNP formulations (LNP l and LNP 2) using ionizable lipids PNI-550, 762, or 516. Fig. 7D shows CAR+TRAC- knock-in efficiency comparing LNP l and LNP 2 formulated with ionizable lipids (PNI-550, 762, or 516), reflecting editing performance with a large donor DNA (3.5 kb) at the TRAC locus.

[0071] Figs. 8A-8B show the HDR efficiency in primary T cells using various LNP formulations with or without Alt-R HDR Enhancer V2. Fig. 8A shows the evaluation of HDR efficiency using LNPs formulated with PNI 550 ionizable lipid, with or without HDR enhancer as indicated. Fig. 8B shows parallel assessment of composition with PNI 762 ionizable lipid.

[0072] Figs. 9A-9B show the assessment of HDR efficiency and cell viability across different LNP l -related formulations. Fig. 9A is a bar graph showing HDR efficiency represented by the percentage of HA+/CD5+ primary T cells (normalized to untreated controls) after treatment with various LNP formulations, including PNI 550-LNP_l and PNI 762-LNP_l. The formulation PNI 762-LNP_l demonstrated the highest HDR efficiency among all the tested compositions. Fig. 9B shows corresponding cell viability (with respect to untreated cells) as determined by flow cytometry. All formulations maintained high viability, indicating that the variations in lipid mix composition do not adversely affect the viability of T cells.

[0073] Figs. 10A-10C show the comparison of HDR knock-in efficiency, post-treatment cell viability, and cell yield between electroporation and LNP-mediated delivery using PNI 550- LNP l in primary T cells. Fig. 10A is a bar graph showing HDR-efficiency as HA+/CD5+ frequency in primary T cells. Both electroporation (EP) and PNI 550-LNP_l LNP delivery methods achieved similar HDR rates, over two healthy donor repeats. Fig. 10B shows the posttreatment cell viability assessed by flow cytometry. Cell viability percentages show PNI 550- LNP l LNP delivery (103% viability with respect to untreated controls) slightly outperforming electroporation (95% viability, with respect to untreated). Fig. 10C shows the yield of edited cells (HA+ cell counts per mL) determined by Acridine Orange/ Propidium Iodide (AO/PI) staining and automated cell counting. PNI 550-LNP_l LNP delivery resulted in a substantially higher yield of HA+ cells (2x10⁵ counts per mL) compared to electroporation (4x10⁴ counts per mL). Error bars represent the standard deviation of measurements, and individual data points for each biological replicate are shown as dots on the bars. No HDR enhancer or similar was included in the experiments.

[0074] Figs. 11A-11E show the comparison of multi-donor HDR editing outcomes using LNP-mediated versus electroporation (EP) delivery in primary T cells. Fig. 11A is a bar graph showing the percentage of CD 19 CAR+/TCR- live cells following HDR editing via PNI 762- LNP l or EP. Fig. 11B shows corresponding post-treatment cell viability (normalized to untreated controls) measured by flow cytometry across multiple donors. Fig. 11C shows the fold expansion of T cell populations by day 7, relative to the starting cell count. Fig. 11D shows the yield of CD 19 CAR+ cells, showing the total cell count post-treatment. Fig. HE shows the cytotoxic activity of LNP l edited CAR T cells co-incubated with CD 19+ SUP-B15 target cells or CD 19- K562 controls at various effector-to-target ratios, measured over 48 hours. For all, error bars denote the standard deviation of biological replicates.

DETAILED DESCRIPTION

[0075] In accordance with an embodiment of the invention, there are provided methods for transforming T cells with HDR elements without disruptive physical methods such as electroporation.

[0076] In another aspect, the invention provides lipid mix compositions including ionizable lipid, one or more phospholipid(s), and stabilizing agent.

[0077] In another aspect, the lipid mix compositions according to the invention are provided for formulating ex vivo cell therapy products, where a modified cell is the drug product.

[0078] In another aspect, the invention provides lipid mix compositions for formulating mRNA LNPs. [0079] In embodiments, the invention provides a composition for transfecting a cell of hematopoietic lineage comprising a lipid nanoparticle (LNP) encapsulating HDR template DNA. In embodiments, the cell is a T cell.

[0080] In embodiments, the LNP comprises an ionizable lipid, a phospholipid and a stabilizing agent. In embodiments, the ionizable lipid has a cyclopentyl headgroup or a tetrahydrofuranyl headgroup. In embodiments, the LNP comprises an ionizable lipid includes PNI 516, PNI 550, PNI 580, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or a combination thereof.

[0081] In embodiments, the DNA comprises a Homology Directed Repair (HDR) template. In embodiments, the mRNA comprises a gene-editing element. In embodiments, the gene editing element includes a Cas9 system element. In embodiments, the invention provides a composition comprising a combination of the LNP compositions as described herein.

[0082] In embodiments, the invention provides a modified cell of hematopoietic lineage comprising a cell of hematopoietic lineage modified by the methods described herein. In embodiments, the invention provides a method of treatment comprising administering to a subject in need thereof an effective amount of a composition of the modified cells described herein.

[0083] Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of the compositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.

[0084] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [0085] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[0086] The practice of the present invention may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature and more current editions thereof, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993- 1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F M. Ausubel et al , eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (IE. Coligan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA. Janeway and P. Travers, 1997); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al, eds., J.B. Lippincott Company, 1993). Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure. [0087] In this disclosure, the recitation of numerical ranges by endpoints includes all numbers subsumed within that range including all whole numbers, all integers, and all fractional intermediates (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5 etc.). In this disclosure the singular forms an “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a composition containing “a compound” includes a mixture of two or more compounds.

[0088] In this disclosure, term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise.

[0089] As used herein, the term “about” is defined as meaning 12.5% plus or minus the recited number. It is used to signify that the desired target concentration might be, for example, 40 Mol%, but that through mixing inconsistencies, the actual percentage might differ by +/- 5 Mol%. [0090] As used herein, the term “substantially” is defined as being 5% plus or minus the recited number. It is used to signify that the desired target concentration might be, for example, 40 Mol%, but that through mixing inconsistencies, the actual percentage might differ by +/- 2 Mol%.

[0091] In this disclosure, the word “comprising” is used in a non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It will be understood that in embodiments which comprise or may comprise a specified feature or variable or parameter, alternative embodiments may consist, or consist essentially of such features, or variables or parameters. A reference to an element by the indefinite article “a” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements.

[0092] In this disclosure, “transfection” means the transfer of nucleic acid into cells. In some cases, it is for the purpose of inducing the expression of a specific gene(s) of interest in both laboratory and clinical settings. In other cases, it is for the purpose of inhibiting expression or function of a deleterious gene. In yet other, non-limiting cases, it is for the purpose of gene editing. It typically includes an ionizable lipid to associate with nucleic acid, and phospholipids. LIPOFECTIN™ and LIPOFECT AMINE™ are established commercial transfecting reagents sold by ThermoFisher Scientific. These research reagents contain permanently cationic lipid(s) and are not suitable for use in vivo or ex vivo. [0093] In this disclosure, “modified” or “genetically modified” or “transfected” are used interchangeably, wherein a cell has been manipulated by means of molecular reprogramming of a genomic sequence (e.g. by insertion, deletion, or substitution). Said cells include the primary transformed cell and progeny derived therefrom without regard to the number of passages.

Progeny may not be completely identical in nucleic acid content to a parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0094] “Lipid” refers to a structurally diverse group of organic compounds that are fatty acid derivatives or sterols or could be lipid like materials as in lipidoids (example Cl 2-200) and are characterized by being insoluble in water but soluble in many organic solvents.

[0095] “Lipid mix compositions” refers to the components that can be used to prepare the lipid nanoparticles (LNPs) encapsulating a payload. Typically, lipid mix compositions for the manufacture of lipid nanoparticles for nucleic acid delivery comprise cationic or ionizable lipid and one or more of phospholipid, cholesterol, or a stabilizer. The stabilizer can include polyethylene glycol conjugated lipids. The lipid mix composition, as used in the instant disclosure, are free of the payload.

[0096] “Lipid mix formulations” refers to the types of components, ratios of components, and/or the ratio of the total components to the nucleic acid payloads (e.g., LNPs including a pay load).

[0097] The lipid mix compositions, which can be used to be mixed with the nucleic acid components, comprise ionizable lipid as described, a neutral lipid or phospholipid or “structural” lipid which helps with the outer bilayer or monolayer of the LNP, optionally cholesterol and optionally a stabilizer as described above. For each application, certain ratios of these four components may be optimized. For vaccine, a mole percent ratio of 50 for ionizable lipid has been used successfully in clinical products. However, targeting gene delivery to cells while maintaining viability of the cells requires a different approach than that used for intramuscular injection and immediate release. In some embodiments illustrated below, lipid mix compositions comprise ionizable lipid (iL), cholesterol, structural lipid, and a stabilizer. In some embodiments, the stabilizer includes PEG DMG, TPGS, polyoxyethylene (40) stearate, polyoxyethylene (10) stearyl ether, polyoxyethylene (20) stearyl ether, Tridecyl -D-maltoside, Polysorbate 20, or Polysorbate 80. Lipid mix compositions may optionally include triglycerides in some embodiments.

[0098] Non-limiting examples of lipid mix compositions (or referred to as “lipid compositions” or “lipid nanoparticle compositions” interchangeably in the instant disclosure) include those disclosed in PCT Publications 2020210901 and 2024006863, which are incorporated by reference herein in their entireties. “Lipid Particles” or “Lipid Nanoparticles” or “LNP” refers to lipid particles manufactured from the lipid mix composition(s) described above and illustrated below. A therapeutic agent such as a nucleic acid may be encapsulated in the lipid mix composition to provide a nucleic acid-containing lipid nanoparticle or nucleic acid lipid nanoparticle (NALNP, or maybe referenced as “LNP” interchangeably in the instant disclosure). In some embodiments, a lipid nanoparticle is a lipid particle under 300 nanometers (nm) in diameter. Lipid particles are generally spherical assemblies of lipids, nucleic acid, cholesterol, and stabilizing agents. Positive and negative charges, ratios, as well as hydrophilicity and hydrophobicity of the components of lipid mix compositions dictate the physical structure of the lipid particles in terms of size and orientation of components. The structural organization of these lipid particles may lead to an aqueous interior with one or more bilayers as in liposomes or it may have a solid interior as in a solid nucleic acid lipid nanoparticle. There may be phospholipid monolayers or bilayers in single or multiple forms. In certain embodiments, lipid particles are between 1 and 1000 nm in diameter.

[0099] “Viability” when referring to cells in vitro or ex vivo, means the ability to continue to grow, divide, or continue to grow and divide, as is normal for the cell type or tissue culture strain. Cell viability is affected by harsh conditions or treatments. Cell viability is important in ex vivo therapy or parenteral administration.

[00100] The compositions of the invention comprise ionizable lipids as a component. As used herein, the term “ionizable lipid” refers to a lipid that is cationic or becomes ionizable (protonated) as the pH is lowered below the pKa of the ionizable group of the lipid but is more neutral at higher pH values. At pH values below the pKa, the lipid is then able to associate with negatively charged nucleic acids (e.g., oligonucleotides). As used herein, the term “ionizable lipid” includes lipids that assume a positive charge on pH decrease from physiological pH, and any of a number of lipid species that carry a net positive charge at a selective pH. Non-limiting examples of suitable ionizable lipids are found in PCT Pub. Nos. WO20252589 and WO21000041, which are incorporated by reference herein in their entireties for all purposes. The ionizable lipid may be present in the lipid nanoparticle composition in any suitable amount or concentration. In some embodiments, the ionizable lipid is present at a concentration of about 10 to about 90 mol% or about 20 to about 70 mol%, e.g., about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, about 80 mol%, about 85 mol%, about 90 mol%, or a concentration within a range defined by any two of the foregoing values. The ionizable lipid is present in lipid compositions according to other embodiments of the invention, preferably in a ratio of about 10 to about 60 Mol%, (“Mol%” means the percentage of the moles that is of a particular component, while the total moles of all the components in the lipid compositions is 100 mol%). The term “about” in this paragraph signifies a plus or minus range of 5 Mol% at increments of 0.1. For example, 28.7 Mol %, 40 Mol %, 47.5 Mol%, 50 Mol % ionizable lipid would all be in the claimed range of embodiments. In some embodiments, the ionizable lipid is present at about 35 to 50 Mol% of the lipid mix composition. DODMA, or 1,2- dioleyloxy-3 -dimethylaminopropane, is an alternative ionizable lipid, as is DLin-MC3-DMA or O-(Z,Z,Z,Z-heptatriaconta-6,9,26,29-tetraen-19-yl)-4-(N,N-dimethylamino) (“MC3”). LNP may be generated from the lipid compositions including the ionizable lipids of the invention.

