Cellular therapies for cancer and auto-immune disorders using functional gene variants identified by base-editing screening of human t cells

Abstract

Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Here, we perform massively parallel base editing screens to generate thousands of variants at gene loci annotated with known or potential clinical significance. We discover a broad landscape of putative gain- (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and its regulatory subunit PIK3R1, LCK, SOS, AKT1, and RHOA. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific against a melanoma epitope or in different generations of CD19 chimeric antigen receptor T (CAR-T) cells demonstrates that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, we show that generations of CD19 CAR-T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production, and leukemia cell killing, including when benchmarked against other recent strategies.

Description

Attorney Docket 44010.185WO-PCT / / CU24092 CELLULAR THERAPIES FOR CANCER AND AUTO-IMMUNE DISORDERS USING FUNCTIONAL GENE VARIANTS IDENTIFIED BY BASE-EDITING SCREENING OF HUMAN T CELLS RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional patent application 63 / 598,843, filed November 14, 2023, to The Trustees of Columbia University, titled “MASSIVELY PARALLEL BASE-EDITING SCREENS TO MAP VARIANT EFFECTS ON ANTI-TUMOR HALLMARKS OF PRIMARY HUMAN T CELLS,” and U.S. provisional patent application 63 / 603,030, filed November 27, 2023, to The Trustees of Columbia University, titled “MASSIVELY PARALLEL BASE-EDITING SCREENS TO MAP VARIANT EFFECTS ON ANTI-TUMOR HALLMARKS OF PRIMARY HUMAN T CELLS,” the entirety of the disclosures of which are hereby incorporated by this reference. INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY FILED

[0002] Incorporated by reference in its entirety herein is a computer-readable nucleotide / amino acid sequence listing submitted concurrently herewith and identified as follows: One 60,825 byte XML file named “185WO-PCT” created on November 14, 2024. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under CA258829, CA280414, CA266446, and CA274506 awarded by the National Institutes of Health. The government has certain rights in the invention. TECHNICAL FIELD

[0004] The subject matter disclosed herein is generally directed to screens for identifying gain-of-function and loss-of-functions mutations associated with immune function and genetically modified CD8+ T cells with enhanced or suppressed immune function. BACKGROUND

[0005] Cellular cancer immunotherapies, such as adoptive T cell transfer (ACT) and Chimeric Antigen Receptor (CAR) T cell therapies, have become an important tool in the armamentarium for clinical cancer care in selected diseases1. ACT of tumor-infiltrating lymphocytes (TILs) produces responses in a subset of patients with metastatic melanoma whoAttorney Docket 44010.185WO-PCT / / CU24092 have progressed on multiple prior lines of therapies, including immune checkpoint blockade (ICB). CAR-T cells are now widely used in the care of different leukemias and lymphomas, resulting in durable response, including in diseases that have traditionally been resistant to conventional therapies2,3. However, most cancer patients do not benefit from cell therapies, and resistance may be due to cancer cell intrinsic changes (e.g., B2M loss, or CD58 loss / downregulation) or modulation of the cell products in the immunosuppressive tumor- microenvironment (TME)3,4.

[0006] Recent studies indicate that intrinsic properties of T cells, which are used to produce such cell therapies, determine anti-tumor capacity and clinical success5–7. TIL transfer, for example, is more successful when T cells have a progenitor-like cell state5. Similarly, CAR-T products in which pre-infusion T cells expressed a less differentiated memory cell state were more likely to produce clinical responses in patients with large B cell lymphomas6. Interestingly, single-point mutations in central signaling proteins (e.g., mTORC) or immune checkpoints (e.g., CD28)8,9,10are sufficient to engender a potentially favorable cell state by modulating T cell receptor (TCR) signaling strength, memory phenotype and cell proliferation.

[0007] With the advent CRISPR-Cas9 technologies, there has been a series of efforts to genome-edit cell products with the goal to improve T cell-based therapies, such as deletion of PDCD1 or CTLA-4, or co-deletion of the endogenous TCR and B2M to reduce the likelihood for graft-versus-host-disease11. Furthermore, critical studies have extended the powerful discovery tool of genome-scale CRISPR-Cas9 knockout screens to primary human T cells, allowing for unbiased identification of genetic drivers of T cell proliferation and survival12. However, CRISPR-Cas9 mediated gene disruption has several biological and technical limitations, including unknown effects on allosteric protein-protein interactions and changes in the tumor-immune synapse4,13, endogenous immunogenic potential of constitutively expressed foreign proteins (e.g. Cas9)14, a high rate of aneuploidy events in perturbed cells15and limited tunability of endogenous and potentially favorable synthetic proteins.

[0008] Emerging base editing (BE) technologies can overcome several of these challenges and generate precisely engineered T cell products. CRISPR-dependent BE uses cytosine and adenine base editors (CBE and ABE) to induce site-specific deamination of cytosine and adenine, leading to C>T and A>G base transitions, respectively16. Consequently, CBE and ABE can generate 64 distinct amino acid substitutions. Coupled with relaxed protospacer- adjacent motif recognition sites (NG instead of NGG) of single-guide RNAs (sgRNAs), BEAttorney Docket 44010.185WO-PCT / / CU24092 enables mutagenesis of endogenous DNA across a wide range of the genome. However, broad application of these methods to primary human T cells has been limited to date due to low editing efficiency, scalability and cell toxicity ensuing from existing delivery methods.

[0009] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention. SUMMARY

[0010] In certain example embodiments, the present invention provides for genetically modified CD8+ T cells with enhanced or suppressed immune function.

[0011] In one aspect, the present invention provides for an isolated CD8+ T cell genetically modified in vitro to comprise one or more gain-of-function (GOF) or loss-of-function (LOF) mutations in one or more genes selected from the group consisting of PIK3CD, PIK3R1, AKT1, RHOA, LCK, RHOA, CTLA4, ITK, ZAP70, NFKB1, CD40LG, and LAT.

[0012] In certain embodiments, the one or more GOF mutations are in PIK3CD, PIK3R1, AKT1, LCK, CTLA4, ITK, NFKB1, ZAP70, or CD40LG. In certain embodiments, the one or more GOF mutations in PIK3CD is a mutation generating a conversion in residue Cys416 to a positive charged amino acid. In certain embodiments, the one or more GOF mutations in PIK3CD is a mutation generating a conversion in residues Tyr524, Glu525, Glu527, or Asp529. In certain embodiments, the one or more GOF mutations is selected from the group consisting of PIK3CD Cys416Arg, PIK3CD Glu525Gly_His526Arg, PIK3CD Glu525Gly, PIK3CD Ser444Pro, PIK3CD Tyr524His, PIK3CD Tyr524Cys, PIK3CD Glu527Gly_Lys528Glu, PIK3CD Asp529Gly, PIK3CD Tyr524His_Leu523Pro, PIK3CD His526Arg_Glu527Gly, PIK3CD Glu527Gly_Lys528Glu, PIK3R1 Leu570Pro, AKT1 Trp80Arg, AKT1 Ile165Val, AKT1 Glu322Gly, AKT1 Asp323Gly, LCK Tyr505Cys_Gln506Arg, LCK Tyr505His, LCK Tyr505Cys, LCK Gln506Arg, CTLA4 Leu47Pro, CTLA4 Val46Ala, CTLA4 Ile102Thr CTLA4 Asp153Gly, ITK Tyr588Cys, ITK Tyr578His, ZAP70 Ile203Val, NFKB1 Glu439Gly, NFKB1 Asn846Asp, NFKB1 Glu63Gly, CD40LG Gln220Arg, and CD40LG Tyr172His. In certain embodiments, the GOF mutation in PIK3CD is Cys416Arg or Glu525Gly_His526Arg.

[0013] In certain embodiments, the one or more LOF mutations are in PIK3CD, RHOA, ZAP70, or LAT. In certain embodiments, the one or more LOF mutations is selected from the group consisting of PIK3CD Ser281Pro, PIK3CD Tyr440His_Leu439Pro, PIK3CD Ser318Pro, PIK3CD Ile899Val_Met900Val, PIK3CD Lys922Gly, RHOAAttorney Docket 44010.185WO-PCT / / CU24092 Tyr42His_Val43Ala, RHOA Tyr42Cys, RHOA Tyr66His, RHOA Arg70Gly, RHOA Asp65Asp, RHOA Val43Ala, ZAP70 Cys39Arg, ZAP70 Met558Val, ZAP70 Val438Ala, ZAP70 Val589Ala, ZAP70 Asp574Gly, ZAP70 Trp576Arg, ZAP70 Cys575Arg, LAT Leu19Pro, LAT Ile18Thr, LAT Asp125Gly, and LAT Asp126Gly. In certain embodiments, the LOF mutation in PIK3CD is Ser281Pro.

[0014] In certain embodiments, the isolated CD8+ T cell was obtained from a subject and genetically modified in vitro, whereby the genetically modified CD8+ T cell is autologous to the subject.

[0015] In certain embodiments, the subject has a tumor. In certain embodiments, the isolated CD8+ T cell is tumor specific. In certain embodiments, the isolated CD8+ T cell expresses a chimeric antigen receptor (CAR). In certain embodiments, the CAR is specific to a tumor antigen. In certain embodiments, the isolated CD8+ T cell expresses an exogenous T cell receptor (TCR). In certain embodiments, the TCR is specific to a tumor antigen.

[0016] In certain embodiments, the subject has an autoimmune disease. In certain embodiments, the isolated CD8+ T cell is specific to an autoantigen. In certain embodiments, the isolated CD8+ T cell expresses inhibitory cytokines.

[0017] In another aspect, the present invention provides for a population of CD8+ T cells comprising a plurality of CD8+ T cells expanded from the isolated CD8+ T cell of any embodiment herein.

[0018] In another aspect, the present invention provides for a method of treating cancer in a subject in need thereof comprising administering the population of CD8+ T cells of any embodiment herein having a GOF mutation to the subject. In certain embodiments, the CD8+ T cells are autologous to the subject. In certain embodiments, the subject has a loss of function of CD58 expression or activity. In certain embodiments, the subject is resistant to checkpoint blockade therapy.

[0019] In another aspect, the present invention provides for a method of treating an autoimmune disease in a subject in need thereof comprising administering the population of CD8+ T cells of any embodiment herein having a LOF mutation to the subject. In certain embodiments, the CD8+ T cells are autologous to the subject.

[0020] In another aspect, the present invention provides for a method of pooled screening of primary CD8+ T cells for GOF or LOF mutations that modulate T cell function comprising: delivering to a population of primary CD8+ T cells a library of guide RNAs targeting one orAttorney Docket 44010.185WO-PCT / / CU24092 more genes selected from Table 1; expressing a mRNA encoding a guide RNA directed base editor in the CD8+ T cells having a guide RNA, wherein the library of guide RNAs target the base editor to generate a plurality of variants in the one or more genes selected from Table 1; and determining by sequencing, guide RNAs in cells having an altered T cell function, whereby the guide RNAs identify the GOF or LOF mutations. In certain embodiments, the guide RNAs are delivered by using a lentiviral vector encoding each guide RNA. In certain embodiments, the lentiviral vector comprises a pol III and a pol II promoter, and wherein the guide RNA is expressed by the pol III promoter and a sequence identifying the guide RNA is expressed by the pol II promoter. In certain embodiments, the mRNA encoding a guide RNA directed base editor is delivered by electroporation. In certain embodiments, the primary cells are human primary cells. In certain embodiments, the primary cells are obtained from a mouse capable of expressing mRNA encoding a guide RNA directed base editor. In certain embodiments, the guide RNAs and mRNA are delivered by electroporation. In certain embodiments, primary CD8+ T cells receiving a guide RNA are selected followed by delivering the mRNA encoding a guide RNA directed base editor to the selected cells. In certain embodiments, the method further comprises sorting cells of interest based on altered T cell function and identifying enriched and / or depleted guide RNAs in the sorted cells. In certain embodiments, single cell RNA-seq is used to identify both the guide sequence in each single cell and gene expression in each single cell, whereby altered T cell function in single cells can be associated to a guide sequence. In certain embodiments, the T cell function is selected from the group consisting of activation, short-term proliferation, long-term proliferation, and cytokine production. In certain embodiments, the method further comprises assaying CD8+ T cells comprising the GOF or LOF mutations in tumor T cell killing assays.