Representative ionizable lipids of the current disclosure include, but are not limited to, PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combinations thereof, as described herein. Ionizable lipids of the invention include those containing a cyclopentyl or a tetrahydrofuranyl headgroup.

[00101] Phospholipids, as used herein, also known as “helper lipids”, “structural lipids” or “neutral lipids” are incorporated into lipid mix compositions and lipid particles of the invention in embodiments. The structural lipid may be present in the lipid nanoparticle composition in any suitable amount. In some embodiments the structural lipid is present in the lipid nanoparticle composition at a concentration of about 1 to about 75 mol% or about 5 to about 60 mol%, e.g., about 1 mol%, about 5 mol%, about 10 mol%, about 15 mol%, about 20 mol%, about 25 mol%, about 30 mol%, about 35 mol%, about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, about 60 mol%, about 65 mol%, or about 70 mol%, about 75 mol%, or a concentration within a range defined by any two of the aforementioned values. The lipid mix compositions and lipid particles of the invention include one or more phospholipids at about 25 to 60 Mol% of the lipid mix composition. In some embodiments, the one or more phospholipids is present at about 10 to 70 Mol% of the lipid mix formulation. In some embodiments, the one or more phospholipids is present at about 10 to 50 Mol% of the lipid mix formulation. In some embodiments, the one or more phospholipids is present at about 10 to 40 Mol% of the lipid mix formulation. Suitable phospholipids support the formation of particles during manufacture of LNP. Phospholipids refer to any one of several lipid species that exist in either in an anionic, uncharged, or neutral zwitterionic form at physiological pH. Representative phospholipids include, but are not limited to, diacylphosphatidylcholines, diacylphosphatidylethanolamines, diacylphosphatidylglycerols, and although not strictly “phospholipids” in a technical sense, is intended to include sphingomyelins (SM), dihydrosphingomyelins, cephalins, and cerebrosides.

[00102] Representative phospholipids include, but are not limited to, zwitterionic lipids, for example, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl-phosphatidy ethanol amine (SOPE), and 1,2-dielaidoyl-sn- glycero-3-phophoethanolamine (trans DOPE). In one preferred embodiment, the phospholipid is distearoylphosphatidylcholine (DSPC). In preferred embodiments, the phospholipid is DOPE. In preferred embodiments, the phospholipid is DSPC.

[00103] In another embodiment, the phospholipid is any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols such as dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleyolphosphatidylglycerol (POPG), cardiolipin, phosphatidylinositol, diacylphosphatidylserine, diacylphosphatidic acid, and other anionic modifying groups joined to neutral lipids. Other suitable phospholipids include glycolipids (e.g., monosial oganglioside GM1). [00104] “ Stabilizer” or “stabilizing agent” is a term used to identify the agent that is added to the ionizable lipid, the phospholipid, and the sterol that form the lipid mix compositions according to the invention. In some embodiments, the stabilizing agent may include a non-ionic stabilizing agent. Non-limiting examples of non-ionic stabilizing agents include, but are not limited to, Polyethyleneglycol (PEG), DMG-PEG2000 (l,2-dimyristoyl-rac-glycero-3-methoxypoly ethylene glycol-200), Polysorbates (Tweens), TPGS (Vitamin E polyethylene glycol succinate), Brij™ S20 (polyoxyethylene (20) stearyl ether), Brij™35 (Polyoxyethylene lauryl ether, Polyethyleneglycol lauryl ether), Brij™S10 (Polyethylene glycol octadecyl ether, Polyoxyethylene (10) stearyl ether), Myrj™52 (polyoxyethylene (40) stearate), or any combinations thereof. Additional non-limiting examples of stabilizing agents include those disclosed in PCT applications PCT/EP2024/075129, PCT/EP2024/075124, PCT/EP2024/075128, which are incorporated by reference herein in their entireties.

[00105] In some embodiments, the stabilizing agent includes PEGylated lipids including but not limited to PEG-DMG 2000 (“PEG-DMG”). Other polyethylene glycol conjugated lipids may also be used. The stabilizing agent may be used alone or in combination with each other.

[00106] In some embodiments, there is no stabilizing agent. In other embodiments, the stabilizing agent comprises about 0.1 to 5 Mol% of the lipid mix composition. In some embodiments, the stabilizing agent includes about 0.5 to 2.5 Mol% of the mix composition. In preferred embodiments, the stabilizing agent is present at greater than 1.0 Mol%. In some embodiments the stabilizing agent is present at 5 Mol%. In some embodiments the stabilizing agent is present at 10 to 15 Mol%. In some embodiments, the stabilizing agent is present at 2.5 to 10 Mol%. In some embodiments, the stabilizing agent has a mol% of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or a mol% value within a range defined by any two of the aforementioned values. In other embodiments, the stabilizing agent is present at greater than 10 Mol% of the mix composition.

[00107] Sterols are included in some embodiments, lipid mix compositions for certain applications, and lipid particles made therefrom include, but are not limited to, cholesterol, betasitosterol, 20-alpha-hydroxysterol, and/or phytosterol. In the lipid mix compositions of the invention, sterol is present at about 15 to 50 Mol% of the lipid mix composition in some embodiments. In some embodiments, sterol is present at about 15 to 25 Mol% of the lipid mix formulation. In some embodiments, a modified sterol or synthetically derived sterol is present. [00108] In the case of cell therapeutics, delivery is to a particular cell type or population, commonly in vitro or ex vivo. In the case of vaccines, delivery is localized to the skin or muscle. As used herein, the term “nucleic acid” is meant to include any oligonucleotide or polynucleotide whose delivery into a cell causes a desirable effect. The definition includes diagnostic agents and research reagents which follow the same physical principles afforded by the invention. Fragments containing up to 50 nucleotides are generally termed oligonucleotides, and longer nucleotides are called polynucleotides. In particular embodiments, oligonucleotides of the present invention are 20-50 nucleotides in length. In embodiments of the invention, polynucleotides are 996 to 4500 nucleotides in length, as in the case of messenger RNA. In particular embodiments, polynucleotides of the invention include up to 14,000 nucleotides.

[00109] The term “nucleic acid” refers to ribonucleotides, deoxynucleotides, modified ribonucleotides, modified deoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can be single stranded, double stranded, or contain portions of both double stranded and single stranded sequence, as appropriate. Messenger RNA (mRNA) can be modified or unmodified, base modified, and may include different type of capping structures, such as Capl. In some embodiments nucleic acid refers to self-amplifying RNA (“saRNA”). In some embodiments, nucleic acid refers to a ssODNA and mRNA.

[00110] As used herein, the terms “polynucleotide” and “oligonucleotide” are used interchangeably and mean single-stranded and double-stranded polymers of nucleotide monomers, including 2'-deoxyribonucleotides (DNA), land ribonucleotides (RNA) linked by internucleotide phosphodiester bond linkages, e.g., 3'-5' and 2'-5', inverted linkages, e.g., 3'-3' and 5'-5', branched structures, or internucleotide analogs. Polynucleotides have associated counter ions, such as H⁺, NH4⁺, trialkylammonium, Mg²⁺, Na⁺, and the like. A polynucleotide may be composed entirely of deoxyribonucleotides (ssODNs), entirely of ribonucleotides (RNA), or chimeric mixtures thereof.

[00111] HDR (Homology-Directed Repair) is a gene editing process of repairing doublestranded DNA breaks by using a template DNA with homologous sequences. The template may be single or double stranded. In the case that it is single stranded, it may be called ssODNs. It is commonly used to introduce precise changes in the genome, such as gene editing, gene knockout, or introducing specific mutations. HDR is primarily used for genome editing applications. It allows for the precise modification of specific genes by introducing changes at the DNA level.

This is commonly used in techniques like CRISPR-Cas9 and CRISPR-Casl2a gene editing. HDR allows for highly precise changes in the genome because it relies on homologous sequences to guide the repair process. HDR permits the copying of a donor strand template of DNA into the region of a double-strand break (DSB).

[00112] Transcription Activator-Like Effector Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs) are precise gene editing technologies designed to introduce specific double-strand breaks (DSBs) in DNA. TALENs utilize customizable DNA-binding domains from Xanthomonas bacteria for targeted DNA sequence recognition, while ZFNs employ engineered zinc finger motifs, each binding to a distinct 3-nucleotide sequence. Both are fused to a FokI nuclease for DNA cleavage. Their specificity makes them suitable for targeted gene editing when used alongside a donor DNA template, facilitating Homology -Directed Repair (HDR) for genomic modifications such as gene correction or mutation introduction.

[00113] ‘ ‘Cell therapeutics,” as defined herein, encompass a diverse array of medical treatments utilizing living cells for therapeutic purposes. This definition broadly includes, but is not limited to: Immunotherapies, such as Chimeric Antigen Receptor T-cell (CAR-T) therapy, T Cell Receptor (TCR) therapy, and Tumor-Infiltrating Lymphocytes (TIL) therapy, which modify and reinfuse immune cells to target neoplastic diseases; Hematopoietic cell therapies, involving the transplantation and manipulation of hematopoietic stem cells (HSCs) for treating hematological disorders, including leukemia and lymphoma, and especially genetic disorders, using either autologous or allogeneic stem cells; Natural Killer (NK) Cell Therapies: These therapies utilize NK cells, a type of cytotoxic lymphocyte critical to the innate immune system, for targeting tumor cells and cells infected by pathogens; and Mesenchymal Stem Cell (MSC) Therapies and Others: Covering treatments with MSCs, applicable in regenerative medicine and autoimmune diseases. This last category also includes therapies using neural stem cells, induced Pluripotent Stem Cells (iPSCs), and other cell types for diverse therapeutic applications.

[00114] Examples of nucleic acid pay load that can be encapsulated in the lipid mix composition of the instant disclosure include, but are not limited to, an antisense oligonucleotide (ASO), a ribozyme, a microRNA (miRNA), a messenger RNA (mRNA), a transfer RNA (tRNA), a transactivating CRISPR RNA (tracrRNA), a guide RNA, a single guide RNA, a self-amplifying RNA (SAM or saRNA), a small nuclear RNA (snRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a circular RNA (circRNA), a non-coding RNA (ncRNA), a self-replicating DNA, a locked nucleic acid (LNA), a DNA, a replicon, a pre-condensed DNA, a transposon, a single gene, a vector, a plasmid or a pDNA, an aptamer, or a combination thereof. In some embodiments, the nucleic acid is an antigen encoded mRNA for prophylactic or therapeutic vaccine, a nucleic acid for gene therapy, or a nucleic acid for immunogenic cell incorporation, wherein the immunogenic cell is a T cell, natural killer cell, dendritic cell, macrophage, or tumorinfiltrating leukocyte. In some embodiments, the incorporation is performed in vitro, ex vivo, or in vivo. In some embodiments, the nucleic acid payload is an mRNA, or saRNA. In some embodiments, the therapeutic agent includes a nucleic acid. In some embodiments, a nucleic acid payload includes deoxyribonucleic acid, complementary deoxyribonucleic acid, or complete genes for gene therapies targeting a variety of diseases, such as cancer, infectious diseases, genetic disorders and neurodegenerative diseases. As described herein, a nucleic acid payload including but not limited to a nucleic acid therapeutic (NAT) or nucleotide of interest, is incorporated into the lipid particle during its formation. More than one nucleic acid therapeutic may be incorporated in this way. The nucleic acid payload may be derived from natural sources, or more commonly, synthesized or grown in culture.

[00115] In embodiments, nucleic acid reagents or payloads are used to silence genes (with for example siRNA), express genes (with for example mRNA), edit genomes (with for example CRISPR/Cas9), and reprogram cells for return to the originating organism (for example ex vivo cell therapy to reprogram immune cells for cancer therapy; autologous transfer or allogenic transfer).

[00116] The nucleic acid that is present in a lipid particle according to this invention may include any form of nucleic acid that is currently known or later developed. The nucleic acids used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or DNA- RNA hybrids. Examples of ssODN include homology arm at desired gene locus along with desired insert. Examples of double-stranded RNA include siRNA and other RNA interference reagents. Single-stranded nucleic acids include antisense oligonucleotides, guide RNA, including CRISPR-Cas9 gRNA, ribozymes, microRNA, mRNA, and triplex-forming oligonucleotides. In embodiments, more than one nucleic acid may be incorporated into the lipid particle, for example mRNA and guide RNA together, or different types of each, or in combination with protein.