[0021] The foregoing and other aspects, features, and advantages will be apparent from the DESCRIPTION and DRAWINGS, and from the CLAIMS if any are included. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.Attorney Docket 44010.185WO-PCT / / CU24092

[0023] An understanding of the features and advantages of the present invention will hereinafter be described in conjunction with the appended and / or included DRAWINGS, where like designations denote like elements, and:

[0024] FIG. 1 – Establishment of methods for high-efficiency base editing in primary human T cells. a, Overview of approach for targeted base editing in primary human T cells. T cells isolated from deidentified human donors are CD3 / CD28-stimulated for 48 hours, and then co-electroporated with a single guide RNA (sgRNA) and in vitro transcribed mRNA encoding the base editor (ABE = adenine base editor; CBE = cytosine base editor). ABE / CBE are translated to a functional protein in the target cell and performs sgRNA specified base edits. b,c, Target sites of sgRNAs against CD2 and beta-2-microglobulin (B2M) sites resulting in splice donor / acceptor mutations or mutations in start codons that results in early termination (SPLd = splice donor mutation, SPLa = splice acceptor mutation, SM = start codon mutation). d, Representative flow cytometry histograms showing ABE-mediated knockout of CD2 and B2M using sgRNAs indicated in (b) and (c). e, Representative flow cytometry histograms demonstrating CBE-mediated knockout of the T cell receptor (TCRab) and B2M (ES = early stop codon mutation). f, Approach for base editing primary human T cells with lentiviral integration of individual or pooled sgRNAs. T cells were isolated and activated as in (a), transduced with sgRNA (coupled with fluorescent mTurquoise reporter in a CropSeq lentiviral vector), and then electroporated with base editor mRNA 72 hours post-transduction. g, Representative flow cytometry dot-plots and histograms demonstrating ABE-mediated knockout of B2M with the workflow described in (f). mTurquoise fluorescence is used as a proxy for sgRNA integration. For histograms, red indicates gated mTurquoise-negative cells, and blue indicates gated mTurquoise-positive cells. h, Editing efficiency (measured by B2M loss on flow cytometry) and viability of T cells electroporated with B2M_SM_1 sgRNA and varying doses of the adenine base editor ABE8e. Timepoint is 4 days post-electroporation of ABE8e. Arrow represents ABE dosing selected for screening experiments.

[0025] FIG. 2 – Design and execution of massively parallel base editor screens in T cells. a, Target gene set for ClinVar library ABE screens. Genes were selected from the “T cell receptor signaling” KEGG pathway, excluding secreted proteins. Visualization was performed in STRING with k-means clustering, n=8 clusters. Lines represent functional interactions. All sgRNAs generating either known ClinVar variants, or different point mutations at the same amino acid residue, were selected for the final library. b, Target gene set for 12-gene tilingAttorney Docket 44010.185WO-PCT / / CU24092 library ABE screens. Genes were selected on the basis of their central functions in T cell signaling and differentiation. The sgRNA library was designed to tile the exons and splice sites of each gene to generate every possible ABE-mediated edit. c, Library-edited primary T cells were isolated (using FACS or serial collections of repeatedly stimulated T cells) for desired phenotypes and functions. Enrichment and depletion of guides in these readouts was assessed by next-generation sequencing (NGS). d, T cell numbers following base editing and chronic restimulation and long-term expansion arm of the ClinVar library screen. Arrows represent T cell restimulation points. At each restimulation point, 20e6T cells were carried forward and restimulated with CD3 / CD28 beads. T cells were sampled for NGS at days 7, 12, 15, 28, 35, and 40 following initial base editor electroporation. e, Representative histograms from the carboxyfluorescein succinimidyl ester (CFSE) short-term proliferation screen arm. Cells were sorted from the dimmest (most proliferative) and brightest (least proliferative) CFSE populations and sgRNA distribution was assessed by NGS. f, Representative contour plots from the cytokine production screen arm. T cells were CD3 / CD28 stimulated for 6 hours and sorted for presence (either single- or double-positive) or absence (double-negative) of TNFα and IFNγ. Sorted populations were compared by WGS.

[0026] FIG. 3 – Global results of base editing screens across libraries, donors, and readouts. a, Density plots showing log2 fold changes (LFC) values of different categories of guides from the ClinVar library at Day 35 post-electroporation of the long-term expansion screen arm. Dotted line represents the bottom 5% of combined empty window and silent mutation controls. Percentages represent the percentage of guides in each category falling below this threshold. All guides generating variants in CD3D, CD3E, CD3G, or CD3Z were binned into the “CD3 complex” category. One representative donor is shown. SgRNA distributions from all long-term proliferation timepoints are compared to a “Day 0” control T cell population which was library-transduced but not electroporated with base editor. b, Scatter plot showing LFC values of essential gene or CD3 complex gene perturbations in both donors from the ClinVar Library at Day 28 post-electroporation in the long-term expansion screen arm, indicating high degree of robustness of the screens across two independent donor input T cells. c, Lollipop plot showing LFC of sgRNAs tiling IL2RG in the 12-gene tiling screen, mapped to the canonical IL2RG isoform. Indicated types of enriched and depleted mutations introduced by sgRNAs and their position in the amino acid sequence are shown. Selected sgRNAs generating clinically identified immunodeficiency variants (red boxes), alternativeAttorney Docket 44010.185WO-PCT / / CU24092 mutations at amino acids with known clinical variants (orange boxes), or variants in the signal peptide (blue boxes) that likely interfere with shuttling of IL2RG protein to the cell membrane are annotated. Timepoint shown is Day 26 post-nucleofection in the long-term expansion screen arm. d, Scatter plot showing LFC of sgRNAs across both donors (x vs y axis) in the long-term expansion ClinVar screen arm. Timepoint shown for all panels is Day 28 post- electroporation. Highlighted are positions of sgRNAs resulting in indicated amino acid changes for selected target genes and protein products (PIK3CD, AKT1, PIK3R1 / 3 and RHOA). e, Volcano plot showing LFC (x axis) and –log10(FDR) (y axis) for sgRNAs in the CFSE short- term proliferation arm of the ClinVar screen. Positive LFC represents enrichment (red) or depletion (blue) of the sgRNA in the highly proliferative population compared to the least proliferative. Indicated are selected amino acid changes in gene products from highly enriched / depleted base edits. FDR cutoff <0.05. Data from one representative donor is shown.

[0027] FIG. 4 – Structure-function analysis of PI3K pathway variants resulting in polyfunctional T cell phenotypes contrasted with LCK mutations predominantly driving isolated T cell proliferation. a, Lollipop plot showing LFC of variants produced in base editing screens following repeated stimulation. Indicated are selected variants and their amino acid positions across the gene product of PIK3CD (p110δ) and its highlighted domains. b, Predicted structural assembly of PIK3CD (p110δ, green) and PIK3R1 (p85, blue) gene products contributing subunits to active PI3K-δ enzyme. Mutated residues discovered in base editing screens are highlighted (PIK3CD mutations in red, PIK3R1 mutations in yellow). Selected regions of the p110δ / p85 interface are highlighted and contrasted between the wild- type (WT) and mutant (mut) gene products. Mutations Y524C, E525G, D527G, D529G in PIK3CD (green) are predicted to be adjacent to residues K379, K382, K419 (dark blue) in PIK3R1. C416R is adjacent to L567 and L570P localizes to a predicted coiled coil region of PIK3R1. c, Representative Sanger sequencing trace from one donor demonstrating adenine base editing of PIK3CD generating the C416R mutation compared to unedited control cells from the same donor. sgRNA protospacer and the targeted base are annotated. The targeted adenine (position 4 in the protospacer) is converted to a guanine, leading to a conversion from thymine to cytosine on the antiparallel strand (represented in the sanger traces). d, Quantification of base editing efficiency at 10 different loci targeted for validation experiments, as assessed by EditR analysis of Sanger sequencing. n=3 independent human donors. e, Representative flow cytometry histograms of phosphorylated S6 (pS235 / S326) andAttorney Docket 44010.185WO-PCT / / CU24092 phosphorylated AKT (pS473) in base edited T cells with indicated mutations following 10 minutes of CD3 / CD28 stimulation. Dotted lines represent the approximate median of the histogram in the silent mutation control. Data are representative of one donor; findings were replicated across three independent donors. f, Fold change (y axis) of percent IL2-positive T cells with indicated genotypes (x axis) following CD3 / CD28 bead stimulation in three independent donors. g, Fold change of TNFα mean fluorescence intensity (MFI) in TNFα- positive T cells across indicated genotypes (x axis) following 6-hour CD3 / CD28 stimulation. For each donor, the fold change of each variant is normalized to that of the control silent mutation. n=3 human donors. h, Proliferation of base edited T cell variants as measured by CFSE dilution following two days of CD3 / CD28 stimulation and four days of expansion. Indicated is the frequency (in %) of highly proliferative T cells (CFSElow) across indicated genotypes (x axis) in three independent donors. i, Representative histograms from flow cytometry experiments in the CFSE experiment (shown in h) for selected mutations in PIK3CD compared to silent controls of deleterious mutation in CD3D (M92T). The vertical dotted line indicates the CFSElowgate for highly proliferative cells. One-way ANOVA with Dunnett’s test for multiple comparisons (panel f, g, h.).

[0028] FIG. 5 – Improving cell-based immunotherapies through base editing of antigen-specific human T cell products. a, Schematic representing antigen-matched T cell- melanoma co-culture system leveraged to interrogate variant effects on multiple axes of T cell polyfunctionality and antigen-specific antitumor activity. Primary human T cells engineered to express the NY-ESO-1 T cell receptor (TCR) are co-cultured with NY-ESO-1-presenting A375 melanoma cells. NY-ESO-1 TCR T cells were modified either with a silent mutation or with several mutations identified in base editing screens and predicted to either enhance or diminish T cell polyfunctionality (e.g. PIK3CD variants) or predominantly influence isolated T cell functions, such as proliferation (e.g., AKT1 or LCK variants). b, TNFα expression in NY-ESO- 1-TCR T cells with indicated base edits (x axis) after 8 hours of co-culture with A375, with or without MHC Class I blocking antibody (aMHC) and at an effector to target ratio of 1:1. c, Representative flow cytometry histograms of TNFα intensity from variants as in (b). d,e, Same as (b) and (d) but showing (d) IL2 positive and (e) representative flow cytometry histograms of NY-ESO-1 specific T cells following co-culture. f, Integrated analysis of multiple flow cytometry readouts (using PaCMAP) to show changes on T cell polyfunctionality. Density plots show distribution of cells with indicated variants across multiple measured cytokineAttorney Docket 44010.185WO-PCT / / CU24092 readouts from co-culture experiments. Each density plot represents 3 replicates for the indicated variant. g, Quantification of T cell polyfunctionality measurement across all flow- cytometry measurements in three independent donors and across indicated tumor-specific T cell genotypes following co-culture with cognate epitope-expressing melanoma cells. h, Counts of viable (dsRed-expressing) A375 melanoma cells (y axis) over time (x axis, in hours) plated either alone or co-cultured at an effector-to-target ratio of 1:1 with NY-ESO-1 T cells harboring indicated mutations. T cells were added 4 hours after initial plating, and imaging was performed using an Incucyte® instrument at approximately 1-hour intervals. n=3 replicates. i, Relative change in cell numbers of A375-dsRed 48 hours after co-culture with NY-ESO-1 variant T cells (effector-to-target ratio 1:1) from an independent experiment. n=3 replicates. One-way ANOVA with Dunnett’s test for multiple comparisons (panels b, d, g).

[0029] FIG.6 – Optimization of workflows for base editing in primary human T cells. a, Schematic of in vitro transcription (IVT) method to generate base editor mRNA. Base editor constructs are cloned into an IVT production vector, amplified and poly-T tailed in a single PCR step, and then in vitro transcribed using modified capping and dNTPs to improve stability and reduce immunogenicity. b, Representative electropherogram trace of in vitro transcribed base editor mRNA indicating high purity and appropriate size of the mRNA products. c, Single- and multi-plex base editing of B2M and CD2 with sgRNAs designed to generate gene knockout as described in Figure 1. d, Knockout editing of CD2 with lentiviral integration of a CD2 knockout guide followed by electroporation of Cas9 mRNA. sgRNAs were delivered in a CROP-seq vector with mTurquoise reporter, and mTurquoise expression shown in this histogram indicates sgRNA expression of Cas9 perturbed cells. e, Representative histograms demonstrating similar base editing efficiency of B2M in both CD4 and CD8 T cells using lentiviral guide integration followed by base editor mRNA electroporation. f, Cytosine base editor-mediated knockout of T cell receptor (TCRab) surface expression by introduction of an early stop codon in the TRBC locus.

[0030] FIG. 7 – Library design and screen preparation. a, Distribution of sgRNAs by category in the ClinVar ABE library. For pathogenic, likely pathogenic, and variant of unknown significance (VUS) categories, sgRNAs are predicted to either make the exact edit present in ClinVar, or an alternative missense mutation in the same amino acid. b, Distribution of sgRNAs by category in the 12-gene tiling ABE library. c, Schematic of sgRNA library cloning and high-titer library lentivirus production for base editor screens. d, Schematic forAttorney Docket 44010.185WO-PCT / / CU24092 generation of library base-edited T cells. T cells were first isolated from healthy human donors, transduced with high-titer sgRNA library pools that were cloned into the CROP-seq mTurquoise lentiviral vector, followed by electroporation with ABE mRNA 72 hours later (see Fig. 1h). e, Transduction efficiency of ClinVar base editor library in two independent human donors T cells, which was performed at an optimized multiplicity of infection to achieve high frequency of cells receiving only one sgRNA. f, Representative histograms of IL2RA (CD25) sort in base edited T cells. For each donor, cells were sorted to select the top and bottom 15% of CD25 expression.

[0031] FIG. 8 – Metrics for rigor and reproducibility of large-scale base editing screens. a, Density plots showing LFC values of different categories of guides from the ClinVar library at Day 35 post-electroporation of the long-term expansion screen arm. Dotted line represents the bottom 5% of combined empty window and silent mutation controls. Indicated are the percentages of guides in each category falling below this threshold. All guides generating variants in CD3D, CD3E, CD3G, or CD3Z were binned into the “CD3 complex” category. The second donor from the screen is shown (in companion to Fig. 3a). SgRNA distributions from all long-term proliferation timepoints are compared to a “Day 0” control T cell population which was library-transduced but not electroporated with base editor. b, Scatter plot showing LFC values of negative control sgRNAs (including both empty window and silent mutations) in both donors from the ClinVar Library at Day 28 post-electroporation in the long- term expansion screen arm with no significant enrichment or depletion of negative control guides is detected. c, Distribution of ranked relative abundance (RRA) scores for gene-wise dropout analysis in the CD25 sort arm of the ClinVar library, across both donors. The top 5 negatively selected genes (in top 15% of CD25 expression compared bottom 15% of CD25 expression) are listed. d, Distribution of ranked relative abundance (RRA) scores for gene-wise dropout analysis in the CFSE sort arm of the ClinVar library, across both donors. The top 5 negatively selected genes (in highly proliferative compared to least proliferative, for CFSE sort). e, Lollipop plot showing LFC of sgRNAs tiling IL2RG in the 12-gene tiling screen, mapped to the canonical IL2RG isoform. Selected sgRNAs generating clinically identified immunodeficiency variants, or alternative mutations at amino acids with known clinical variants, are annotated. Timepoint shown is Day 26 post-nucleofection in the long-term expansion screen arm. The second donor from the screen is shown (in companion to Fig. 3c). f, Scatter plot showing LFC of selected sgRNAs generating mutations in LCK, SOS1, andAttorney Docket 44010.185WO-PCT / / CU24092 PTPRC. Timepoint shown is Day 28 post-electroporation in the ClinVar long-term expansion screen arm. Selected variants (e.g. LCK Y505C) were used for downstream functional validation experiments. g, Lollipop plot for sgRNAs targeting RHOA in one representative donor in the ClinVar screen, mapped to the canonical isoform. Timepoint shown is Day 35 post-electroporation in the long-term proliferation arm of the screen. h, Timecourse line graphs of LFC of sgRNAs targeting RHOA in one representative donor in the long-term expansion arm of the ClinVar screen. i, Volcano plot showing enriched and depleted guides in the CFSE lo vs hi proliferation sort. Positive LFC indicates enrichment of guides in the highly proliferative (CFSE lo) compared to least proliferative (CFSE hi) populations. One representative donor is shown (in companion to Fig. 3e). FDR cutoff <0.05. j, Volcano plot showing enriched and depleted guides in the CD25 hi vs lo proliferation sort. Positive LFC indicates enrichment of guides in the CD25hi compared to CD25lo populations. FDR cutoff <0.05. One representative donor is shown.

[0032] FIG. 9 – Screen results for genes undergoing tiled base editing. a-e, Lollipop plots for genes targeted in the 12-gene tiling screen, with sgRNA editing positions mapped to the canonical isoform. Timepoint shown is Day 26 post-electroporation in the long-term expansion screen arm.

[0033] FIG. 10 – Screen results for additional genes undergoing tiled base editing. a- e, Lollipop plots for genes targeted in the 12-gene tiling screen, with sgRNA editing positions mapped to the canonical isoform. Timepoint shown is Day 26 post-nucleofection in the long- term expansion screen arm.

[0034] FIG. 11 – Enrichment and structure-function relationship of variants promoting T cell proliferation. a, Lollipop plot showing LFC of variants produced base editing screens and enriched following repeated stimulation. Indicated are selected variants and their amino acid positions across the gene product of PIK3CD (p110δ) and its highlighted domains. Representative timepoint shown is 15 days post-electroporation. b,c, Timecourse line graphs of LFC of sgRNAs targeting PIK3CD in both donors in the long-term expansion arm of the ClinVar screen. d,e, Lollipop plots for sgRNAs targeting AKT1 across both donors in the ClinVar screen, mapped to the canonical isoform. Timepoint shown is Day 35 post- electroporation in the long-term proliferation arm of the screen. f,g, Timecourse line graphs of LFC of sgRNAs targeting AKT1 in both donors in the long-term expansion arm of the ClinVar screen. h, Structure and position of mutations in AKT1. (right) Overall predicted structure ofAttorney Docket 44010.185WO-PCT / / CU24092 AKT1 (blue) and mutated residues (red). (left) Wild-type (WT) and mutated (mut) residues (red). D323G is predicted to localize next to L14 (dark blue). i,j, Lollipop plots for sgRNAs targeting LCK across both donors in the 12-gene tiling screen, mapped to the canonical isoform. Timepoint shown is Day 26 post-electroporation in the long-term proliferation arm of the screen. k, Structure and position of mutations in LCK. (top) Overall predicted structure of LCK (blue) and mutated residues (red). (bottom) Wild-type (WT) and mutated (mut) residues (red).

[0035] FIG. 12 – Design and results of co-culture experiments to improve T cell mediated lysis using variants identified in base editing screens. a, Schematic for virus-free engineering and rapid expansion of NY-ESO-1 specific primary human T cells. b, Representative flow cytometry dot plot of NY-ESO-1 TCR-engineered T cells stained with TRAC antibody and NY-ESO-1 dextramer prior to flow sorting. c, Viable count relative to initial timepoint for A375-dsRed cells cultured at varying effector:target ratios of NY-ESO-1 specific T cells for 48 hours. aMHC = MHC class I blocking antibody, which blocks peptide- MHC-specific activation of T cells. n=3 replicates. d, Representative flow cytometry histograms of ABE- or CBE-mediated knockout of B2M or CD2 in NY-ESO-1 specific T cells using sgRNAs shown in Figure 1. e, Mean fluorescence intensity (MFI, y axis) of TNFα expression in NY-ESO-1-TCR T cells with indicated base edits (x axis) after 8 hours of co- culture with A375-dsRED. One-way ANOVA f, Frequency (in %, y axis) of Granzyme B expression in NY-ESO-1-TCR T cells with indicated base edits (x axis) after 8 hours of co- culture with A375-dsRED. g, MFI (y axis) of Granzyme B expression in NY-ESO-1 specific T cells with indicated base edits (x axis) in the presence or absence of aMHC after 8 hours of co-culture with A375-dsRED. h, Flow cytometric evaluation of frequency (y axis) and intensity (x axis) of Granzyme B expression in NY-ESO-1 specific T cells with indicated genotypes from experiment shown in (g). i, Relative change in cell numbers of A375-dsRed 48 hours after co-culture with NY-ESO-1 variant T cells (effector-to-target ratio 0.5:1) from an independent experiment. n=3 replicates. j, Counts of viable A375-dsRed cells (y axis) over time (x axis, in hours) plated either alone or co-cultured at an effector-to-target ratio of 0.5:1 with NY-ESO-1 specific T cells harboring indicated mutations. T cells were added 4 hours after initial plating and imaging was performed using an Incucyte instrument at approximately 1-hour intervals. n=3 replicates. One-way ANOVA with Dunnett’s test for multiple comparisons (panels c, e, f, g, i).Attorney Docket 44010.185WO-PCT / / CU24092

[0036] FIG.13 – Establishment of methods for high-efficiency base editing in primary human T cells. (A)-G)

[0037] FIG. 14 – Design and execution of massively parallel base editor screens in T cells. (A)-(G)

[0038] FIG. 15 – Identification of favorable T cell signaling variants in base editor screens. (A)-(N)

[0039] FIG. 16 – Improvement of T cell-based immunotherapies with base edited signaling variants. (A)-(L)

[0040] FIG. 17 – Optimization of workflows for base editing in primary T cells. (A)- (G)

[0041] FIG. 18 – Library design and screen preparation.

[0042] FIG. 19 – Screen results for genes undergoing tiled base editing.

[0043] FIG. 20 – Design and results of co-culture experiments to improve T cell mediated lysis using variants identified in base editing screens.

[0044] FIG. 21 – Design of screen.

[0045] FIG.22 – Experimental design and phenotypic readouts for massively parallel base editor screens in T cells. a, Approach for base editing primary human T cells with lentiviral integration of sgRNAs. b, Representative flow cytometry dot-plots and histograms demonstrating ABE-mediated knockout of B2M with the workflow described in (a). For histograms, red indicates gated mTurquoise-negative cells, and blue indicates gated mTurquoise-positive cells. c, Quantification of ABE-mediated knockout of B2M with the sgRNA used in (b) (n=3 independent human donors). d, Target gene set for ClinVar library ABE screens. Visualization was performed in STRING with k-means clustering (n=8 clusters). Lines represent functional interactions. e, Library-edited primary T cells were isolated (using FACS or serial collections of repeatedly stimulated T cells) for desired phenotypes and functions. Enrichment and depletion of guides in these readouts was assessed by next- generation sequencing (NGS). f, T cell counts following base editing and repeated restimulation in the long-term expansion arm of the ClinVar library screen. Arrows below graph represent T cell restimulation points, when 20e6T cells were restimulated with CD3 / CD28 beads. Arrows above graph represent timepoints when T cells were collected for next-generation sequencing (NGS), at days 7, 12, 15, 28, 35, and 40 following base editor electroporation. g, Representative histograms from the carboxyfluorescein succinimidyl esterAttorney Docket 44010.185WO-PCT / / CU24092 (CFSE) short-term proliferation screen arm. Cells were sorted from the dimmest (most proliferative) and brightest (least proliferative) CFSE populations. h, Representative histograms from the T cell activation screen arm, in which base edited T cells were sorted to select for cells in the top and bottom 15% of CD25 (IL2RA) expression. i, Representative contour plots from the cytokine production screen arm, in which T cells were CD3 / CD28 stimulated for 6 hours and sorted for presence (either single- or double-positive) or absence (double-negative) of TNFα and IFNγ. Error bars represent mean + / - SD (panel c).