[00117] In some cases, a nucleic acid encodes a genetically engineered receptor that specifically binds to a ligand, such as a recombinant receptor, and a molecule involved in a metabolic pathway, or functional portion thereof. Alternately, the molecule involved in a metabolic pathway is a recombinant molecule, including an exogenous entity. A genetically engineered receptor and the molecule involved in a metabolic pathway may be encoded by one nucleic acid or two or more different nucleic acids. In some examples, a first nucleic acid might encode a genetically engineered receptor that specifically binds to a ligand and a second nucleic acid might encode the molecule involved in a metabolic pathway.

[00118] Gene of Interest (“GOI”) is the nucleic acid molecule intended to be integrated and expressed, or exist on an exosome and be expressed, in the cell or in a bioreactor. Genes of Interest include those encoding insulin, human growth hormone, CFTR, P globin, 5 globin, y globin, BCL11A, KLF1, CCR5, CXCR4, PPP1R12C (AAVS1), HPRT, albumin, Factor VIII, Factor IX, LRRK2, Htt, SOD1, C9orf72, TARDBP, FUS, RHO, CFTR, SFTPB, TRAC, TRBC, PD1, CTLA-4, HLA A, HLA B, HLA C, HLA-DP, HLA-DQ, HLA-DR, LMP7, TAP 1, TAP2, TAPBP, CIITA, DMD, GR, IL2RG, Rag-1, RFX5, FAD2, FAD3, ZP15, KASII, MDH, EPSPS, or a fragment thereof.

[00119] Gene of Interest may encode a bispecific T cell engager (BiTE) molecule; a hormone; a cytokine (e.g., IL-2, insulin, IFN-y, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-a), a chemokine (e.g., MIP-la, MIP-ip, MCP-1, MCP-3, and RANTES), a cytotoxin (e.g., Perforin, Granzyme A, and Granzyme B), a cytokine receptor (e.g., an IL-2 receptor, an IL-7 receptor, an IL- 12 receptor, an IL- 15 receptor, and an IL-21 receptor), or an engineered antigen receptor.

[00120] Nucleic acid payloads may include both coding and non-coding genes of interest. Coding regions are the instructions for building proteins, which are essential molecules for the structure, function, and regulation of the body's cells and tissues. Coding regions, also known as exons, are the segments of DNAthat directly encode the amino acid sequence of a protein. These regions are transcribed into messenger RNA (mRNA), which serves as a template for protein synthesis during translation. Mutations in coding regions can lead to changes in the amino acid sequence of the resulting protein, which may affect its structure or function. Coding regions are highly conserved across species and are crucial for understanding the genetic basis of inherited diseases and the development of therapeutic interventions.

[00121] Noncoding regions, also known as introns and regulatory sequences, are segments of DNAthat do not code for proteins. Introns are spliced out during mRNA processing, and only the exons are retained in the mature mRNA. Regulatory sequences, such as promoters, enhancers, and silencers, play critical roles in controlling gene expression by influencing the transcriptional activity of genes. Noncoding regions are involved in various cellular processes, including gene regulation, chromatin structure, and RNA processing. Mutations in noncoding regions can impact gene expression levels or patterns, leading to phenotypic changes or disease susceptibility. Noncoding regions also contain regions of repetitive DNA, such as transposable elements, which can contribute to genome instability and genetic diversity.

[00122] Noncoding regions of the genome do not directly encode proteins but can code for noncoding RNAs (ncRNAs). Noncoding RNAs are RNA molecules that are transcribed from DNA but are not translated into proteins. Instead, they perform various regulatory and structural functions within the cell. Examples of noncoding RNAs include: tRNA molecules are involved in translating the genetic code from mRNA into amino acid sequences during protein synthesis, rRNA molecules are components of ribosomes, the cellular machinery responsible for protein synthesis. They help catalyze the assembly of amino acids into proteins, miRNAs are small RNA molecules that regulate gene expression by binding to specific mRNA molecules and either inhibiting their translation or promoting their degradation, Long non-coding RNA (IncRNAs) are RNA molecules longer than 200 nucleotides that do not encode proteins. They play diverse roles in gene regulation, chromatin organization, and other cellular processes, Small nuclear RNAs (snRNAs) are involved in the processing of pre-mRNA transcripts, including splicing and other RNA modification processes, Small Nucleolar RNAs (snoRNAs) guide the chemical modification of ribosomal RNA and other RNAs, and PiWi- interacting RNAs (piRNAs) are involved in silencing the activity of transposable elements in the genome, thereby maintaining genomic stability.

[00123] Nucleic acid payload of the instant disclosure may encode an engineered T cell receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor.

[00124] “Therapeutic agents” as used herein include nucleic acids as herein described, or nucleic acid therapeutics (“NAT”), proteins, peptides, polypeptides, and small molecules.

[00125] The term “polypeptides” herein encompasses “oligopeptides” and “proteins” and tertiary and quaternary structures thereof, that are therapeutic agents in some embodiments. An oligopeptide generally consists of from two to twenty amino acids. A polypeptide is a single linear chain of many amino acids of any length held together by amide bonds. A protein consists of one or more and may include structural proteins, energy catalysts, albumin, hemoglobin, immunoglobulins, and enzymes. [00126] The lipid particles of the invention can be assessed for size using devices that size particles in solution, such as the Malvern™ Zetasizer™. The particles generally have a mean particle diameter of from 15 nm to 1000 nm. A subgroup of lipid particles is “lipid nanoparticles” or LNP with a mean diameter of from about 15 to about 300 nm. In some embodiments, the mean particle diameter is greater than 300 nm. In some embodiments, the lipid particle has a diameter of about 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less, 100 nm or less, or 50 nm or less. In one embodiment, the lipid particle has a diameter of from about 50 to about 150 nm. Smaller particles generally exhibit increased circulatory lifetime in vivo compared to larger particles. Smaller particles have an increased ability to reach tumor sites than larger nanoparticles. In one embodiment, the lipid particle has a diameter from about 15 to about 50 nm.

[00127] The lipid particles according to embodiments of the invention can be prepared by standard T-tube mixing techniques, turbulent mixing, trituration mixing, agitation promoting orders self-assembly, or passive mixing of all the elements with self-assembly of elements into nanoparticles. A variety of methods have been developed to formulate lipid nanoparticles (LNP) containing genetic drugs. Suitable methods are disclosed in U.S. Pat. No. 5,753,613, U.S. Pat. No. 6,734,171, and U.S. Pat. No. 7,901,708, by way of example. These methods include mixing preformed lipid particles with nucleic acid therapeutic (NAT) in the presence of ethanol or mixing lipid dissolved in ethanol with an aqueous media containing NAT and result in lipid particles with NAT encapsulation efficiencies of 65-99%. All of these methods rely on the presence of ionizable lipid to achieve encapsulation of NAT and a stabilizing agent to inhibit aggregation and the formation of large structures. The properties of the lipid particle systems produced, including size and NAT encapsulation efficiency, are sensitive to a variety of lipid mix compositions parameters such as ionic strength, lipid and ethanol concentration, pH, NAT concentration and mixing rates.

[00128] Microfluidic two-phase droplet techniques have been applied to produce monodisperse polymeric microparticles for drug delivery or to produce large vesicles for the encapsulation of cells, proteins, or other biomolecules. The use of hydrodynamic flow focusing to create monodisperse liposomes of controlled size has also been demonstrated.

[00129] Parameters such as the relative lipid and NAT concentrations at the time of mixing, as well as the mixing rates are difficult to control using existing formulation procedures, resulting in variability in the characteristics of NAT produced, both within and between preparations. The new lipid mix compositions of the disclosure is unique in that the ratio of ionizable lipid to phospholipid is surprisingly low. Automated micro-mixing instruments such as the NanoAssemblr® instruments (Cytiva, USA) enable the rapid and controlled manufacture of nanomedicines (liposomes, lipid nanoparticles, and polymeric nanoparticles). NanoAssemblr™ instruments accomplish controlled molecular self-assembly of nanoparticles via microfluidic mixing cartridges that allow millisecond mixing of nanoparticle components at the nanoliter, microliter, or larger scale with customization or parallelization. Rapid mixing on a small scale allows reproducible control over particle synthesis and quality that is not possible in larger instruments.

[00130] Preferred methods incorporate instruments such as the microfluidic mixing devices like the NanoAssemblr™ series including Spark™, Ignite™, Blaze™, GMP system or commercial formulation system, in order to achieve nearly 100% of the nucleic acid used in the formation process is encapsulated in the particles in one step. In preferred embodiments, the lipid particles are prepared by a process by which from about 75 to about 100% of the nucleic acid used in the formation process is encapsulated in the particles.

[00131] U.S. Pat. Nos. 9,758,795 and 9,943,846 describe methods of using small volume mixing technology and novel formulations derived thereby. U.S. Pat. No. 10,159,652 describes more advanced methods of using small volume mixing technology and products to formulate different materials. U.S. Pat. No. 9,943,846 discloses microfluidic mixers with different paths and wells to elements to be mixed. PCT Pub. No. WO 2017117647 discloses microfluidic mixers with disposable sterile paths. U.S. Pat. No. 10,076,730 discloses bifurcating toroidal microfluidic mixing geometries and their application to microfluidic mixing. PCT Pub. No. W02018006166 discloses a programmable automated micromixer and mixing chips, therefore. U.S. Design Nos. D771834, D771833, D772427, D803416, D800335, D800336 and D812242 disclose mixing cartridges having microchannels and mixing geometries for mixer instruments sold by Cytiva, USA.

[00132] In embodiments of the invention, devices for biological microfluidic mixing are used to prepare the lipid particles according to embodiments of the invention. The devices include a first and second stream of reagents, which feed into the microfluidic mixer, and lipid particles are collected from the outlet, or emerge into a sterile environment.

[00133] The first stream includes a therapeutic agent in a first solvent. Suitable first solvents include solvents in which the therapeutic agents are soluble and that are miscible with the second solvent. Non-limiting examples of suitable first solvents include aqueous buffers. Representative first solvents include citrate and acetate buffers, or optionally other low pH buffers.

[00134] The second stream includes lipid mix materials in a second solvent. Suitable second solvents include solvents in which the ionizable lipids according to embodiments of the invention are soluble, and that are miscible with the first solvent. Non-limiting examples of suitable second solvents include 1,4-dioxane, tetrahydrofuran, acetone, acetonitrile, dimethyl sulfoxide, dimethylformamide, acids, and alcohols. Representative second solvents include aqueous ethanol 90%, or anhydrous ethanol.

[00135] In one embodiment of the invention, a suitable device includes one or more microchannels (i.e., a channel having its greatest dimension less than 2 millimeters). In one example, the microchannel has a diameter from about 20 to about 300 pm. In another example, the microchannel has a diameter from about 300 to about 1000 pm. In examples, at least one region of the microchannel has a principal flow direction and one or more surfaces having at least one groove or protrusion defined therein, the groove or protrusion having an orientation that forms an angle with the principal direction (e.g., a staggered herringbone mixer), as described in U.S. Pat. No. 9,943,846, or a bifurcating toroidal flow as described in U.S. Pat. No. 10,076,730. To achieve maximal mixing rates, it is advantageous to avoid undue fluidic resistance prior to the mixing region. Thus, one example of a device has non-microfluidic channels having dimensions greater than 1000pm, to deliver the fluids to a single mixing channel.

[00136] Less complex mixing methods and instruments such as those disclosed in, for example, U.S. Published Patent Application No. 20040262223, are also useful in creating lipid particle formulations of the invention.

[00137] The lipid mixes of the present invention may be used to deliver a therapeutic agent to a cell, in vitro, ex vivo, or in vivo. In particular embodiments, the therapeutic agent is a nucleic acid, which is delivered to a cell using nucleic acid-lipid particles of the present invention. The nucleic acid can be an siRNA, miRNA, a LNA replicon (including a vector with antigenic mRNA), a selfamplifying RNA, an mRNA, a guide RNA, long noncoding RNA (IncRNA), a short hairpin RNA (shRNA), an aptamer, a ribozyme, circular RNA, a CRISPR/Cas-based editing cassette, or a DNA molecule such as an antisense oligonucleotide (ASO), a single stranded oligonucleotide (ssODN), plasmid DNA (pDNA), a vector, a single gene, a transposon, PCR amplicons, single- or doublestranded linear DNA, circular DNA, coiled DNA, or supercoiled DNA. [00138] In other embodiments, the therapeutic agent is an oligopeptide, polypeptide, or protein which is delivered to a cell using peptide-lipid particles of the present invention. In other embodiments, the therapeutic agent is a mixture of nucleic acid and protein components, such as Cas9. The methods and lipid mix formulations may be readily adapted for the delivery of any suitable therapeutic agent for the treatment of any disease or disorder that would benefit from such treatment.