[0046] FIG.23 – Results of base editing screens across donors. a, Density plots showing log2(fold change) (LFC) values of different categories of guides from the ClinVar library at Day 35 post-electroporation of the long-term expansion screen arm in one representative donor. Dashed line represents the bottom 5% of the distribution of combined empty window and silent mutation controls. Percentages represent the percentage of guides in each category falling below this threshold. “Proline,” “Splice Acceptor,” and “Splice Donor” represent perturbations in essential genes. sgRNAs generating variants in CD3D, CD3E, CD3G, or CD3Z were binned into the “CD3 complex” category. b, Scatter plot showing LFC and R2values of essential gene or CD3 complex gene perturbations in both donors from the ClinVar Library at Day 28 post- electroporation in the long-term expansion screen arm. c, Lollipop plot showing LFC of sgRNAs tiling IL2RG at Day 26-post electroporation in the long-term expansion arm of the 12-gene tiling screen. Each point represents an sgRNA and is mapped to the position of the targeted residue in the canonical IL2RG isoform. Indicated types of enriched and depleted mutations introduced by sgRNAs and their position in the amino acid sequence are shown. Selected sgRNAs generating clinically identified immunodeficiency variants (red boxes), alternative mutations at amino acids with known clinical variants (orange boxes), or variants in the signal peptide (blue boxes) are annotated. d, Scatter plot showing LFC of sgRNAs across both donors in the long-term expansion ClinVar screen arm. Timepoint shown for all panels is Day 28 post-electroporation. Highlighted are positions of sgRNAs for selected target genes (PIK3CD, AKT1, PIK3R1 and RHOA) resulting in indicated amino acid changes in protein products. e, Volcano plot showing LFC (x axis) and –log10(False Discovery Rate, FDR) (y axis) for sgRNAs in the CFSE short-term proliferation arm of the ClinVar screen. Positive LFC represents enrichment (red) or depletion (blue) of the sgRNA in the highly proliferative population compared to the least proliferative. Indicated are selected amino acid changes inAttorney Docket 44010.185WO-PCT / / CU24092 gene products from highly enriched / depleted sgRNAs. FDR cutoff <0.05. Data from one representative donor is shown.

[0047] FIG. 24 – Structure-function analysis of PIK3CD and other gene variants on T cell signaling and activity. a, Lollipop plot showing LFC of sgRNAs targeting PIK3CD at Day 35 post-electroporation in the long-term proliferation arm of the ClinVar screen. sgRNAs are mapped to the canonical isoform of PIK3CD (p110δ). b, Predicted structural assembly of PIK3CD (p110δ, green) and PIK3R1 (p85, blue) gene products forming the active PI3K-δ enzyme. Mutations discovered in BE screens are highlighted (PIK3CD in red, PIK3R1 in yellow). Selected regions of the p110δ / p85 interface are highlighted and contrasted between the wild-type (WT) and mutant (mut) gene products. c, Sanger sequencing traces from one human donor demonstrating adenine base editing of PIK3CD generating Cys416Arg. d, Quantification of base editing efficiency by 10 sgRNAs using EditR. Horizontal brackets indicate multiple loci edited by the same sgRNA. Parentheses in labels indicate which edit is being assessed. The position of each targeted adenine base within the editing window of the base editor is annotated (n=3 independent human donors). e, Representative flow cytometry histograms of phosphorylated S6 and phosphorylated AKT in T cells with indicated mutations following 10 minutes of CD3 / CD28 stimulation. Dotted lines represent the approximate median of the silent mutation control. Data are representative of one donor, with similar findings in n=3 independent human donors. f, Fold change of percent IL2-positive T cells and g, Fold change of TNFα mean fluorescence intensity (MFI) in TNFα-positive T cells, in T cells with indicated genotypes (x axis) compared to silent controls following 6-hour CD3 / CD28 bead stimulation (n=3 independent human donors). h, Change in frequency (in %) of highly proliferative T cells (CFSElo) across indicated genotypes (x axis) compared to T cell with a control silent mutation (n=3 independent human donors). i, Representative histograms from CFSE experiment in (h) for selected mutations compared to silent control. Dotted line indicates the approximate CFSElowgate cutoff for highly proliferative cells. For each human donor used in f-h, data was normalized to the silent control condition within each donor. One-way ANOVA with Dunnett’s test for multiple comparisons (panel f-h.). Error bars represent mean + / - SD (panel d, f-h).

[0048] FIG. 25 – Enhanced signaling, polyfunctionality and melanoma cell-lytic capacity of epitope-specific T cells engineered with gene variants identified in base editing screens. a, Schematic of T cell-melanoma co-culture system to evaluate efficacy of base editedAttorney Docket 44010.185WO-PCT / / CU24092 T cells. b, TNFα, c, IL2, and d, GrzB expression in NY-ESO-1 TCR T cells with indicated genotype (x axis) after 8 hours of co-culture with A375 cells, with or without MHC Class I blocking antibody (aMHC), at an effector to target ratio of 1:1 (n=3 independent biological replicates). e, Representative flow cytometry histograms of TNFα, IL2, and GrzB expression from variants shown in (b-d). f, Integrated analysis of multiple flow cytometry readouts (using PaCMAP). Heatmaps represent relative cytokine expression level, with red representing higher expression. Density plots show distribution of cells with each genotype across multiple cytokine readouts from co-culture experiments. Each density plot represents n=3 combined biological replicates for the indicated variant. g, Quantification of T cells co-expressing TNFα, IL2, and GrzB across indicated tumor-specific T cell genotypes following 8 hours of co-culture with A375 cells (n=3 independent biological replicates). h, Relative cell count (normalized to time t0) of A375-dsRed after 48 hours of co-culture with NY-ESO-1 TCR T cells with indicated genotypes (x axis) at a 1:1 effector to target ratio (n=3 independent biological replicates.) i, Following the 48-hour co-culture in (h), NY-ESO-1 TCR T cells from this co- culture were then carried forward and plated onto fresh A375 cells, and killing in this repeat co-culture was assessed after 48 hours in the same manner (n=3 independent biological replicates). j, Relative cell count (normalized to time t0) of A375-BFP CD58-KO cells after 72 hours of co-culture with NY-ESO-1 TCR T cells of the indicated genotype (x axis) at a 1:1 effector to target ratio. NT = non-targeting control sgRNA (n=3 independent biological replicates). For (b-d, g), dotted lines represent mean of control (silent) values. For (h-j), dotted line represents relative A375 cell count at time t0. One-way ANOVA with Dunnett’s test for multiple comparisons (panels b-d, g-j). Error bars represent mean + / - SD (panels b- d, g-j).

[0049] FIG. 26 – Enhanced signaling, cytokine production and leukemia cell-lytic capacity of CD19 CAR-T cells equipped with PIK3CD or PIK3R1 variants identified in base editing screens. a, Quantification of AKT phosphorylation (pS473) in CD19-BBz CAR T cells after 15 minutes of culture with either Nalm6 leukemia (+) or media alone (-) (n=3 independent biological replicates). b, Quantification of CD19-BBz CAR T cell intracellular expression of TNF⍺, c, IL2, and d, IFNγ after 8-hour culture with Nalm6 leukemia at an 0.5:1 effector to target ratio (+) or in media only (-) (n=3 independent biological replicates). e, ELISA quantification of CD19-BBz CAR T cell production of TNF⍺, f, IFNγ, and g, IL2 expression after 24-hour co-culture with WT (+) or CD19-KO(-) Nalm6 leukemia at an 0.5:1 effector to target ratio (n=3 independent biological replicates). h, Representative flow cytometry contourAttorney Docket 44010.185WO-PCT / / CU24092 plots of data from (b-d). GFP is a proxy for CAR expression. i, Quantification of Nalm6 leukemia cell killing after 48-hour co-culture with CD19-BBz CAR T cells at a 0.125:1 effector-to-target ratio (n=3 independent biological replicates). For (a-g), dotted lines represent mean of control (NT) values. For i, dotted line represents relative Nalm6 cell count at time t0. One-way ANOVA with Dunnett’s test for multiple comparisons (panels a-g, i). Error bars represent mean + / - SD (panels a-g, i).

[0050] FIG. 27 – Optimization of workflows for base editing in primary human T cells. a, Overview of approach for targeted base editing in primary human T cells. b-d, Target sites of sgRNAs against CD2, B2M, and TRBC1 / 2 sites predicted to generate gene knockout through several mechanisms (SPLd = splice donor site mutation, SPLa = splice acceptor site mutation, SM = start codon mutation, ES = conversion to early stop codon). e, Representative flow cytometry histograms from one human donor showing ABE-mediated knockout of CD2 and B2M using sgRNAs indicated in (b-c), and f, CBE-mediated knockout of CD2, TRBC1 / 2, and B2M using sgRNAs indicated in (b-d). g, Quantification of base editing efficiency in (e) (n = 3 independent human donors). h, Quantification of base editing efficiency in (f), (n = independent human 4 donors for B2M_ES and TRBC1 / 2_ES; n = 2 independent human donors for B2M_SPLd and CD2_SM). i, Representative flow cytometry dotplots and histograms demonstrating CBE-mediated knockout of TCRab. For histograms, red indicates gated mTurquoise-negative cells, and blue indicates gated mTurquoise-positive cells. j, Quantification of ABE-mediated knockout of B2M with lentiviral integration of B2M_SM_1 sgRNA and electroporation of ABE mRNA in CD4 and CD8 T cell subsets (n = 2 independent human donors). k, Editing efficiency (measured by % B2M loss on flow cytometry) and viability of T cells transduced with B2M_SM_1 sgRNA and electroporated with varying doses of ABE. Vertical dotted line represents ABE dose selected (per 1e6 T cells) for screens. Error bars represent mean + / − SD (panels g, h, j).

[0051] FIG. 28 – Tiling screen targets, library transduction, and pooled base editing of T cells. a, Classification of sgRNAs in the ClinVar library based on mutation subtype. b, Schematic of gene targets for the 12-Gene tiling screen and their function in T cells. c, Classification of sgRNAs in the 12-Gene tiling library based on mutation subtype. d, Schematic for generation of library base-edited T cells. e, Transduction efficiency of ClinVar base editor library in n = 2 independent human donors.Attorney Docket 44010.185WO-PCT / / CU24092

[0052] FIG. 29 – Metrics for rigor and reproducibility of large-scale base editing screens. a, Density plots showing LFC values of different categories of guides from the ClinVar library at Day 35 post-electroporation of the long-term expansion screen arm. Dashed line represents the bottom 5% of the distribution of combined empty window and silent mutation controls. Indicated are the percentages of guides in each category falling below this threshold. sgRNAs generating variants in CD3D, CD3E, CD3G, or CD3Z were binned into the ‘CD3 complex’ category. The second donor from the screen is shown (in companion to Fig. 2a). b, Scatter plot showing LFC values of negative control sgRNAs (including both empty window and silent mutations) in both donors from the ClinVar Library at Day 28 post- electroporation in the long-term expansion screen arm. c-d, Distribution of robust rank aggregation (RRA) scores for gene-wise dropout analysis in the c, CD25 hi vs lo (activation) sort and d, CFSE lo vs hi (short-term proliferation) sort arms of the ClinVar library across both donors. The top 5 negatively selected genes in CD25 hi vs lo and in CFSE lo vs hi are listed. e, Shared positive control sgRNA (n = 600) were identified between the ClinVar and 12-gene tiling screens and sgRNA LFCs from matched long-term proliferation arm timepoints (Day 28 of ClinVar Screen, Day 26 of 12-gene Tiling Screen) are plotted. For each screen, the average LFC of each sgRNA across both donors is plotted. Simple linear regression with two-sided Pearson test (panel e).

[0053] FIG. 30 – Analysis of ClinVar screen across readouts. a, Scatterplot showing LFC of selected sgRNAs generating mutations in LCK, SOS1, and PTPRC. Timepoint shown is Day 28 post-electroporation in the ClinVar long-term expansion screen arm. b, Volcano plot showing enriched and depleted guides in the CFSE lo vs hi proliferation sort. For visualization purposes, one mutation for each labeled sgRNA is shown. One representative donor is shown. False discovery rate (FDR) cutoff <0.05. c, Volcano plot showing enriched and depleted guides in the CD25 hi vs lo proliferation sort. For visualization purposes, one mutation for each labeled sgRNA is shown. FDR cutoff <0.05. One representative donor is shown.

[0054] FIG.31 – Characterization of variant effects by ClinVar classification. a, (Top) distribution of negative control sgRNAs in the ClinVar library at day 28 of the long-term proliferation screen arm. (Bottom) sgRNA LFC distributions for selected genes targeted in the ClinVar library. Red lines indicate sgRNAs generating amino acid mutations which are identical to ClinVar-annotated pathogenic variants. b-j, Scatterplots of sgRNAs targeting selected genes in the ClinVar library at day 28 of the long-term proliferation screen arm. DottedAttorney Docket 44010.185WO-PCT / / CU24092 lines represent top and bottom 5% cutoffs of negative control sgRNA distribution. sgRNAs are binned into four distinct categories: predicted to generate an identical mutation to a ClinVar ‘VUS’ (‘Same VUS’; dark blue), predicted to generate a different mutation at an amino acid with a ClinVar ‘VUS’ (‘Diff VUS’; light blue), predicted to generate an identical mutation to a ClinVar ‘pathogenic’, ‘pathogenic / likely pathogenic’, or ‘likely pathogenic’ variant (‘Same P’; red), and sgRNAs predicted to generate a different mutation at an amino acid with a ClinVar ‘pathogenic’, ‘pathogenic / likely pathogenic’, or ‘likely pathogenic’ variant (‘Diff P’; yellow). Selected sgRNAs are annotated. Simple linear regression (panels b-j).

[0055] FIG. 32 – Independent analysis of 12-gene tiling screen and integration of results with ClinVar screen. a, sgRNA LFCs across both donors at Day 26 of the long-term proliferation arm of the 12-gene tiling screen are plotted. Dotted lines represent top and bottom 10% cutoffs of the distribution of negative control sgRNAs (empty window and silent only sgRNAs) for each donor. Selected sgRNAs, with predicted target gene and mutation, are shown. b, sgRNA LFCs as in a, with blue overlay filtered by target gene. c, Shared sgRNAs (n = 325) were identified between the ClinVar and 12-gene tiling screens and sgRNA LFCs from matched long-term proliferation arm timepoints (Day 28 of ClinVar Screen, Day 26 of 12-gene Tiling Screen) are plotted. For each screen, the average LFC of each sgRNA across both donors is plotted. Selected sgRNAs with shared enrichment / depletion patterns across donors and screens are annotated. Simple linear regression (panel c).

[0056] FIG. 33 – Enrichment and structure-function relationship of variants promoting T cell proliferation. a, Lollipop plot showing LFC of sgRNAs targeting PIK3CD at Day 15 post-electroporation in the long-term proliferation arm of the ClinVar screen. sgRNAs are mapped to the targeted region of the canonical isoform of PIK3CD (p110δ) and functional domains of the protein are annotated. Selected variants and their predicted mutational consequences are annotated. b,c, Timecourse line graphs of LFC of sgRNAs targeting PIK3CD in both donors in the long-term proliferation arm of the ClinVar screen. d, Lollipop plot for sgRNAs targeting AKT1 at Day 35 post-electroporation in the long- term proliferation arm of the ClinVar screen, mapped to the canonical AKT1 isoform. e, Timecourse line graphs of LFC of sgRNAs targeting AKT1 in the long-term expansion arm of the ClinVar screen. f, Structure and position of mutations in AKT1. (right) Overall predicted structure of AKT1 (blue) and mutated residues (red). (left) Wild-type (WT) and mutated (mut) residues (red). D323G is predicted to localize next to L14 (dark blue). g, Lollipop plots forAttorney Docket 44010.185WO-PCT / / CU24092 sgRNAs targeting LCK at day 26 post-electroporation in the long-term proliferation arm of the 12-gene tiling screen, mapped to the canonical LCK isoform. h, Structure and position of mutations in LCK. (top) Overall predicted structure of LCK (blue) and mutated residues (red). (bottom) Wild-type (WT) and mutated (mut) residues (red).

[0057] FIG. 34 – Signaling and impact of subtle differences in editing efficiency and phenotypic readouts. a, Quantification of S6 phosphorylation (pS235 / S236) and b, AKT phosphorylation (pS473) measured by flow cytometry in T cells with indicated genotypes (x axis) after 10 minutes of stimulation with anti-CD3 / CD28 antibodies. c, For all validated sgRNAs targeting PIK3CD (that is, Cys416Arg, Tyr524Cys, Glu525Gly_His526Arg, and Glu527Gly_Lys528Glu), sgRNA editing efficiency and effect size on AKT phosphorylation (pS473), d, TNFα MFI, and e, IL2 expression are plotted for each of the 3 donors used in initial validation experiments in Fig. 3. In cases where sgRNAs generated multiple edits within the editing window (for example, PIK3CD Glu525Gly_His526Arg), the average editing efficiency across all targeted bases in the editing window was used. Data in (a-b) was generated from n = 3 independent human donors. Within each donor this data was normalized to the silent control condition. One-way ANOVA with Dunnett’s test for multiple comparisons (panels a,b). Simple linear regression (panels c-e). Error bars represent mean + / − SD (panels a, b).

[0058] FIG. 35 – Experimental design, functional assays, and melanoma co-culture experiments with NY-ESO-1 TCR T cells engineered with variants identified in base editing screens. a, Schematic for engineering and expanding NY-ESO-1 specific T cells. b, Representative flow cytometry dotplot of NY-ESO-1 specific T cells prior to sorting. c, Viable A375-dsRed cells relative to time t0 after culture with NY-ESO-1 specific T cells for 48 hours at varying effector to target ratios. aMHCI = MHC class I-blocking antibody (n = 3 independent biological replicates). d, Representative flow cytometry histograms of ABE-mediated knockout of B2M or CD2 in NY-ESO-1 specific T cells. e, Representative contour plots of single or multiplexed base editing of B2M and CD2. f, AKT phosphorylation (pS473), in NY- ESO-1 specific T cells after either 15 minutes of co-culture with A375 cells (+) or media alone (-) (n = 3 independent biological replicates). g, MFI of TNFα and h, GrzB in NY-ESO-1 specific T cells with indicated base edits after 8-hour co-culture with A375-dsRed cells at a 1:1 effector to target ratio (n = 3 independent biological replicates). i, Frequency of NY-ESO-1 specific T cells with indicated genotypes co-expressing TNFα, IL2, and GrzB after 8 hours of co-culture with A375 cells at a 1:1 effector to target ratio (n = 3 independent biologicalAttorney Docket 44010.185WO-PCT / / CU24092 replicates.). NT = non-targeting control sgRNA j, Viable wild-type (WT) or CD58-KO A375 cells relative to time t0 after 48 hours of co-culture with NY-ESO-1 specific T cells at a 1:1 effector to target ratio (n = 3 independent biological replicates.) k, Viable B2M-KO A375 cells relative to time t0after 48 hours of co-culture with NY-ESO-1 specific T cells with indicated genotypes. Dotted lines in (f-i) represent mean of the control. Dotted lines in (j-k) represent relative viable cell count at time t0. One-way ANOVA with Tukey’s test for multiple comparisons (panel c). One-way ANOVA with Dunnett’s test for multiple comparisons (panels f-i, k). Student’s t test (panel j). Error bars represent mean + / − SD (panels c, f-k).

[0059] FIG. 36 – Design and results of leukemia co-culture with CD19 CAR-T cells equipped with variants identified in base editing screens. a, Representative histograms of GFP expression, indicating transduction efficiency of first- and second-generation CD19-CAR constructs (CD19-CD3z or CD19-BBz, respectively) in primary human T cells. Blue histograms represent untransduced control T cells. b, Expression of CTLA4 on CD19-CAR T cells, edited with a control non-targeting sgRNA (NT) or CTLA4-KO sgRNA, following 48- hour co-culture with Nalm6 leukemia cells at an 0.5:1 effector to target ratio. c, Relative cell numbers of Nalm6 cells 48 hours after co-culture with CD19-BBz CAR T variants at several E:T ratios, compared to time t0. d, Relative number of CD19-KO Nalm6 cells 48 hours after co-culture with CD19-BBz CAR T cells at an 0.25:1 effector to target ratio, compared to time t0. e, Quantification of CD19-CD3z CAR T cell AKT phosphorylation (pS473) by flow cytometry after 15 minutes of culture with either Nalm6 leukemia (+) or media alone (−) with representative flow histograms. f, Quantification of CD19-CD3z CAR T cell intracellular expression of TNFα and g, IL2 after 8-hour culture with Nalm6 leukemia (+) or in media only (-). h, Relative cell numbers of wild-type and i, CD19-KO Nalm6 cells 48 hours after co- culture with CD19-CD3z CAR T cells at an 0.5:1 effector to target ratio, compared to time t0. Dotted lines in (e-g) represent mean of the control population. Dotted lines in (panel c, d, h, i) represent relative viable cell count at time t0. One-way ANOVA with Tukey’s test for multiple comparisons (panel b), one-way ANOVA with Dunnett’s test for multiple comparisons (panels d-i). Error bars represent mean + / - SD (panels b-i).

[0060] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.Attorney Docket 44010.185WO-PCT / / CU24092 DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0061] Detailed aspects and applications of the disclosure are described below in the following drawings and detailed description of the technology. Unless specifically noted, it is intended that the words and phrases in the specification and the claims be given their plain, ordinary, and accustomed meaning to those of ordinary skill in the applicable arts.

[0062] In the following description, and for the purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the various aspects of the disclosure. It will be understood, however, by those skilled in the relevant arts, that embodiments of the technology disclosed herein may be practiced without these specific details. It should be noted that there are many different and alternative configurations, devices and technologies to which the disclosed technologies may be applied. The full scope of the technology disclosed herein is not limited to the examples that are described below.

[0063] The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a step” includes reference to one or more of such steps.

[0064] The words “exemplary,” “example,” or various forms thereof are used herein to mean serving as an example, instance, or illustration. Any aspect or design described herein as “exemplary” or as an “example” is not necessarily to be construed as preferred or advantageous over other aspects or designs. Furthermore, examples are provided solely for purposes of clarity and understanding and are not meant to limit or restrict the disclosed subject matter or relevant portions of this disclosure in any manner. It is to be appreciated that a myriad of additional or alternate examples of varying scope could have been presented, but have been omitted for purposes of brevity.

[0065] When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable. The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0066] The terms “about” or “approximately” as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of + / -10% or less,Attorney Docket 44010.185WO-PCT / / CU24092 + / -5% or less, + / -1% or less, and + / -0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier “about” or “approximately” refers is itself also specifically, and preferably, disclosed.

[0067] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, mean “including but not limited to”, and are not intended to (and do not) exclude other components.

[0068] As required, detailed embodiments of the present disclosure are included herein. It is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the disclosure to be better understood. However, they are given merely by way of guidance and do not imply any limitation.

[0069] The present disclosure may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that this disclosure is not limited to the specific materials, devices, methods, applications, conditions, or parameters described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed inventions. The term “plurality”, as used herein, means more than one. When a range of values is expressed, another embodiment includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment. All ranges are inclusive and combinable.

[0070] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Definitions of common terms and techniques in molecular biology may be found in Molecular Cloning: A Laboratory Manual, 2ndedition (1989) (Sambrook, Fritsch, and Maniatis); Molecular Cloning: A Laboratory Manual, 4thedition (2012) (Green and Sambrook); Current Protocols in Molecular Biology (1987) (F.M. Ausubel et al. eds.); the series Methods in Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (1995)Attorney Docket 44010.185WO-PCT / / CU24092 (M.J. MacPherson, B.D. Hames, and G.R. Taylor eds.): Antibodies, A Laboratory Manual (1988) (Harlow and Lane, eds.): Antibodies A Laboratory Manual, 2ndedition 2013 (E.A. Greenfield ed.); Animal Cell Culture (1987) (R.I. Freshney, ed.); Benjamin Lewin, Genes IX, published by Jones and Bartlet, 2008 (ISBN 0763752223); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0632021829); Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 9780471185710); Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992); and Marten H. Hofker and Jan van Deursen, Transgenic Mouse Methods and Protocols, 2ndedition (2011).