[00139] In certain embodiments, the present invention provides methods for introducing a nucleic acid into a cell (i.e., transfection). Transfection is a technique commonly used in molecular biology for the introduction of nucleic acid cargo (or NATs) from the extracellular to the intracellular space for the purpose of transcription, translation and expression of the delivered nucleic acid therapeutic (NAT) for production of some gene product or for down regulating the expression of a disease-related gene. Transfection efficiency is commonly defined as either the i) percentage of cells in the total treated population showing positive expression of the delivered gene, as measured by live or fixed cell imaging (for detection of fluorescent protein), and flow cytometry or ii) the intensity or amount of protein expressed by treated cell(s) as analyzed by live or fixed cell imaging or flow cytometry or iii) using protein quantification techniques such as ELISA, or western blot. These methods may be carried out by contacting the lipid particles or lipid mix formulations of the present invention with the cells for a period of time sufficient for intracellular delivery to occur.

[00140] Typical applications include using well known procedures to provide intracellular delivery of siRNA to knock down or silence specific cellular targets in vitro and in vivo. Alternatively, applications include delivery of DNA or mRNA sequences that code for therapeutically useful polypeptides. In this manner, therapy is provided for genetic diseases by supplying deficient or absent gene products. Methods of the present invention may be practiced in vitro, ex vivo, or in vivo. For example, the lipid mix formulations of the present invention can also be used for delivery of nucleic acids to cells in vivo, using methods which are known to those of skill in the art. In another example, the lipid mix formulations of the invention can be used for delivery of nucleic acids to a sample of patient cells that are ex vivo, then are returned to the patient.

[00141] The delivery of nucleic acid cargo by a lipid particle of the invention is described below. For in vivo administration, the pharmaceutical compositions are preferably administered parenterally (e.g., intraarticularly, intravenously, intraperitoneally, subcutaneously, intrathecally, intradermally, intratracheally, intraosseous, intramuscularly or intratumorally). In particular embodiments, the pharmaceutical compositions are administered intravenously, intramuscularly, intrathecally, or intraperitoneally by a bolus injection. Other routes of administration include topical (skin, eyes, mucus membranes), oral, pulmonary, intranasal, sublingual, rectal, and vaginal. [00142] For ex vivo applications, the pharmaceutical compositions are preferably administered to biological samples that have been removed from the organism, then the cells are washed and restored to the organism. The organism may be a mammal, and in particular may be human. This process is used for cell reprogramming, genetic restoration, or immunotherapy, for example. [00143] In one embodiment, the present invention provides a method of modulating the expression of a target polynucleotide or polypeptide. These methods generally comprise contacting a cell with a lipid particle of the present invention that is associated with a nucleic acid capable of modulating the expression of a target polynucleotide or polypeptide. As used herein, the term “modulating” refers to altering the expression of a target polynucleotide or polypeptide. Modulating can mean increasing or enhancing, or it can mean decreasing or reducing.

[00144] ‘ ‘Cells of the hematopoeitic lineage” includes hematopoietic stem cells, precursor immune cells such as T cells and B cells, macrophages, or natural killer cells. The term is intended to encompass cells of both the innate and adaptive immune system.

[00145] B cells can be isolated from whole blood by cells sorting or magnetic activated bead cell sorting. Moore DK, Motaung B, du Plessis N, Shabangu AN, Loxton AG; SU-IRG Consortium. Isolation of B-cells using Miltenyi MACS bead isolation kits. PLoS One. 2019 Mar 20; 14(3). White cells in general can be separated from whole blood using density. Immune cell isolation includes methods that enable the enrichment of immune cell subsets using antibody- mediated recognition of specific cell surface antigens, followed by sorting or separation with techniques such as flow cytometry, density centrifugation or magnetic isolation.

[00146] A T cell, or T lymphocyte, is a lymphocyte subtype that has the lead role in cell- mediated immunity. T cells can be distinguished from other white blood cells, (for example, B cells or natural killer cells), by the existence of a T cell receptor on the cell surface. The main categories of T cells include Helper (CD4+), Cytotoxic (CD8+), Memory and Regulatory T cells. [00147] The log phase of growth with reference to T cell cultures means, for example, the time that the cells undergo a rapid expansion, around day 5 or day 6 post activation. Log phase can be observed through a sudden increase in cell count, this rapid expansion can be used as a time point to begin preparing LNPs for T cell treatment. In embodiments of the invention, T cells may be activated in different ways. The triple activation method using anti-CD3/CD28/CD2 antibodies is exemplified below, but dual activation was also effective in our studies. Dual activation is performed using anti CD3/CD28 antibodies. Current clinically used protocols employ the dual activation protocol.

[00148] T cells may in some cases be derived from differentiated from induced pluripotent stem cells (iPSC) or Embryonic Stem Cells (ESC).

[00149] Preparation of T cells for transformation by methods of the invention includes one or more culture and/or preparation steps. The T cells are usually isolated from biological tissue (such as peripheral blood or arterial blood) derived from a mammalian subject. In some embodiments, the subject from which the cell is isolated has a disease or condition or in need of a cell therapy or to which cell therapy will be administered. In some embodiments, the subject from which the cell is isolated is a healthy human donor or volunteer.

[00150] The cells in some embodiments are primary cells, such as primary human cells. The tissue sources include blood, tissue, lymph, or other tissue sources taken directly from the subject, and samples resulting from one or more processing steps, such as separation, centrifugation, washing, and/or incubation.

[00151] The tissue source from which the T cells are derived may be a blood or a blood-derived tissue source, or an apheresis or leukapheresis product. Exemplary tissue sources include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, lymph node, spleen, or other lymphoid tissues. The cells in some embodiments are obtained from a different species than the eventual subject needing therapy.

[00152] Isolation of the cells may include more preparation or non-affinity-based cell separation. In some cases, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove or enrich for certain components.

[00153] In some cases, cells from the circulating blood of a subject are obtained by apheresis or leukapheresis. The blood cells may be washed to remove the plasma fraction, and an appropriate buffer or media is used for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, a washing step is performed by tangential flow filtration (TFF) according to the manufacturer's instructions (Spectrum Krosflo, GE Akta Flux, for example). In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca⁺⁺/Mg⁺⁺ free PBS.

[00154] Unmodified cells mean cells that have not been treated to any structural or genetic changes after removal from a living body. Modified cells mean cells that have been augmented or changed in some way during or after removal from a living body.

[00155] Separating the T cells from tissue sources may involve density-based cell separation methods, including the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll™ or Ficoll™ gradient. Other methods include the separation of different cell types based on the expression or presence in the cell of one or more specific surface markers.

[00156] 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, can be isolated by positive or negative selection techniques. As one example, CD3⁺, CD28⁺ T cells can be positively selected using CD3/CD28 conjugated magnetic beads (e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander). A CD4⁺ or CD8⁺ selection step can be used to separate CD4⁺ helper and CD8⁺ cytotoxic T cells. Memory T cells are present in both CD62L⁺ and CD62L" subsets of CD8⁺ peripheral blood lymphocytes. Alternatively, a selection for CD4⁺ helper cells may be undertaken. In some cases, naive CD4⁺ T lymphocytes are CD45RO", CD45RA⁺, CD62L⁺, CD4⁺ T cells. In others, central memory CD4⁺ cells are CD62L⁺ and CD45RO⁺. In still other cases, effector CD4⁺ cells are CD62L" and CD45RO.

[00157] Cell populations can also be isolated using affinity magnetic separation techniques. The cells to be separated are incubated with magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads™ (Clontech) or MACS™ (Miltenyi) beads). The magnetically responsive material is attached to a binding partner that specifically binds to a surface marker, present on the cell, cells, or population of cells that it is desired to separate. T cells may be isolated by positive or negative selection processes from tissue sources depending on preference. Kits for both are available, for example, from StemCell Technologies in Vancouver, Canada.

[00158] For therapeutic purposes, isolation or separation is carried out using an apparatus that carries out one or more of the isolation, cell preparation, separation, processing, an incubation, required to transform the T cells. In some aspects, the system is used to carry out each of these steps in a closed or sterile environment. In one example, the system is a system as described in United States Patent Pub. No. 20110003380 Al. Separation and/or other steps may be accomplished using the CliniMACS system (Miltenyi Biotec). 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. A desired cell population can be collected and enriched via flow cytometry, in which cells stained for multiple cell surface markers are carried in a fluid stream. Other methods include FACS or microelectromechanical systems (MEMS) chips in combination with a FACS-based detection system (see, e.g., WO 2010/033140).

[00159] T cell incubation and treatment may be carried out in a culture vessel, such as a chamber, well, column, tube, tubing set, valve, vial, culture dish, bag, tank, or other container for culture or cultivating cells. Stimulating conditions or agents include one or more agent, such as a ligand, capable of activating an intracellular signaling domain of a TCR complex. Incubation may be carried out as described in U.S. Pat. No. 6,040,177 to Riddell et al. T cell cultures can be expanded by adding 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.

[00160] T cell stimulating conditions include temperatures suitable for the growth of human T lymphocytes, for example, from 25 to 37 degrees Celsius. Optionally, the incubation may further include a supportive population of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells, at a ratio to initial T cells of 10 to 1.

[00161] In other embodiments, the present invention provides a method of treating a disease or disorder characterized by over expression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from a ssODN accompanied by endonuclease and gene editing protein and guide RNA. [00162] In still other embodiments, the present invention provides a method of treating a disease or disorder characterized by under-expression of a polypeptide in a subject, comprising providing to the subject a pharmaceutical composition of the present invention, wherein the therapeutic agent is selected from a plasmid or DNA which includes a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof. In certain embodiments, the therapeutic agent is selected from an mRNA, a self-amplifying RNA (saRNA), or a ssODNA, includes a nucleic acid therapeutic that specifically encodes or expresses the under-expressed polypeptide, or a complement thereof. Examples include RNA vaccines, and more particularly self-amplifying mRNA vaccines.

[00163] For delivery of a biologically active agent (e.g., RNA encoding an immunogen) to cells of the immune system (e.g., antigen-presenting cells, including professional antigen presenting cells), in one embodiment formulation of the invention is delivered intramuscularly, after which immune cells can infiltrate the delivery site and process delivered RNA and/or process encoded antigen produced by non-immune cells, such as muscle cells. Such immune cells can include macrophages (e.g., bone marrow derived macrophages), dendritic cells (e.g., bone marrow derived plasmacytoid dendritic cells and/or bone marrow derived myeloid dendritic cells), T-cells, and monocytes (e.g., human peripheral blood monocytes), etc. (for example, see W02012/006372). [00164] The DNA is delivered with a lipid formulation of the invention (e.g., formulated as a liposome or LNP). In some embodiments, the invention utilizes LNPs within which immunogenencoding ssODNs is encapsulated. Encapsulation within LNPs can protect ssODNs from DNAse digestion. The encapsulation efficiency does not have to be 100%. Presence of external bp ssODNs molecules (e.g., on the exterior surface of a liposome or LNP) or “naked” DNA molecules (DNA molecules not associated with a liposome or LNP) is acceptable. Preferably, for a formulation comprising lipids and DNA molecules, at least half of the DNA molecules (e.g., at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least about 96%, at least about 97%, at least about 98%, or at least 99% of the ssODNs molecules) are encapsulated in LNPs or complexed with LNPs.

[00165] Some lipid nanoparticles may comprise a lipid core (e.g., the formulation may comprise a mixture of LNPs and nanoparticles with a lipid core). In such cases, the DNA or ssODNs molecules may be encapsulated by LNPs that have an aqueous core or cores and complexed with the LNPs that have a lipid core by noncovalent interactions (e.g., ionic interactions between negatively charged DNA and cationic lipid). Encapsulation and complexation with LNPs (whether with a lipid or aqueous core) can protect DNA from DNase digestion. The encapsulation/complexation efficiency does not have to be 100%. Presence of “naked” ssODNs molecules (DNA molecules not associated with the LNP) is acceptable. Preferably, for a formulation comprising a population of LNPs and a population of DNA molecules, at least half of the population of DNA molecules (e.g., at least e.g., at least 50 %, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the DNA molecules) are either encapsulated in LNPs or complexed with LNPs.

[00166] Some lipid nanoparticles have multilamellar components such as phospholipid bilayers and aqueous pockets.

[00167] For delivery of immunogen-coding HDR template DNA or ssODNs, the preferred range of LNP diameters is in the range of 60-180 nm, and in more particular embodiments, in the range of 80-160 nm. An LNP can be part of a composition comprising a population of LNPS, and the LNPS within the population can have a range of diameters. For a composition comprising a population of LNPs with different diameters, it is preferred that (i) at least 80% by number of the LNP have diameters in the range of 60-180 nm, e.g., in the range of 80-160 nm, (ii) the average diameter (by intensity, e.g., Z-average) of the population is ideally in the range of 60-180 nm, e.g., in the range of 80-160 nm; and/or the diameters within the plurality have a polydispersity index <0.2. To obtain LNPs with the desired diameter(s), mixing can be performed using a process in which two feed streams of aqueous DNA solution are combined in a single mixing zone with one stream of an ethanolic lipid solution, all at the same flow rate e.g., in a microfluidic channel. See other description relating to NanoAssemblr® microfluidic mixers sold by Cytiva, USA.

[00168] In some embodiments, the DNA codes for specific HDR templates with desired molecular changes neoantigens in cancer cells or solid tumours. In some embodiments, the RNA is an mRNA to a tumor antigen selected from: (a) cancer-testis antigens such as NY-ESO-I, SSX2, SCPI as well as RAGE, BAGE, GAGE and MAGE family polypeptides, for example, GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (which can be used, for example, to address melanoma, lung, head and neck, NSCLC, breast, gastrointestinal, and bladder tumors; (b) mutated antigens, for example, p53 (associated with various solid tumors, e.g., colorectal, lung, head and neck cancer), p21/Ras (associated with, e.g., melanoma, pancreatic cancer and colorectal cancer), CDK4 (associated with, e.g., melanoma), MUMI (associated with, e.g., melanoma), caspase-8 (associated with, e.g., head and neck cancer), CIA 0205 (associated with, e.g., bladder cancer), HLA-A2-R1701, beta catenin (associated with, e.g., melanoma), TCR (associated with, e.g., T-cell non-Hodgkins Lymphoma), BCR-abl (associated with, e.g., chronic myelogenous leukemia), triosephosphate isomerase, KIA 0205, CDC-27, and LDLRFUT; (c) over-expressed antigens, for example, Galectin 4 (associated with, e.g., colorectal cancer), Galectin 9 (associated with, e.g., Hodgkin’s disease), proteinase 3 (associated with, e.g., chronic myelogenous leukemia), WT I (associated with, e.g., various Leukemias), carbonic anhydrase (associated with, e.g., renal cancer), aldolase A (associated with, e.g., lung cancer), PRAME (associated with, e.g., melanoma), HER-2/neu (associated with, e.g., breast, colon, lung and ovarian cancer), mammaglobin, alpha-fetoprotein (associated with, e.g., hepatoma), KSA (associated with, e.g., colorectal cancer), gastrin (associated with, e.g., pancreatic and gastric cancer), telomerase catalytic protein, MUC-I (associated with, e.g., breast and ovarian cancer), G-250 (associated with, e.g., renal cell carcinoma), p53 (associated with, e.g., breast, colon cancer), and carcinoembryonic antigen (associated with, e.g., breast cancer, lung cancer, and cancers of the gastrointestinal tract such as colorectal cancer); (d) shared antigens, for example, melanoma-melanocyte antigens such as MART-1/ Melan A, gplOO, MCIR, melanocytestimulating hormone receptor, tyrosinase, tyrosinase related protein-I/TRPI and tyrosinase related protein- 2/TRP2 (associated with, e.g., melanoma); (e) prostate associated antigens such as PAP, PSA, PSMA, PSH-PI, PSM-PI, PSM-P2, associated with e.g., prostate cancer; (f) immunoglobulin idiotypes (associated with myeloma and B cell lymphomas, for example). In certain embodiments, tumor immunogens include, but are not limited to, pl 5, Hom/Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens, including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigens, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23HI, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, pl 6, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29&BCAA), CA 195, CA 242, CA-50, CAM43, CD68&KPI, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY- CO-I, RCASI, SDCCAG16, TA-90 (Mac-2 binding protein/cyclophilin C-associated protein), TAAL6, TAG72, TLP, TPS, and the like.

[00169] Compositions in accordance with the present disclosure comprise an effective amount of the lipid formulations described herein (e.g., LNP), as well as any other components, as needed. In embodiments, the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of associating the active ingredient with an excipient and/or one or more other accessory ingredients.

[00170] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one- third of such a dosage.

[00171] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1 percent and 99 percent (w/w) of the active ingredient.

[00172] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams and Wilkins, Baltimore, MD, 2006). The use of a conventional excipient medium is contemplated herein, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition.

[00173] In some embodiments, the particle size of the lipid particles may be increased and/or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the NAT delivered to mammals by changing biodistribution. Size may also be used to determine target tissue, with larger particles being cleared quickly and smaller one reaching different organ systems. The following is a description of representative lipid particles prepared with nucleic acid (LNP), how they are made, evidence of their advantages, and methods for using them to deliver therapeutic benefits. EXAMPLES

[00174] General considerations: All solvents and reagents were commercial products and used as such unless noted otherwise. Temperatures are given in degrees Celsius.

ABBREVIATIONS ng = nanogram g = gram h = hour(s)

MFI = Median Fluorescence Intensity min = minute(s) mL = milliliter(s) mmol = millimole(s)

N/P ratio = the ratio of positively chargeable lipid amine (N = nitrogen) groups to negatively charged nucleic acid phosphate (P) groups

PBS = phosphate buffered solution

C21 -200 = 1 , 1 ' - [ [2- [4- [2- [ [2- [bis(2-hy droxydodecy l)amino] ethyl] (2- hydroxydodecyl)amino]ethyl]-l-piperazinyl]ethyl]imino]bis-2-dodecanol

MC3 = 4-(dimethylamino)-butanoic acid, (10Z,13Z)-l-(9Z,12Z)-9,12-octadecadien-l-yl-10,13- nonadecadien-l-yl ester

SM-102 = 9-Heptadecanyl 8- {(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino} octanoate

[00175] ‘ ‘Gene of interest” (GOI) signifies a genetic element or elements intended for expression to achieve a therapeutic goal, including immunization. Chimeric antigens, cancer- associated antigens, autoimmune associated antigens, and epidermal growth factor (EPO) are examples of a GOI, but GOI is not limited to these examples.

[00176] iL = ionizable lipid, a lipid that is cationic at lower pH, and converts to uncharged at higher pH. ILs are commonly used in formulations of nucleic acid cargo.

[00177] UT = untreated

[00178] Components of the Lipid Mixes include the ionizable lipid, phospholipid, cholesterol, and stabilizing agent. Low pH buffers (3-6) may be used. For ionizable lipids, the pH of the buffer is typically below the pKa of the lipid. Table 1. Ionizable Lipids (iL) Examples

[00179] The ionizable lipids of the present disclosure have asymmetric centers. It is to be understood that while specific configurations of non-limiting examples of ionizable lipids were shown in Table 2, in some embodiments, the ionizable lipids may occur as racemates, racemic mixtures, individual enantiomers, enantiomeric mixtures, individual diastereomers, or as diastereomeric mixtures, with all possible isomers like tautomers and mixtures thereof.

Methods and Materials

Example 1

Isolation of T cells

[00180] T cells were isolated from whole human blood, buffy coats, leukapheresis products, or other sources obtained from healthy or patient donors. Blood or apheresis material was treated with anticoagulants such as ACDA, EDTA, or heparin. Purification was performed using immunomagnetic selection methods (e.g., positive selection for CD3 or CD4/CD8 cells or negative selection of non-T-cell populations), such that the resulting fraction typically contained CD4+ and CD8+ T cells.

T Cell Cryopreservation and Recovery

[00181] Isolated T cells were cryopreserved in a cryoprotectant solution (for example, 10% DMSO or a proprietary commercial formulation such as the CryoStor® CS10 medium by STEMCELL Technologies) and stored in liquid or vapor-phase nitrogen. On the day of use, cryovials were thawed at about 37 °C. Thawed cells were transferred to a basal medium and washed at least once (by centrifugation) to remove residual cryoprotectant. Cell count was performed by automated fluorescent cell counter using acridine orange/propidium iodide solution to obtain live cell count.

Primary T Cell Preparation

[00182] T cells must be activated to proliferate and differentiate into effector cells. In the lab, activation can be mimicked by a specific set of cytokines (signalling proteins) such as IL-2 and other proteins like CD2, CD3, CD28. Upon activation, T cells will grow rapidly. Immediately after thaw, or within 24 hours, T cells were activated using bead-bound, plate-bound, or soluble activators that engage CD3/CD28 and optionally with additional co-stimulatory molecules (such as CD2). Activation was performed in flasks, multi-well plates, G-Rex vessels (Wilson Wolf Manufacturing, USA), or Xuri Cell Expansion System W25 (Cytiva, USA), depending on the scale. Cytokines such as IL-2, IL-7, and/or IL- 15 were added to support proliferation. The cell density was maintained between about 0.1 * 10^A6 and 2 * 10^A6 cells/mL, and cultures were incubated at 37 °C with 5% CO2. Media changes or perfusion was performed as necessary to sustain growth and viability.

T Cell Expansion

[00183] Following the initial activation phase (typically 1-5 days), T cells may be further expanded in flasks, G-Rex vessels, or Xuri Cell Expansion System to accommodate for large-scale production. The cell density was monitored regularly, and sub-culturing or media addition/perfusion was performed to maintain predefined cell densities.

T Cell Gene Modification

A. LNP-Based Delivery

[00184] During the 0-4 days post-activation period, T cells were treated with LNP formulations containing mRNA(s) encoding proteins of interest, and/or gene-editing components (such as CRISPR systems). Typical dosing was approximately 1-10 pg of total nucleic acid per

1 x 1O^A6 cells, but higher or lower doses could be used depending on the target and experimental design. T cells were incubated for 24 - 48 hours for transient protein expression or 72 - 96 hours for gene editing / insertion techniques, throughout which culture media was refreshed or perfused as needed.

B. Electroporation or Nucleofection

[00185] As a comparison, in certain examples, T cells were electroporated or nucleofected with equivalent cargoes to that of LNPs (i.e. 4D-Nucleofector® by Lonza). Commercial electroporation protocols were employed (such as EH- 140 or EO-100 using recommended buffers), and transfected T cells were immediately returned to culture in an appropriate T cell medium to recover for the appropriate length of time.

T Cell Analysis and Downstream Processing

[00186] Gene-modified T cells were collected at timepoints ranging from Day 1 to Day 10 (post-modification), or beyond, for analysis by flow cytometry and/or functional tests (e.g., cytotoxicity, cytokine secretion). If required, cells were further expanded, harvested, and/or re-cryopreserved.

LNP Preparation

[00187] Lipid nanoparticles, LNPs, could be prepared on the NanoAssemblr® Spark™, Ignite™, Blaze™, GMP or commercial formulation system (Cytiva, USA) for testing. In a nonlimiting example, genetic materials including messenger RNA (mRNA), single/synthetic guide RNA (sgRNA), DNA, or combinations thereof, were diluted in an aqueous phase (e.g., 100 mM sodium acetate buffer (pH 4)) to a predefined concentration (e.g., 0.1 to 0.5 mg/mL), depending on the amount of ionizable lipid and lipid mix concentration, and desired N/P ratio. N/P ratio typically ranged from 2-20. Non-limiting examples of the effective ratio of RNA to DNA is exemplified in Example 2.

[00188] An organic phase including lipid mix composition dissolved in an organic solvent is also used. In some cases, a concentration of 12.5, 25, 37.5 or 50 mM was used. However, other concentrations of lipid mix composition solution can be used as well. In some cases, ethanol is used as the organic solvent but other organic solvents can be used instead of ethanol. LNP were then prepared by running both fluids, namely, aqueous phase including RNAZDNA payload and organic phase including the lipid mix composition at a predefined flow ratio (e.g., 2:1, 3: 1, or 5: 1 (aqueous : organic phases)) and at a predefined total flow rate (e.g., 8-20 mL/min) in the microfluidic mixer. Other flow ratios and/or flow rates can also be used.

[00189] In some cases, following mixing in the microfluidic device, the post cartridge lipid nanoparticle sample including the pay load was diluted into RNAse free tubes containing about 10 to about 40 volumes of PBS, pH 7.4. Ethanol was removed through either dialysis in phosphate buffered saline (PBS), pH 7.4, or using Amicon™ centrifugal filters (Millipore, USA) at 2500- 4000 RCF, or using TFF systems. Once the required concentration was achieved, the lipid nanoparticles were filter sterilized using 0.2 pm filters in aseptic conditions.