[0071] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0072] As used herein, a “biological sample” may contain whole cells and / or live cells and / or cell debris. The biological sample may contain (or be derived from) a “bodily fluid”. The present invention encompasses embodiments wherein the bodily fluid is selected from amniotic fluid, aqueous humour, vitreous humour, bile, blood serum, breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from a mammal organism, for example by puncture, or other collecting or sampling procedures.

[0073] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0074] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broaderAttorney Docket 44010.185WO-PCT / / CU24092 aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment(s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily – but may be - all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0075] Reference is made to Walsh ZH, Shah P, Kothapalli N, et al. Mapping variant effects on anti-tumor hallmarks of primary human T cells with base-editing screens. Nat Biotechnol. Published online May 23, 2024; and Walsh ZH, Shah P, Kothapalli N, et al. Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells. Preprint. bioRxiv. 2023;2023.12.13.571465. Published 2023 Dec 14.

[0076] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference. OVERVIEW

[0077] Embodiments disclosed herein provide for more efficient cellular cancer immunotherapies. Single-nucleotide variants (SNVs) in key T cell genes can drive clinical pathologies and could be repurposed to improve cellular cancer immunotherapies. Thousands of germline single-nucleotide variants (SNVs) exist in genes important for central T cell functions that cause clinical immune syndromes that can manifest on a spectrum from autoimmunity to immunodeficiency. A fraction of these mutations have well-defined pathogenicity and characterized clinical manifestations, but thousands of variants of uncertainAttorney Docket 44010.185WO-PCT / / CU24092 significance (VUSs) exist whose presence is not definitively or mechanistically linked to a clinical phenotype. Thus, Applicants reasoned that precisely engineering T cells with specific SNVs could enable several clinically relevant discoveries including identifying SNVs that enhance desirable T functions and thereby form a basis for more efficient cellular cancer immunotherapies and identifying previously unclassified or unstudied SNVs with strong functional consequences, nominating them for further study and potential reclassification.

[0078] Cellular therapies like T cell transfer and CAR-T cell therapies involve different immunotherapies to treat cancer and auto-immune disorders. However, many patients are irresponsive or suffer from various side effects that may be fatal. Furthermore, current genome- editing tools often have low editing efficiency particularly in primary human T cells. This, in addition to potential toxicity of or lack of response to therapies like adoptive T cell transfer and CAR-T cell therapies, poses difficulties in treating cancer and certain auto-immune disorders.

[0079] Base editing enables generation of single nucleotide variants, but large-scale screening in primary human T cells is limited due to low editing efficiency, among other challenges1. The present invention describes a method and system to achieve ultra-high efficiency base-editing using adenine and cysteine base-editors in primary human T cells with high efficacy and efficiency. Here, Applicants overcome existing challenges through improved production, delivery, and scalability of BE and achieve unprecedented single and multiplex editing efficiency in primary human T cells, while finding optimal balances between genome editing and maintaining cell viability. The base editor is produced entirely using in vitro transcription, resulting in a single encoding RNA transcript, delivered as a single RNA molecule translated by the host cell (rather than transduction with a virus), and enables timed delivery of single-guide RNAs for individual gene edits. This approach can be applied to perform large-scale genomic screens as well as in developing alternative cellular therapies against cancer and auto-immune disorders. Overall, the present invention can be used to generate natural or synthetic variants, that either alone or in combination with other edits, can be applied to treat cancer patients and individuals with auto-immune disorders, and reduce toxicity associated with cellular products.

[0080] Specifically, Applicants sought to leverage emerging CRISPR-dependent base editing (BE), which uses cytosine or adenine base editors (CBE and ABE) to induce site- specific deamination of cytosine or adenine, leading to C>T and A>G base transitions, respectively18. Applicants developed a high-throughput approach for high-efficiency andAttorney Docket 44010.185WO-PCT / / CU24092 massively parallel adenine and cytosine base-editor screening in primary human T cells. Coupled with relaxed protospacer-adjacent motif recognition sites (NG instead of NGG), BE enables mutagenesis of endogenous DNA across a wide range of the genome. Building on prior reports showing feasibility of high-efficiency BE of primary human T cells (PHTs)19–23, Applicants optimized methods to enable scalable BE screening, and performed multiple BE screens in PHTs, generating thousands of variants across 102 genes important for T cell functions. Applicants further performed full-length tiling mutagenesis of selected genes. Applicants further read out variant effects on hallmarks of T cell anti-tumor immunity, including activation, proliferation, and cytokine production. Applicants performed several large-scale BE screens in multiple donors under acute and chronic T cell activation conditions and found novel variants that improve T cell polyfunctionality across multiple donors. Applicants mapped the impact of base edits to hallmarks of T cell-mediated anti-tumor immunity, and harnessed variants that scored favorably across these readouts to enhance cellular immunotherapies. Applicants discovered a broad landscape of putative gain- (GOF) and loss-of-function (LOF) mutations, including in PIK3CD and its regulatory subunit PIK3R1, LCK, AKT1, CTLA-4, JAK1, SOS, AKT1, and RHOA. Applicants discovered variants that affected many (e.g., PIK3CD C416R) or only selected (e.g. LCK Y505C) hallmarks of T cell activity, and functionally validated several hits by probing downstream signaling nodes and testing their impact on T cell polyfunctionality and proliferation.

[0081] Using primary human T cells in which Applicants engineered a T cell receptor (TCR) specific to a commonly presented tumor testis antigen as a model for cellular immunotherapy, Applicants demonstrate that base edits identified in the screens can tune specific or broad T cell functions and ultimately improve tumor elimination while exerting minimal off-target activity. Base editing of PIK3CD and PIK3R1 variants in T cells with an engineered T cell receptor specific against a melanoma epitope or in different generations of CD19 chimeric antigen receptor T (CAR-T) cells demonstrated that discovered GOF variants, but not LOF or silent mutation controls, enhanced signaling, cytokine production and lysis of cognate melanoma and leukemia cell models, respectively. Additionally, Applicants show that generations of CD19 CAR-T cells engineered with PIK3CD GOF mutations demonstrate enhanced antigen-specific signaling, cytokine production, and leukemia cell killing, including when benchmarked against other recent strategies. Specifically, T cells with engineered melanoma epitope-specific T cell receptors (TCRs) or CD19 CAR-T cells, equipped withAttorney Docket 44010.185WO-PCT / / CU24092 favorable variants identified in these screens, exhibited enhanced activation signaling, cytokine production, and tumor cell-lytic activity against cognate melanoma and leukemia models, respectively. Enhanced activity also extended to contexts that typically confer resistance to cell therapy (such as CD58 loss) and when benchmarked against other recently demonstrated strategies to improve CAR-T cell therapy, such as CRISPR-Cas9 KO of CTLA-4. Through completely virus-free production and protein-free base editing of specific epitope-reactive human T cells that harbor base edits nominated from these screens, Applicants engineered T cell products with superior, on-target tumor-lytic ability.

[0082] In summary, Applicants present the first large-scale base editing screen in primary human T cells and provide a framework for scalable and targeted base editing at unprecedented efficiency. Coupled with multi-modal phenotypic mapping, Applicants accurately nominate variants that produce a broadly desirable T cell state and leverage these synthetic proteins to improve models of cellular cancer immunotherapies. Lastly, by comparing variant effects against the effects of known pathogenic mutations, this approach may enable re-classification of previously described VUS. Thus, this study paves the way for rapid identification and characterization of variants that could improve existing and future cellular cancer immunotherapies. Terminology and Definitions

[0083] The terms “therapeutic agent”, “therapeutic capable agent” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0084] As used herein, “treatment” or “treating,” or “palliating” or “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and / or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. For prophylactic benefit, the compositions may be administered to a subject at risk of developing a particular disease, condition, or symptom, or to a subject reporting one or more of the physiological symptoms of a disease, even though theAttorney Docket 44010.185WO-PCT / / CU24092 disease, condition, or symptom may not have yet been manifested. As used herein “treating” includes ameliorating, curing, preventing it from becoming worse, slowing the rate of progression, or preventing the disorder from re-occurring (i.e., to prevent a relapse).

[0085] The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will provide an image for detection by any one of the imaging methods described herein. The specific dose may vary depending on one or more of: the particular agent chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to be imaged, and the physical delivery system in which it is carried.

[0086] A “pharmaceutical composition” refers to a composition that usually contains an excipient, such as a pharmaceutically acceptable carrier that is conventional in the art and that is suitable for administration to cells or to a subject.

[0087] All gene name symbols refer to the gene as commonly known in the art. The examples described herein that refer to the human gene names are to be understood to also encompasses genes in any other organism, for example mouse genes or any other gene used in a model of disease (e.g., homologous, orthologous genes). Any reference to the gene symbol is a reference made to the entire gene or variants of the gene. Any reference to the gene symbol is also a reference made to the gene product (e.g., protein). The term, homolog, may apply to the relationship between genes separated by the event of speciation (e.g., ortholog). Orthologs are genes in different species that evolved from a common ancestral gene by speciation. Normally, orthologs retain the same function in the course of evolution. Gene symbols may be those referred to by the HUGO Gene Nomenclature Committee (HGNC) or National Center for Biotechnology Information (NCBI). METHODS OF TREATMENT

[0088] In example embodiments, cancer can be treated by administering T cells to a subject in need thereof with T cells comprising a gain of function mutation as described herein. For example, T cells having the gain of function mutations have enhanced anti-tumor immuneAttorney Docket 44010.185WO-PCT / / CU24092 activity. In example embodiments, subjects resistant to cancer treatments can be treated with the T cells described herein (e.g., resistance to immunotherapies, such as immune checkpoint inhibitors). For example, subjects that have tumors with decreased or abolished antigen presentation (e.g., mutations in B2M) can be treated (see, e.g., Sade-Feldman M, Jiao YJ, Chen JH, et al. Resistance to checkpoint blockade therapy through inactivation of antigen presentation. Nat Commun.2017;8(1):1136. Published 2017 Oct 26). For example, tumors that do not express CD58 can evade the immune system (see, e.g., US patent application publication US20240294643A1) and can be treated.

[0089] In example embodiments, T cells comprising loss of function mutations that reduce T cell immune responses are used to treat autoimmune diseases. Not being bound by a theory, T cells having the loss of function mutations described herein can generate a suppressive immune environment when contacting an autoantigen. For example, the T cells may express suppressive cytokines that reduce the immune response of other immune cells not comprising the loss of function mutations. In example embodiments, the genetically engineered T cells of the instant invention are different from any autologous T cells obtained from a subject. Adoptive Cell Therapy

[0090] In example embodiments, the mutations described herein are introduced to T cells using a genetic modification agent to improve adoptive cell therapy. As used herein, “ACT”, “adoptive cell therapy” and “adoptive cell transfer” may be used interchangeably. In certain embodiments, Adoptive cell therapy (ACT) can refer to the transfer of cells to a patient with the goal of transferring the functionality and characteristics into the new host by engraftment of the cells (see, e.g., Mettananda et al., Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia, Nat Commun.2017 Sep 4;8(1):424). As used herein, the term "engraft" or "engraftment" refers to the process of cell incorporation into a tissue of interest in vivo through contact with existing cells of the tissue. Adoptive cell therapy (ACT) can refer to the transfer of cells, most commonly immune-derived cells (e.g., T cells or NK cells), back into the same patient or into a new recipient host with the goal of transferring the immunologic functionality and characteristics into the new host. If possible, use of autologous cells helps the recipient by minimizing GVHD issues. The adoptive transfer of autologous tumor infiltrating lymphocytes (TIL) (Zacharakis et al., (2018) Nat Med. 2018 Jun;24(6):724-730; Besser et al., (2010) Clin. Cancer Res 16 (9) 2646–55; Dudley et al., (2002) Science 298 (5594): 850–4; and Dudley et al., (2005) Journal of Clinical Oncology 23 (10):Attorney Docket 44010.185WO-PCT / / CU24092 2346–57.) or genetically re-directed peripheral blood mononuclear cells (Johnson et al., (2009) Blood 114 (3): 535–46; and Morgan et al., (2006) Science 314(5796) 126-9) has been used to successfully treat patients with advanced solid tumors, including melanoma, metastatic breast cancer and colorectal carcinoma, as well as patients with CD19-expressing hematologic malignancies (Kalos et al., (2011) Science Translational Medicine 3 (95): 95ra73). In certain embodiments, allogenic cells immune cells are transferred (see, e.g., Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266). As described further herein, allogenic cells can be edited to reduce alloreactivity and prevent graft-versus-host disease. Thus, use of allogenic cells allows for cells to be obtained from healthy donors and prepared for use in patients as opposed to preparing autologous cells from a patient after diagnosis.