[00190] Final encapsulation efficiency was measured by the Ribogreen™ assay and Quant-iT™ RiboGreen® RNA Reagent and Kit (Invitrogen, USA) following manufacturer directions.

Observed particle attributes were generally sized from 50 - 200 nm for mRNA, depending on lipid composition and genetic therapeutic payload.

Single Guide RNAs and DNA Template Sequences

[00191] Various homology directed repair (HDR) templates (in the form of a single-stranded oligodeoxynucleotides, ssODN, or plasmid DNA or double stranded linear DNA or single stranded linear DNA) were encapsulated as part of the LNPs, separately or along with Cas9 mRNA and single guide RNA (sgRNA). Encapsulation approaches are shown in Example 3.

[00192] LNP-mediated HDR optimization in primary T cells utilizes hemagglutinin (HA) tagging of CD5 based on the following publication Shy et al. 2023: Shy, Brian R., et al. "High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.

Nature biotechnology 41.4 (2023): 521-531.

Table 2: CD5 sgRNA and HA-tag Donor

[00193] Other constructs include the use of a CD 19 chimeric antigen receptor (CAR) plasmid DNA to the TRAC (T cell receptor) locus. The CD 19 insert sequence was designed as previously published by Kath et al. 2022, with the homology arms modified to fit a different the sgRNA: Kath, J, Du, W., Pruene, A., Braun, T., Thommandru, B., Turk, R., . & JVagner, D. L. (2022). Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells. Molecular Therapy-Methods & Clinical Development, 25, 311-330.

Table 3. TCR sgRNA and CD19 CAR and GFP Donor (both donors to the same target site)

NHEJ Inhibitors, HDR Enhancers and DNA Sensor Inhibitors

[00194] Small molecule or nucleic-acid based HDR modulators (non-limiting list in Table 4) were either directly added to the T cell culture or co-encapsulated within the lipid nanoparticles for delivery. Depending upon the small molecule properties, co-encapsulation may be achieved by inclusion within the aqueous or organic phase during LNP production. For small molecules, such as V2 HDR enhancer, 1-20 pM of the HDR enhancer was added to the cell mixture containing 1 pg/mL of ApoE at the time of, or up to 12 hours before treatment with LNPs.

Table 4. Examples of small molecule or nucleic acid-based HDR enhancers.

Detection Performed at Day 7 After Thaw for Gene Editing:

[00195] Wells were counted AO/PI on an automatic fluorescent cell counter.

Table 5. Stain matrix

[00196] The plate was centrifuged at 300 x g for 5 minutes at room temperature (RT), after which the supernatant was removed. A viability stain, specifically FVS660 (BD Bioscience, 564405) diluted 1 : 1000 in PBS, was added to a volume of 200 pL to the treated, untreated, and various fluorescence minus one (FMO) wells. For all other wells, 200 pL of PBS was added. The plate was incubated for 10 minutes in the dark at RT, then centrifuged again at 300 x g for 5 minutes, and the supernatant was discarded. Subsequently, each well received 200 pL of BSA stain buffer (BD Biosciences, 554657).

[00197] The plate underwent two wash cycles, each involving centrifugation at 300 x g for 5 minutes at RT and removal of the supernatant. Antibody staining was then conducted for CD5, HA, TCR, or CAR markers. For CD5 and HA staining, the respective antibodies were added to the treated, untreated, and viability FMO wells, along with BSA stain buffer to the unstained and FMO controls. Similarly, TCR and CAR primary antibodies were added to the relevant wells, followed by incubation, the addition of BSA stain buffer, wash, and secondary CAR antibody staining.

[00198] After a final 15 -minute incubation in the dark at RT, the wells were washed with 200 pL of BSA stain buffer and centrifuged. The cell pellets were resuspended in either 0.2% BSA staining buffer or RPMI + 2% FBS, with the volume dependent on the cell count per well. The plate was then kept covered, protected from light, and on ice until acquisition, with the final resuspension volumes optimized for the CytoFLEX™ flow cytometer (Beckman Coulter), though alternative volumes were recommended for use with other cytometers.

Tumor Cell Killing Assay

[00199] To evaluate antigen-specific cytotoxic activity of engineered T cells, a co-culture assay was performed using labeled target cells (SUP-B15, CD 19+ or K562, CD19-) and the effector anti-CD19 CAR T cells. Target cells were RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 1 mM Sodium Pyruvate, lx MEM and lx GlutaMax (R+FBS media) for at least 7 days after thaw and used for up to 12 passages. Prior to co-culture, the target cells were pre- labeled with a metabolic viability dye, violet proliferation dye 450 (VPD450), to enable downstream identification and quantification of cell viability by flow cytometry. Following the 24 h or 48 h co-culture with effector cells at various effector-to-target (E:T) ratios, total cell viability was assessed at the time of detection using a second viability stain (FVS660). Cytotoxicity was quantified based on the loss of viable target cells. Specific lysis values were calculated compared to untreated target cell controls to determine relative cytotoxic potency of the engineered CAR T cell. Comparison between CD 19+ and CD 19- target cells allows determining crowding effects or non-specific non-CD19 mediated killing.

Example 1

[00200] Primary T cells were cultured at densities of 0.25, 0.5, or 1 million cells per milliliter prior to or at the time of LNP addition. Post-transfection with LNPs containing a Cas9 mRNA, sgRNA and a HDR template (96h), HA+ T cells were quantified. At all densities tested, significant HDR rates were achieved, between 15-28% relative to untreated controls.

[00201] Figs. 1A-1B show the cell density for LNP-Mediated HDR editing in primary T cells. Fig. 1A shows the percent HA+/CD5+ live T cells normalized to wild-type untreated controls at the indicated cell densities (0.1, 0.25, 0.5, and 1.0 million cells/mL). Significant HDR efficiencies, 15% to 28%, were achieved at both all tested densities, 15% to 28%. Error bars represent three technical replicates. Error bars represent three technical replicates. Fig. IB shows representative 2D flow plots of comparing the frequency of HA-tagged CD5+ cells at two contrasting cell densities, 0.1 and 1.0 million cells/mL.

Example 2

[00202] Treatment kinetics for LNP-mediated HDR in primary T cells was conducted.

Simultaneous or staggered delivery were used for the delivery of the ssODN, and the Cas9 mRNA + sgRNA. HDR efficiency was assessed by the percentage of HA+ and CD5+ cells. Shown in Figs. 2A and 2B, optimal LNP treatment point was observed when all cargoes were delivered simultaneously on all days tested, Day 1, Day 2 and Day 3 (DX all), with a significant decrease in efficiency when ssODN-LNP was added 24 hours post Cas9 mRNA/sgRNA delivery (e.g. “D3 all” vs. “D3 Cas9/sgRNA + 24hr ssODN”, Fig. 2B). Furthermore, among the samples tested in Fig. 2B, the condition of 5 pg Cas9 mRNA+sgRNA : 10 pg ssODN provided the highest %KI for HDR efficiency. These results show a flexible temporal window for LNP-mediated delivery of HDR components. Regarding the specific treatment dose, we theorize that the elevated dose (e.g., 15 pg total nucleic acid / million cells compared to other tested samples as shown in Fig. 2B), along with the excess DNA, are beneficial for homology directed repair in cells.

[00203] Figs. 2A-2B show LNP treatment kinetics of HDR efficiency in primary T cells with temporal separation of Cas9 mRNA + sgRNA and DNA (e.g., ssODN) delivery. Fig. 2A is a schematic representation of experiment timeline for days of delivery of CRISPR components. All components (Cas9 mRNA, sgRNA, and ssODN) were delivered on Dayl (DI), Day 2(D2), or Day 3 (D3). Additional set included the delivery of Cas9 mRNA and sgRNA first, then the staggered delivery of the ssODN 24 hours later. Fig. 2B shows raw HA+CD5+ levels, with background levels indicated as untreated (UT), across various time points (DI, D2, D3) and conditions: simultaneous delivery of all components (DX all), and staggered delivery of ssODN added 24 hours post Cas9/sgRNA treatment (Dx Cas9 + 24hr ssODN). Treatments are further broken down by the weight of Cas9 mRNA/sgRNA and ssODN added per million T cells (5 pg : 1 pg, 1 pg : 1 pg, 5 pg : 5 pg, 5 pg : 10 pg). Optimal HDR efficiency is observed with simultaneous delivery on all days, Day 1 to Day 3 (DX all), at the largest tested dose (5 pg Cas9/sgRNA : 10 pg ssODN per million T cells) with efficiency decreasing with delayed ssODN addition. Error bars represent standard deviation across technical replicates.

Example 3

[00204] Figs. 3A-3C show comparison of HDR efficiency and cell viability between different LNP configurations. Fig. 3A shows encapsulation strategy, either Cas9 mRNA and sgRNA in one LNP and DNA in another LNP (“separate” encapsulation), or all three components in the same LNP (“all-in-one”). Fig. 3B shows HDR efficiency represented by the percentage of HA+ CD5+ primary T cells after treatment with LNPs encapsulating CRISPR/Cas9 components either in a segregated manner (Cas9 mRNA and sgRNA in one LNP, ssODN in another), or as a combined LNP (‘All in one’). Significance evaluated using student t-test of select groups, n.s. (nonsignificant) is p > 0.05. Error bars represent n=3 or n=6 technical replicates. Knock in percentage was normalized to untreated controls. 1 pM HDR enhancer V2 was added to cell culture in these examples. Fig. 3C shows cell viability post-LNP treatment (with respect to untreated controls) with the corresponding LNP configurations. [00205] In this non-limiting example, a comparative study was conducted to evaluate HDR efficiency in primary T cells utilizing two different encapsulation strategies for the delivery of Cas9 mRNA, sgRNA, and ssODN. Separate LNPs for RNA and DNA components or the combined encapsulation in a single PNI 550-LNP_l LNP formulation all the components (see Fig 3A). The results, normalized to untreated controls, indicated that HDR efficiency did not significantly differ between the two encapsulation methods (Fig. 3B). By maintaining equivalent nucleic acid doses across both delivery strategies, the percentage of HA+/CD5+ cells were comparable when either the all-in-one LNP formulation or when separate LNPs were used. Cell viabilities between the two strategies remains the same, showing no impact on cell viability compared to untreated controls (Fig. 3C). These findings suggest that the physical co-packaging of the RNA and DNA components into a single nanoparticle is not critical for HDR outcome, thus providing flexibility in LNP design and application for gene editing in primary T cells.

Example 4

[00206] Figs. 4A-4F show evaluation of HDR efficiency and cell viability with different concentrations of Alt-R HDR enhancer V2. Figs. 4A-4C show LNPs produced on the NanoAssmblr Spark platform, without further purification. Figs. 4D-4F show LNPs produced on the NanoAssmblr Ignite platform, with complete buffer exchange and cleanup. Fig. 4A shows HDR efficiency of PNI 550-LNP_l LNPs containing Cas9 mRNA, sgRNA and ssODN, with varying concentrations of HDR Enhancer V2, as indicated in the x-axis. Fig. 4B shows corresponding cell viability normalized to untreated controls, at the indicated HDR Enhancer concentration. Fig. 4C shows representative 2D flow cytometry plots showing the frequency of HA+CD5+ T cells at 0 pM and 4 pM HDR Enhancer V2, respectively. Fig. 4D shows HDR efficiency of PNI 550-LNP_l LNPs produced on the Ignite platform containing Cas9 mRNA, sgRNA and ssODN, with varying concentrations of HDR Enhancer V2, as indicated. Fig. 4E shows corresponding cell viability normalized to untreated controls, at the indicated HDR Enhancer concentration. Fig. 4F shows representative 2D flow cytometry plots showing the frequency of HA+/CD5+ T cells at 0 pM and 4 pM HDR Enhancer V2. Error bars represent technical replicates.

[00207] Dose- response analysis of IDT’ s Alt-R HDR Enhancer V2 was conducted to assess the impact on HDR efficiency. For Spark-produced PNI 550-LNP_l LNPs, without added LNP cleanup and downstream processing, the addition of the HDR enhancer V2 significantly increased HDR efficiency in a dose-dependent manner, with the highest HDR efficiency observed at a 4 pM enhancer concentration. In contrast, LNPs produced on the Ignite platform, which incorporates a cleanup step, demonstrated marginal improvements with enhancer concentrations above 1 -2 pM. Importantly, both platforms allow for significant HDR efficiency without added enhancers, 26% and 41% for the Spark and Ignite respectively. However, the significantly higher editing results using the Ignite highlight the importance of LNP purification in conjunction with HDR enhancers for optimizing gene editing efficiency. HDR Enhancer V2 mitigated limitations associated with non-purified LNPs, though its efficacy was less pronounced with purified LNPs.

Example 5

[00208] Figs. 5A-5B show the impact of small molecule and nucleic acid modulators on HDR efficiency and cell viability with PNI 550-LNP_l LNPs. Fig. 5A shows the knock-in efficacy (% KI) of PNI 550-LNP_l LNPs at various modulator doses (0-20 pM), represented as HA+/CD5+ cells normalized to untreated controls. Modulators tested include RU521 alone, Alt-R HDR Enhancer V2 alone, a combination of RU521 with V2, BX795, XL413, and A151. Fig. 5B shows the corresponding cell viability determined through live/ dead flow cytometry staining after treatment with the indicated modulators.

[00209] This example assesses the impact of small molecule modulators and a DNA oligonucleotide (Al 51) on HDR efficiency and cell viability. The molecules include the non- homologous end joining (NHEJ) Alt-R HDR Enhancer V2, cGAS DNA sensor inhibitor RU521, cGAS competitive inhibitor Al 51, the TANK-binding kinase inhibitor BX795, and the ATP competitive inhibitor XL413. PNI 550 LNP l LNPs for HA tagged CD5 was used as the model for HDR in primary T cells. It was observed that BX795 improved knock-in efficiency in a dosedependent manner without compromising cell viability. We hypothesized that its mechanism, potentially involving inhibition of DNA sensors, is advantageous for HDR applications. The HDR Enhancer V2, while significantly improved HDR editing rates, has significantly reduced cell viability, particularly at concentrations exceeding 10 pM, which may correlate with its role as an NHEJ inhibitor. RU521, XL413, and Al 51 showed negligible effects on HDR, while the combination of RU521 with V2 indicated a slight enhancement. Among the modulators tested in Figs. 5A and 5B, BX795 emerged as the most effective modulator for HDR applications in terms of efficacy and viability.

Example 6

[00210] Figs. 6A-6B show proprietary PNI ionizable lipids compared to benchmark lipids. Fig. 6A) HDR knock-in efficiency is shown as the percentage of HA+/CD5+ T cells normalized to wild-type untreated (UT). The ionizable lipid PNI 550 in LNP l demonstrates the high HDR efficiency (9.5%), significantly exceeding that of SM-102 in Moderna’s Spikevax® composition, or various benchmark lipids in the LNP l composition. Error bars represent the standard deviation of n=4 technical replicates. Fig. 6B) HDR knock-in efficiency shown as the percentage of GFP+/TCR- T cells normalized to wild-type untreated (UT). PNI ionizable lipids in LNP l show higher GFP insertion rate than MC3 in the Onpattro® composition and SMI 02 in Spikevax® composition. Error bars represent deviation among technical and biological (primary T cell donor) replicates. Table 6 for Example 6: Lipid compositions with mol% ratios that were used to encapsulate Cas9 mRNA, sgRNA and ssODN in a single LNP.

[00212] In the LNP l compositions shown in Figs. 6A-6B, ionizable lipids were changed, with the same mol ratios maintained, by using PNI 550, PNI 580, PNI 728, PNI 768, BOCHD-C3- DMA, SM-102, or MC3. Benchmark LNP samples with SM-102 in Spikevax® lipid composition or MC3 in Onpattro lipid composition were also prepared.

Table 6

[00213] To evaluate the potency of PNI ionizable lipids, comparative analysis of non-limiting examples of ionizable lipids was conducted for LNP-mediated HDR efficiency in primary T cells against known literature benchmarks. In Fig. 6A, ionizable lipid PNI 550 was used as the proprietary PNI lipid in the composition termed LNP l . Additionally, comparative ionizable lipids included BOCHD-C3-DMA, MC3 and SM-102, which were both tested in the same LNP l composition. Furthermore, SM-102 was assessed as a constituent of Moderna's Spikevax composition (see Table 6, “Spikevax (SM-102)”).) Fig. 6A displays the knock-in (KI) efficiency expressed as a percentage of HA+CD5+ cells normalized to untreated (UT) controls. The proprietary PNI 550 lipid outperformed the conventional lipids with a knock-in efficiency of 9.5%, while BOCHD-C3-DMA and both instances of SM-102 (in LNP l or the Spikevax composition) displayed significantly lower efficacies. MC3 demonstrated the lowest efficiency, suggesting less compatibility with the LNP-mediated delivery of HDR components in primary T cells. Fig. 6B further expands on HDR insertion with PNI lipids through the insertion of a much larger GFP (~2.5 kb) target. As shown in Fig. 6B, PNI lipids significantly outperformed SM-102 (in Spikevax composition) and MC3 (in Onpattro composition). Testing was conducted in three healthy primary T cell donor replicates, except for PNI 728 which was done in two of the three healthy primary T cell donor replicates.

Example 7

[00214] Figs. 7A-7C show HDR efficiency of different ionizable lipids in primary T cells. Fig. 7A shows the percent HA+/CD5+ HDR rate using two LNP formulations (LNP l vs. LNP 2) with various ionizable lipids (PNI-550, 659, 762, 768 or 769). Fig. 7B shows corresponding 2D flow cytometry plots depicting the expression of HA tag versus CD5 on T cells, treated with LNPs including ionizable lipids (PNI 550, PNI 762, and PNI 659) in LNP l composition. Fig. 7C shows percent HA+/CD5+ HDR rate using two LNP formulations (LNP l vs. LNP 2) with various ionizable lipids (PNI-550, 762 or 516), with no enhancer added. Error bars indicate standard deviations of >3 technical replicates. Fig. 7D shows the percent CAR+/TRAC — HDR rate using two LNP formulations (LNP l vs. LNP 2) with the ionizable lipids (PNI-550, 762 or 516) under various doses of nucleic acid (NA), with no enhancer added, for pDNA-mediated (~3.5 kb) HDR of the chimeric antigen receptor (CAR), targeted to the TRAC locus. Error bars indicate standard deviations of 2 technical replicates.

[00215] In further pursuit of developing lipid compositions for HDR, a series of proprietary PNI ionizable lipids were evaluated in both LNP l and LNP 2 compositions. Ionizable lipids PNI 550, PNI 516, PNI 659, PNI 762, PNI 768, and PNI 769 were tested with CD5 HA knock ins over multiple experiments and cargoes. Both LNP l and LNP 2 performed well with all lipids, achieving meaningful HDR in primary T cells for small gene insertions (Figs. 7A-7D). All LNPs prepared using the ionizable lipids PNI 550, PNI 659, PNI 762, PNI 768, and PNI 769 as part of the LNP l composition were tested in two primary T cell donors. In both donors all ionizable lipids show significant HDR efficiency for HA insertion, showing similar performance (data not shown). Example flow cytometry plots are shown in Fig. 7B for PNI 550, PNI 762 and PNI 659. When switching LNP compositions, shown in Fig 7C, both LNP l and LNP 2 perform well with PNI lipids, for example, PNI 516, 550 and 762, in which all compositions achieving meaningful HDR in primary T cells for small gene insertions. Finally, similar to Fig. 7C, lipid nanoparticles comprising LNP l and LNP 2 with either PNI 550, PNI 762 and PNI 516 were tested for pDNA- mediated (~3.5 kb) HDR of the chimeric antigen receptor (CAR), targeted to the TRAC locus. As shown in Fig. 7D, both LNP l and LNP 2 achieve equivalent HDR rates, and with all PNI lipids showing meaningful CAR expression. Collectively, these findings suggest that the PNI ionizable lipids facilitate efficient delivery of the gene editing complex and DNA, thereby improving the HDR outcome. This enhancement could be attributed to factors such as enhanced release of the encapsulated genetic material into the cytoplasm, increased stability of the LNPs (for example in the cell culture media) and/or enhanced cellular uptake of the LNPs.

[00216] Table 7 for Example 7: Lipid composition mol% ratios that were used to encapsulate Cas9 mRNA, sgRNA and DNA (ssODN or pDNA) in a single LNP. In the LNP 1 or LNP 2 compositions shown in Figs. 7A-7C, ionizable lipids were changed while the same mol ratios were maintained, by using PNI 550, PNI 516, PNI 659, PNI 728, PNI 762, PNI 768, or PNI 769. Table 7.

Example 8

[00217] Figs. 8A-8B show the HDR efficiency in primary T cells using various LNP formulations with or without Alt-R HDR Enhancer V2. Fig. 8A shows the evaluation of HDR efficiency using LNPs formulated with PNI 550 ionizable lipid, with or without HDR enhancer as indicated. Fig. 8B shows parallel assessment of composition with PNI 762 ionizable lipid. Error bars represent two technical replicates. [00218] Building on the foundational data outlined in Examples 1-7, which established parameters for cell density, treatment kinetics delivery, encapsulation strategies, and ionizable lipid efficacy, we sought to further refine the LNP composition.

[00219] The analysis focused on a series of LNP compositions, each designed to test the synergy between ionizable lipids and helper lipid components. The compositions were as follows, with composition ratios by mole percent.

[00220] Table 8 for Example 8: Lipid composition mol% ratios that were used to encapsulate

Cas9 mRNA, sgRNA and ssODN in a single LNP.

Table 8

[00221] In Figs. 8A and 8B, PNI 550 and PNI 762 lipid were used as ionizable lipid in various compositions according to their respective descriptions specified in Table 8. Among the samples tested, LNP l yielded the highest HDR efficiency. Other compositions (e.g, LNP3 in Fig. 8 A, LNP 2, and DDM in Fig. 8B) also achieved significant HDR efficiency.

Example 9

[00222] For further optimization of LNP l LNP compositions for HDR in primary T cells, a design of experiment (DoE) approach was employed to investigate the interplay between PNI 550 and other lipid components that may further enhance gene editing efficiency. The following compositions were tested in primary T cells.

[00223] Table 9 for Example 9: Lipid composition mol% ratios that were used to encapsulate Cas9 mRNA, sgRNA and ssODN in a single LNP. LNP l was tested as PNI 550-LNP_l and PNI 762-LNP_l.

Table 9

[00224] Figs. 9A-9B show assessment of HDR efficiency and cell viability across different LNP formulations. Fig. 9A shows HDR efficiency represented by the percentage of HA+/CD5+ primary T cells after treatment with various LNP formulations, including the PNI 550-LNP_l and previously identified PNI 762-LNP_l. The formulation PNI 762-LNP_l demonstrates the highest HDR efficiency among the tested compositions. Fig. 9B shows corresponding cell viability as determined by flow cytometry. Viability is normalized to untreated wild-type controls. Error bars represent technical replicates. No HDR enhancer or similar was included in the experiment. [00225] All the formulations with PNI ionizable lipids have significantly out-performed conventional lipid formulations, as shown in Fig. 9A. Among the tested samples including PNI ionizable lipids, the highest HA+/CD5+ population was observed with LNP l (40:20:37.5:2.5 mol%), HDR_DoEl_S3 (50: 10:37.5:2.5 mol%), and HDR DoEl SlO (40:10:48.83:1.17 mol%). The high efficacy of high cholesterol content within the LNP formulations may be attributed to cholesterol’s ability to reinforce membrane rigidity, allowing for enhanced LNP stability and protection against premature payload release during cellular delivery.

[00226] The benchmark lipid formulation used in Figs. 9A-9B include the literature published, optimized lipid formulations for DNA delivery to T cells, “7-CAR, 9-CAR, 11 -CAR” (Prazeres, P. et al. Delivery of plasmid DNA by ionizable lipid nanoparticles to induce CAR expression in T cells, International Journal of Nanomedicine, 5891-5904, 2023). 7-CAR, 9-CAR, and 11-CAR LNPs were prepared according to the prescribed ratio of Cl 2-200, DOPE, cholesterol, and C14- PEG2000 (shown in the table for Example 9). In the experiments, the resulting particles showed agreeable size and encapsulation efficacy for 7-CAR, 9-CAR, 11-CAR to those of the literature findings, though no meaningful HDR insertion was observed in primary T cells with these benchmark compositions. The resulting T cell viability post-LNP transfection remained high for the samples tested (Fig. 9B).

Example 10

[00227] PNI 550-LNP_l LNPs were benchmarked to electroporation, a commonly used technique for gene editing of T cells. HA tagging of CD5 was used for knock-in evaluation, along with cell viability through the flow cytometer, and cell counts using acridine orange/propidium iodide (AO/PI) staining. The results represent 2 biological and 3 technical replicates each.