[0091] Aspects of the invention involve the adoptive transfer of immune system cells, such as T cells or NK cells, specific for selected antigens, such as tumor associated antigens or tumor specific neoantigens (see, e.g., Maus et al., 2014, Adoptive Immunotherapy for Cancer or Viruses, Annual Review of Immunology, Vol. 32: 189-225; Rosenberg and Restifo, 2015, Adoptive cell transfer as personalized immunotherapy for human cancer, Science Vol.348 no. 6230 pp.62-68; Restifo et al., 2015, Adoptive immunotherapy for cancer: harnessing the T cell response. Nat. Rev. Immunol. 12(4): 269-281; and Jenson and Riddell, 2014, Design and implementation of adoptive therapy with chimeric antigen receptor-modified T cells. Immunol Rev. 257(1): 127–144; and Rajasagi et al., 2014, Systematic identification of personal tumor- specific neoantigens in chronic lymphocytic leukemia. Blood. 2014 Jul 17;124(3):453-62).

[0092] In certain embodiments, an antigen (such as a tumor antigen or self-antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: MR1 (see, e.g., Crowther, et al., 2020, Genome-wide CRISPR–Cas9 screening reveals ubiquitous T cell cancer targeting via the monomorphic MHC class I-related protein MR1, Nature Immunology volume 21, pages178–185), B cell maturation antigen (BCMA) (see, e.g., Friedman et al., Effective Targeting of Multiple BCMA-Expressing Hematological Malignancies by Anti- BCMA CAR T Cells, Hum Gene Ther. 2018 Mar 8; Berdeja JG, et al. Durable clinical responses in heavily pretreated patients with relapsed / refractory multiple myeloma: updated results from a multicenter study of bb2121 anti-Bcma CAR T cell therapy. Blood. 2017;130:740; and Mouhieddine and Ghobrial, Immunotherapy in Multiple Myeloma: The Era of CAR T Cell Therapy, Hematologist, May-June 2018, Volume 15, issue 3); PSA (prostate-Attorney Docket 44010.185WO-PCT / / CU24092 specific antigen); prostate-specific membrane antigen (PSMA); PSCA (Prostate stem cell antigen); Tyrosine-protein kinase transmembrane receptor ROR1; fibroblast activation protein (FAP); Tumor-associated glycoprotein 72 (TAG72); Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); Mesothelin; Human Epidermal growth factor Receptor 2 (ERBB2 (Her2 / neu)); Prostase; Prostatic acid phosphatase (PAP); elongation factor 2 mutant (ELF2M); Insulin-like growth factor 1 receptor (IGF-1R); gplOO; BCR-ABL (breakpoint cluster region-Abelson); tyrosinase; New York esophageal squamous cell carcinoma 1 (NY-ESO-1); κ-light chain, LAGE (L antigen); MAGE (melanoma antigen); Melanoma-associated antigen 1 (MAGE-A1); MAGE A3; MAGE A6; legumain; Human papillomavirus (HPV) E6; HPV E7; prostein; survivin; PCTA1 (Galectin 8); Melan-A / MART- 1; Ras mutant; TRP-1 (tyrosinase related protein 1, or gp75); Tyrosinase-related Protein 2 (TRP2); TRP-2 / INT2 (TRP-2 / intron 2); RAGE (renal antigen); receptor for advanced glycation end products 1 (RAGE1); Renal ubiquitous 1, 2 (RU1, RU2); intestinal carboxyl esterase (iCE); Heat shock protein 70-2 (HSP70-2) mutant; thyroid stimulating hormone receptor (TSHR); CD123; CD171; CD19; CD20; CD22; CD26; CD30; CD33; CD44v7 / 8 (cluster of differentiation 44, exons 7 / 8); CD53; CD92; CD100; CD148; CD150; CD200; CD261; CD262; CD362; CS-1 (CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1- 1)Cer); Tn antigen (Tn Ag); Fms-Like Tyrosine Kinase 3 (FLT3); CD38; CD138; CD44v6; B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2); Interleukin 11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); Protease Serine 21 (PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet- derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); Mucin 1, cell surface associated (MUC1); mucin 16 (MUC16); epidermal growth factor receptor (EGFR); epidermal growth factor receptor variant III (EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); ephrin type-A receptor 2 (EphA2); Ephrin B2; Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2- 3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; high molecular weight-melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor alpha; Folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); G protein-coupled receptor class C group 5, member DAttorney Docket 44010.185WO-PCT / / CU24092 (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR- 1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (HAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie 2); CT (cancer / testis (antigen)); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53; p53 mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-Acetyl glucosaminyl-transferase V (NA17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B1; Cyclin D1; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS); Squamous Cell Carcinoma Antigen Recognized By T Cells-1 or 3 (SART1, SART3); Paired box protein Pax- 5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint-1, -2, -3 or -4 (SSX1, SSX2, SSX3, SSX4); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); mouse double minute 2 homolog (MDM2); livin; alphafetoprotein (AFP); transmembrane activator and CAML Interactor (TACI); B-cell activating factor receptor (BAFF-R); V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS); immunoglobulin lambda-like polypeptide 1 (IGLL1); 707-AP (707 alanine proline); ART-4 (adenocarcinoma antigen recognized by T4 cells); BAGE (B antigen; b-catenin / m, b-catenin / mutated); CAMEL (CTL- recognized antigen on melanoma); CAP1 (carcinoembryonic antigen peptide 1); CASP-8Attorney Docket 44010.185WO-PCT / / CU24092 (caspase-8); CDC27m (cell-division cycle 27 mutated); CDK4 / m (cycline-dependent kinase 4 mutated); Cyp-B (cyclophilin B); DAM (differentiation antigen melanoma); EGP-2 (epithelial glycoprotein 2); EGP-40 (epithelial glycoprotein 40); Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4); FBP (folate binding protein); , fAchR (Fetal acetylcholine receptor); G250 (glycoprotein 250); GAGE (G antigen); GnT-V (N- acetylglucosaminyltransferase V); HAGE (helicose antigen); ULA-A (human leukocyte antigen-A); HST2 (human signet ring tumor 2); KIAA0205; KDR (kinase insert domain receptor); LDLR / FUT (low density lipid receptor / GDP L-fucose: b-D-galactosidase 2-a-L fucosyltransferase); L1CAM (L1 cell adhesion molecule); MC1R (melanocortin 1 receptor); Myosin / m (myosin mutated); MUM-1, -2, -3 (melanoma ubiquitous mutated 1, 2, 3); NA88-A (NA cDNA clone of patient M88); KG2D (Natural killer group 2, member D) ligands; oncofetal antigen (h5T4); p190 minor bcr-abl (protein of 190KD bcr-abl); Pml / RARa (promyelocytic leukaemia / retinoic acid receptor a); PRAME (preferentially expressed antigen of melanoma); SAGE (sarcoma antigen); TEL / AML1 (translocation Ets-family leukemia / acute myeloid leukemia 1); TPI / m (triosephosphate isomerase mutated); CD70; and any combination thereof. In example embodiments, claudins are targeted by ACT (see, e.g., US20210347847A1).

[0093] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-specific antigen (TSA).

[0094] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a neoantigen.

[0095] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a tumor-associated antigen (TAA).

[0096] In certain embodiments, an antigen to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) is a universal tumor antigen. In certain preferred embodiments, the universal tumor antigen is selected from the group consisting of: a human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1B 1 (CYP1B), HER2 / neu, Wilms' tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonicAttorney Docket 44010.185WO-PCT / / CU24092 antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (Dl), and any combinations thereof.

[0097] In certain embodiments, an antigen (such as a tumor antigen) to be targeted in adoptive cell therapy (such as particularly CAR or TCR T-cell therapy) of a disease (such as particularly of tumor or cancer) may be selected from a group consisting of: CD19, BCMA, CD70, CLL-1, MAGE A3, MAGE A6, HPV E6, HPV E7, WT1, CD22, CD171, ROR1, MUC16, and SSX2. In certain preferred embodiments, the antigen may be CD19. For example, CD19 may be targeted in hematologic malignancies, such as in lymphomas, more particularly in B-cell lymphomas, such as without limitation in diffuse large B-cell lymphoma, primary mediastinal b-cell lymphoma, transformed follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia including adult and pediatric ALL, non- Hodgkin lymphoma, indolent non-Hodgkin lymphoma, or chronic lymphocytic leukemia. For example, BCMA may be targeted in multiple myeloma or plasma cell leukemia (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic Chimeric Antigen Receptor T Cells Targeting B Cell Maturation Antigen). For example, CLL1 may be targeted in acute myeloid leukemia. For example, MAGE A3, MAGE A6, SSX2, and / or KRAS may be targeted in solid tumors. For example, HPV E6 and / or HPV E7 may be targeted in cervical cancer or head and neck cancer. For example, WT1 may be targeted in acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), chronic myeloid leukemia (CML), non- small cell lung cancer, breast, pancreatic, ovarian or colorectal cancers, or mesothelioma. For example, CD22 may be targeted in B cell malignancies, including non-Hodgkin lymphoma, diffuse large B-cell lymphoma, or acute lymphoblastic leukemia. For example, CD171 may be targeted in neuroblastoma, glioblastoma, or lung, pancreatic, or ovarian cancers. For example, ROR1 may be targeted in ROR1+ malignancies, including non-small cell lung cancer, triple negative breast cancer, pancreatic cancer, prostate cancer, ALL, chronic lymphocytic leukemia, or mantle cell lymphoma. For example, MUC16 may be targeted in MUC16ecto+ epithelial ovarian, fallopian tube or primary peritoneal cancer. For example, CD70 may be targeted in both hematologic malignancies as well as in solid cancers such as renal cell carcinoma (RCC), gliomas (e.g., GBM), and head and neck cancers (HNSCC). CD70 is expressed in both hematologic malignancies as well as in solid cancers, while its expression in normal tissues is restricted to a subset of lymphoid cell types (see, e.g., 2018 American Association for Cancer Research (AACR) Annual meeting Poster: Allogeneic CRISPR Engineered Anti-CD70 CAR-Attorney Docket 44010.185WO-PCT / / CU24092 T Cells Demonstrate Potent Preclinical Activity Against Both Solid and Hematological Cancer Cells).