[00228] Figs. 10A-10C show the Comparison of HDR Efficiency, Cell Viability, and Yield between Electroporation and PNI 550-LNP_l LNP Delivery. Fig. 10A shows HDR-mediated HA tag knock-in efficiency in primary T cells. Both electroporation (EP) and PNI 550-LNP_l LNP delivery methods achieved similar HDR rates, as indicated by the percentage of HA+/CD5+ cells, normalized to untreated control (wt UT). Fig. 10B shows post-treatment cell viability assessed by flow cytometry. Cell viability percentages show PNI 550-LNP_l LNP delivery (103% viability relative to untreated control) slightly outperforming electroporation (95% viability). Fig. 10C shows yield of edited cells (HA+ cell counts per mL) determined by AO/PI staining and automated cell counting. PNI 550-LNP_l LNP delivery resulted in a substantially higher yield of HA+ cells (2x10⁵ counts per mL) compared to electroporation (4x10⁴ counts per mL). Error bars represent the standard deviation of measurements, and individual data points for each biological replicate are shown as dots on the bars. No HDR enhancer was included in the experiments. [00229] The results show that delivery of HDR nucleic acids using LNP matched electroporation in terms of percent HA knock-in, in a given population, along with comparable cell viability confirmed through the flow cytometer (with the LNPs show marginally higher cell viability). However, the most pronounced effect between the two delivery systems arose in the yield of edited cells. Automated cell counting post-AO/PI staining shows a substantial increase in HA+ cell yield with LNP delivery, achieving over 5-fold increase in cell recovery with PNI 550 LNP l LNPs, as opposed to electroporation. It was hypothesized that electroporated cells either reduce their proliferation rate compared to LNP treated cells (hence yielding lower cell numbers in the population), or the cells died shortly after electroporation, with the debris being washed off during culture/staining procedure (such that percent viability was unaffected by the Day 4-post treatment readout).

Example 11

[00230] Figs. 11A-11E show multi-donor performance comparing LNP or electroporation (EP) mediated HDR. PNI 762-LNP_l containing Cas9 mRNA, TRAC7 sgRNA and CD 19 nanoplasmid were added to primary T cells, on day 3 post-thaw and post-activation. On day 7 post-thaw, performance metrics were evaluated. Fig. 11A shows anti-CD19-CAR+/TCR- live cells when constructs were inserted with LNPs or EP. Fig. 11B shows the corresponding cell viability as determined through flow cytometry. Fig. 11C shows the fold expansion of the gene inserted T cells on day 7 with respect to starting cell numbers. Fig. 11D shows CD 19 CAR+ cell yield per 10,000 cells treated, as determined through total live cells and gene insertion rate. Figs. 11A-11D represent average performance of at least 2 technical replicates within n=6 unique experiments over n=4 unique T cell donors and for EP, n=3 unique T cell donors. Fig. HE shows HDR CAR T cells with PNI 762-LNP_l incubated with SUP-B15 cells (solid) at the indicated effector to target ratio (for 48 hours). CD 19 negative K562 cells (dashed) were incubated with the CAR T cells at equivalent doses as a negative control. Data represents mean of n=2 technical replicates in n=l healthy T cell donor. [00231] Electroporation was further compared with LNPs for the delivery of the 3.5 kb antiCD 19 CAR construct for HDR in primary T cells. By quantifying CD19-CAR knock-in efficiency, cell viability, cell proliferation and CAR+ cell yields, the relative efficacies were assessed for the delivery methods (Figs. 11A-11D). The experiments contain no HDR enhancer. [00232] The multi-donor analysis of the data, as shown in the provided figure, indicates that LNP delivery achieves comparable knock-in efficiencies to electroporation (Fig. 11 A). Notably, significant donor-to- donor variability was found with electroporation efficiency, ranging from 3- 30%, while LNPs maintain much better donor to donor repeatability. LNPs show higher cell viability on day 7 post-thaw and allow for 10-fold higher proliferation of the T cell population. Collectively, LNP treated cells yield significantly larger numbers of CD19-CAR positive cells compared to electroporation.

[00233] The LNP-mediated CD 19 CAR T cells were functionally evaluated for CD 19+ B cell clearance (SUP-B15 cell line), with CD 19- (K562 cell line) serving as the negative control. Shown in Fig. HE, the resulting LNP- engineered CAR T cells are functional and clear virtually all CD 19+ cells within 48h at the lowest tested effector to target ratio. At the lower ratios K562 (CD19-) cells remained unperturbed, with small amounts of cell death occurred in the higher ratios, likely due to crowding within the assay well-plate.

[00234] These findings suggest that LNPs offer a superior platform for the delivery of large gene constructs like CD 19 CAR into primary T cells. The high knock-in efficiency, especially combined with high cell yield highlight the advantages of LNPs for cell-based therapeutics development.

[00235] While specific embodiments of the invention have been described and illustrated, such embodiments should be considered illustrative of the invention only and not as limiting the invention as construed in accordance with the accompanying claims.

Claims

CLAIMS:

1. A lipid nanoparticle (LNP) for transfecting a cell of hematopoietic lineage, the lipid nanoparticle (LNP) comprising a lipid mix composition comprising an ionizable lipid, the lipid mix composition encapsulating an HDR template DNA comprising a gene of interest for insertion into a target chromosomal locus.

2. The lipid nanoparticle of claim 1, wherein the HDR template DNA is double stranded.

3. The lipid nanoparticle of claim 1, wherein the HDR template DNA is single stranded.

4. The lipid nanoparticle of any one of claims 1 to 3, wherein the HDR template DNA comprises a chimeric antigen receptor.

5. The lipid nanoparticle of any one of claims 1 to 4, further comprising an endonuclease or an mRNA encapsulated by the lipid mix composition, wherein the mRNA encodes the endonuclease.

6. The lipid nanoparticle of claim 5, wherein the endonuclease is a CRISPR-associated endonuclease.

7. The lipid nanoparticle of any one of claims 1 to 6, further comprising a single guide RNA.

8. The lipid nanoparticle of any one of claims 1 to 4, wherein the HDR template DNA is accompanied by a guide RNA in combination with an endonuclease or an mRNA encoding the endonuclease.

9. The lipid nanoparticle of any one of claims 1 to 8, wherein the lipid mix composition provides a higher knock-in efficiency in an LNP-mediated delivery of the HDR template DNA in primary T cells compared to a lipid mix composition comprising MC3 (4- (dimethylamino)-butanoic acid, ( 1 OZ, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -y 1- 10, 13 - nonadecadien-l-yl ester) or SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino} octanoate).

10. The lipid nanoparticle of any one of claims 1 to 9, wherein the ionizable lipid comprises a tetrahydrofuranyl or a cyclopentyl head group.

11. The lipid nanoparticle of any one of claims 1 to 10, wherein the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combination thereof.

12. The lipid nanoparticle of any one of claims 1 to 11, wherein the lipid mix composition further comprises a structural lipid, a sterol, a stabilizer, or any combinations thereof.

13. The lipid nanoparticle of any one of claims 1 to 12, wherein the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijSlO), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof

14. The lipid nanoparticle of claim 12 or 13, wherein the lipid mix composition comprises about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.

15. The lipid nanoparticle of any one of claims 12 to 14, wherein the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijSlO).

16. The lipid nanoparticle of any one of claims 12 to 14, wherein the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).

17. A lipid mix composition for encapsulating a HDR template DNA including a gene of interest for insertion into a desired chromosomal locus, the lipid mix composition comprising an ionizable lipid.

18. The lipid mix composition of claim 17, wherein the lipid mix composition provides a higher knock-in efficiency in an LNP-mediated delivery of the HDR template DNA in primary T cells compared to a lipid mix composition comprising MC3 (4- (dimethylamino)-butanoic acid, ( 10Z, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -y 1- 10, 13 - nonadecadien-l-yl ester) or SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino} octanoate).

19. The lipid mix composition of claim 17 or 18, wherein the ionizable lipid comprises a tetrahydrofuranyl or a cyclopentyl head group.

20. The lipid mix composition of any one of claims 17 to 19, wherein the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combinations thereof.

21. The lipid mix composition of any one of claims 17 to 20, wherein the lipid mix composition further comprises a structural lipid, a sterol, a stabilizer, or any combinations thereof

22. The lipid mix composition of claim 21, wherein the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijSlO), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof.

23. The lipid mix composition of claim 21 or 22, comprising about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.

24. The lipid mix composition of any one of claims 21 to 23, wherein the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijSlO).

25. The lipid mix composition of any one of claims 21 to 23, wherein the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).

26. A method for transfecting a cell of hematopoietic lineage, the method comprising: contacting cells of hematopoietic lineage with a lipid nanoparticle (LNP) including a lipid mix composition comprising an ionizable lipid, the lipid mix composition encapsulating an HDR template DNA comprising a gene of interest for insertion into a desired chromosomal locus, thereby transfecting the cells; and culturing the cells in a cell culture media.

27. The method of claim 26, further comprising isolating the cells from the cell culture media.

28. The method of claim 26 or 27, wherein the HDR template DNA is double stranded.

29. The method of claim 26 or 27, wherein the HDR template DNA is single stranded.

30. The method of any one of claims 26 to 29, wherein the HDR template DNA comprises a chimeric antigen receptor.

31. The method of any one of claims 26 to 30, wherein the HDR template DNA is accompanied by an endonuclease or an mRNA encoding the endonuclease.

32. The method of claim 31, wherein the endonuclease is a CRISPR-associated endonuclease.

33. The method of any one of claims 26 to 32, wherein the HDR template DNA is accompanied by a single guide RNA.

34. The method of any one of claims 26 to 30, wherein the cells are contacted with the LNP containing the HDR template DNA accompanied by a guide RNA in combination with an endonuclease or an mRNA encoding the endonuclease.

35. The method of any one of claims 26 to 30, wherein the cells are contacted with a plurality of LNPs, each LNP of the plurality of LNPs containing the HDR template DNA, a guide RNA and/or endonuclease.

36. The method of any one of claims 26 to 35, further comprising contacting a homology directed repair enhancer with the cells, wherein the homology directed repair enhancer comprises an HDR enhancer, NHEJ inhibitor, DNA sensor inhibitor, or cell cycle syncing molecule.

37. The method of any one of claims 26 to 36, wherein a target cell density is between 0.1 to 1 million cells/mL.

38. The method of any one of claims 26 to 37, wherein contacting cells of hematopoietic lineage includes transfecting primary T cells with the lipid nanoparticle (LNP), wherein the LNP provides a higher knock-in efficiency in delivery of the HDR template DNA in primary T cells compared to a lipid nanoparticle comprising MC3 (4-(dimethylamino)- butanoic acid, ( 10Z, 13Z)- 1 -(9Z, 12Z)-9, 12-octadecadien- 1 -yl- 10, 13 -nonadecadien- 1 -yl ester) or SM-102 (9-Heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6- (undecyloxy)hexyl]amino} octanoate).

39. The method of any one of claims 26 to 38, wherein the ionizable lipid comprises a tetrahydrofuranyl or a cyclopentyl head group.

40. The method of any one of claims 26 to 39, wherein the ionizable lipid comprises PNI 516, PNI 550, PNI 580, PNI 659, PNI 714, PNI 726, PNI 728, PNI 761, PNI 762, PNI 768, PNI 769, PNI 771, or any combinations thereof.

41. The method of any one of claims 26 to 40, wherein the lipid mix composition of the LNP further comprises a structural lipid, a sterol, a stabilizer, or any combinations thereof.

42. The method of claim 41, wherein the stabilizer comprises polyoxyethylene (10) stearyl ether (BrijSlO), tocopherol polyethelyne glycol succinate (TPGS), PEG-DMG, dodecyl maltoside, sucrose monolaurate, or any combinations thereof.

43. The method of claim 41 or 42, wherein the lipid mix composition comprises about 20 - 50 Mol% ionizable lipid, about 10 - 70 Mol% structural lipid, about 10 - 70 Mol% sterol, and about 0.5 - 3 Mol% stabilizer, wherein the total mol% of components in the lipid mix composition is 100 mol%.

44. The method of any one of claims 41 to 43, wherein the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 37.5 Mol% cholesterol, and about 2.5 Mol% polyoxyethylene (10) stearyl ether (BrijSlO).

45. The method of any one of claims 41 to 43, wherein the lipid mix composition comprises about 40 Mol% ionizable lipid, about 20 Mol% distearoylphosphatidylcholine (DSPC), about 39 Mol% cholesterol, and about 0.75 Mol% Tocopherol polyethylene glycol 1000 succinate (TPGS).