[0098] In example embodiments, Yescarta is an FDA-approved CAR-T cell therapy that is particularly used to treat relapsed or refractory large B-cell lymphoma. Yescarta has shown relatively high efficacy in terms of patient response and preventing cancer progression or the need for additional cancer treatment. In example embodiments, Abecma is an FDA-approved CAR T-cell therapy used to treat adult patients with relapsed or refractory multiple myeloma following other therapeutic approaches. In example embodiments, Tecartus is an FDA- approved CAR T-cell therapy to treat relapsed or refractory mantle cell lymphoma as well as B-cell precursor acute lymphoblastic leukemia. It is used following previous therapies and has previously been reported to show relatively high complete and overall response rate in treating lymphoma. In example embodiments, Kymriah is an FDA-approved CAR T-cell therapy to treat diffuse large B-cell lymphoma and relapsed or refractory acute lymphoblastic leukemia. Novartis has relatively long-term data showing durable remission and long-term survival in children and young adults with this form of leukemia. Furthermore, Kymriah has a relatively good safety profile according to currently reported results. In example embodiments, Carvykti is an FDA-approved CAR T-cell therapy to treat relapsed or refractory multiple myeloma following four lines of treatment. In prior studies involving a follow-up of approximately 18 months, patients showed a relatively high overall response rate as well as in previous pilot studies. It was also shown to reduce risk of progression and relapse

[0099] Various strategies may for example be employed to genetically modify T cells by altering the specificity of the T cell receptor (TCR) for example by introducing new TCR α and β chains with selected peptide specificity (see U.S. Patent No. 8,697,854; PCT Patent Publications: WO2003020763, WO2004033685, WO2004044004, WO2005114215, WO2006000830, WO2008038002, WO2008039818, WO2004074322, WO2005113595, WO2006125962, WO2013166321, WO2013039889, WO2014018863, WO2014083173; U.S. Patent No. 8,088,379).

[0100] As an alternative to, or addition to, TCR modifications, chimeric antigen receptors (CARs) may be used in order to generate immunoresponsive cells, such as T cells or natural killer cells (NK), specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see U.S. Patent Nos.5,843,728; 5,851,828;Attorney Docket 44010.185WO-PCT / / CU24092 5,912,170; 6,004,811; 6,284,240; 6,392,013; 6,410,014; 6,753,162; 8,211,422; and, PCT Publication WO9215322).

[0101] In general, CARs are comprised of an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises an antigen- binding domain that is specific for a predetermined target (see, e.g., Gong Y, Klein Wolterink RGJ, Wang J, Bos GMJ, Germeraad WTV. Chimeric antigen receptor natural killer (CAR-NK) cell design and engineering for cancer therapy. J Hematol Oncol. 2021;14(1):73; Guedan S, Calderon H, Posey AD Jr, Maus MV. Engineering and Design of Chimeric Antigen Receptors. Mol Ther Methods Clin Dev. 2018;12:145-156; Petersen CT, Krenciute G. Next Generation CAR T Cells for the Immunotherapy of High-Grade Glioma. Front Oncol. 2019;9:69; Lu H, Zhao X, Li Z, Hu Y, Wang H. From CAR-T Cells to CAR-NK Cells: A Developing Immunotherapy Method for Hematological Malignancies. Front Oncol. 2021; and Miliotou AN, Papadopoulou LC. CAR T-cell Therapy: A New Era in Cancer Immunotherapy. Curr Pharm Biotechnol. 2018;19(1):5-18). While the antigen-binding domain of a CAR is often an antibody or antibody fragment (e.g., a single chain variable fragment, scFv), the binding domain is not particularly limited so long as it results in specific recognition of a target. For example, in some embodiments, the antigen-binding domain may comprise a receptor, such that the CAR is capable of binding to the ligand of the receptor. Alternatively, the antigen- binding domain may comprise a ligand, such that the CAR is capable of binding the endogenous receptor of that ligand.

[0102] The antigen-binding domain of a CAR is generally separated from the transmembrane domain by a hinge or spacer. The spacer is also not particularly limited, and it is designed to provide the CAR with flexibility. For example, a spacer domain may comprise a portion of a human Fc domain, including a portion of the CH3 domain, or the hinge region of any immunoglobulin, such as IgA, IgD, IgE, IgG, or IgM, or variants thereof. Furthermore, the hinge region may be modified so as to prevent off-target binding by FcRs or other potential interfering objects. For example, the hinge may comprise an IgG4 Fc domain with or without a S228P, L235E, and / or N297Q mutation (according to Kabat numbering) in order to decrease binding to FcRs. Additional spacers / hinges include, but are not limited to, CD4, CD8, and CD28 hinge regions.

[0103] The transmembrane domain of a CAR may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membraneAttorney Docket 44010.185WO-PCT / / CU24092 bound or transmembrane protein. Transmembrane regions of particular use in this disclosure may be derived from CD8, CD28, CD3, CD45, CD4, CD5, CDS, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, TCR. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0104] Alternative CAR constructs may be characterized as belonging to successive generations. First-generation CARs typically consist of a single-chain variable fragment of an antibody specific for an antigen, for example comprising a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8α hinge domain and a CD8α transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3ζ or FcRγ (scFv-CD3ζ or scFv-FcRγ; see U.S. Patent No. 7,741,465; U.S. Patent No. 5,912,172; U.S. Patent No. 5,906,936). Second-generation CARs incorporate the intracellular domains of one or more costimulatory molecules, such as CD28, OX40 (CD134), or 4-1BB (CD137) within the endodomain (for example scFv-CD28 / OX40 / 4-1BB-CD3ζ; see U.S. Patent Nos. 8,911,993; 8,916,381; 8,975,071; 9,101,584; 9,102,760; 9,102,761). Third-generation CARs include a combination of costimulatory endodomains, such a CD3ζ-chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, CD2, CD7, LIGHT, LFA-1, NKG2C, B7-H3, CD30, CD40, PD-1, or CD28 signaling domains (for example scFv-CD28- 4-1BB-CD3ζ or scFv-CD28-OX40-CD3ζ; see U.S. Patent No. 8,906,682; U.S. Patent No. 8,399,645; U.S. Pat. No. 5,686,281; PCT Publication No. WO2014134165; PCT Publication No. WO2012079000). In certain embodiments, the primary signaling domain comprises a functional signaling domain of a protein selected from the group consisting of CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCERIG), FcR beta (Fc Epsilon R1b), CD79a, CD79b, Fc gamma RIIa, DAP10, and DAP12. In certain preferred embodiments, the primary signaling domain comprises a functional signaling domain of CD3ζ or FcRγ. In certain embodiments, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-Attorney Docket 44010.185WO-PCT / / CU24092 associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, and NKG2D. In certain embodiments, the one or more costimulatory signaling domains comprise a functional signaling domain of a protein selected, each independently, from the group consisting of: 4-1BB, CD27, and CD28. In certain embodiments, a chimeric antigen receptor may have the design as described in U.S. Patent No. 7,446,190, comprising an intracellular domain of CD3ζ chain (such as amino acid residues 52- 163 of the human CD3 zeta chain, as shown in SEQ ID NO: 14 of US 7,446,190), a signaling region from CD28 and an antigen-binding element (or portion or domain; such as scFv). The CD28 portion, when between the zeta chain portion and the antigen-binding element, may suitably include the transmembrane and signaling domains of CD28 (such as amino acid residues 114-220 of SEQ ID NO: 10, full sequence shown in SEQ ID NO: 6 of US 7,446,190; these can include the portion of CD28 as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3). Alternatively, when the zeta sequence lies between the CD28 sequence and the antigen-binding element, intracellular domain of CD28 can be used alone (such as amino sequence set forth in SEQ ID NO: 9 of US 7,446,190). Hence, certain embodiments employ a CAR comprising (a) a zeta chain portion comprising the intracellular domain of human CD3ζ chain, (b) a costimulatory signaling region, and (c) an antigen-binding element (or portion or domain), wherein the costimulatory signaling region comprises the amino acid sequence encoded by SEQ ID NO: 6 of US 7,446,190.

[0105] Alternatively, costimulation may be orchestrated by expressing CARs in antigen- specific T cells, chosen so as to be activated and expanded following engagement of their native αβTCR, for example by antigen on professional antigen-presenting cells, with attendant costimulation. In addition, additional engineered receptors may be provided on theAttorney Docket 44010.185WO-PCT / / CU24092 immunoresponsive cells, for example to improve targeting of a T-cell attack and / or minimize side effects

[0106] By means of an example and without limitation, Kochenderfer et al., (2009) J Immunother.32 (7): 689-702 described anti-CD19 chimeric antigen receptors (CAR). FMC63- 28Z CAR contained a single chain variable region moiety (scFv) recognizing CD19 derived from the FMC63 mouse hybridoma (described in Nicholson et al., (1997) Molecular Immunology 34: 1157–1165), a portion of the human CD28 molecule, and the intracellular component of the human TCR-ζ molecule. FMC63-CD828BBZ CAR contained the FMC63 scFv, the hinge and transmembrane regions of the CD8 molecule, the cytoplasmic portions of CD28 and 4-1BB, and the cytoplasmic component of the TCR-ζ molecule. The exact sequence of the CD28 molecule included in the FMC63-28Z CAR corresponded to Genbank identifier NM_006139. To encode the anti-CD19 scFv component of the vector, the authors designed a DNA sequence which was based on a portion of a previously published CAR (Cooper et al., (2003) Blood 101: 1637–1644). This sequence encoded the following components in frame from the 5’ end to the 3’ end: an XhoI site, the human granulocyte-macrophage colony- stimulating factor (GM-CSF) receptor α-chain signal sequence, the FMC63 light chain variable region (as in Nicholson et al., supra), a linker peptide (as in Cooper et al., supra), the FMC63 heavy chain variable region (as in Nicholson et al., supra), and a NotI site. A plasmid encoding this sequence was digested with XhoI and NotI. To form the MSGV-FMC63-28Z retroviral vector, the XhoI and NotI-digested fragment encoding the FMC63 scFv was ligated into a second XhoI and NotI-digested fragment that encoded the MSGV retroviral backbone (as in Hughes et al., (2005) Human Gene Therapy 16: 457–472) as well as part of the extracellular portion of human CD28, the entire transmembrane and cytoplasmic portion of human CD28, and the cytoplasmic portion of the human TCR-ζ molecule (as in Maher et al., 2002) Nature Biotechnology 20: 70–75). The FMC63-28Z CAR is included in the KTE-C19 (axicabtagene ciloleucel) anti-CD19 CAR-T therapy product in development by Kite Pharma, Inc. for the treatment of inter alia patients with relapsed / refractory aggressive B-cell non-Hodgkin lymphoma (NHL). Accordingly, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may express the FMC63- 28Z CAR as described by Kochenderfer et al. (supra). Hence, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element (or portion orAttorney Docket 44010.185WO-PCT / / CU24092 domain; such as scFv) that specifically binds to an antigen, an intracellular signaling domain comprising an intracellular domain of a CD3ζ chain, and a costimulatory signaling region comprising a signaling domain of CD28. Preferably, the CD28 amino acid sequence is as set forth in Genbank identifier NM_006139 (sequence version 1, 2 or 3). Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the anti-CD19 scFv as described by Kochenderfer et al. (supra).

[0107] Additional anti-CD19 CARs are further described in WO2015187528. More particularly Example 1 and Table 1 of WO2015187528, incorporated by reference herein, demonstrate the generation of anti-CD19 CARs based on a fully human anti-CD19 monoclonal antibody (47G4, as described in US20100104509) and murine anti-CD19 monoclonal antibody (as described in Nicholson et al. and explained above). Various combinations of a signal sequence (human CD8-alpha or GM-CSF receptor), extracellular and transmembrane regions (human CD8-alpha) and intracellular T-cell signalling domains (CD28-CD3ζ; 4-1BB-CD3ζ; CD27-CD3ζ; CD28-CD27-CD3ζ, 4-1BB-CD27-CD3ζ; CD27-4-1BB-CD3ζ; CD28-CD27- FcεRI gamma chain; or CD28-FcεRI gamma chain) were disclosed. Hence, in certain embodiments, cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may comprise a CAR comprising an extracellular antigen-binding element that specifically binds to an antigen, an extracellular and transmembrane region as set forth in Table 1 of WO2015187528 and an intracellular T-cell signaling domain as set forth in Table 1 of WO2015187528. Preferably, the antigen is CD19, more preferably the antigen-binding element is an anti-CD19 scFv, even more preferably the mouse or human anti-CD19 scFv as described in Example 1 of WO2015187528. In certain embodiments, the CAR comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13 as set forth in Table 1 of WO2015187528.

[0108] By means of an example and without limitation, chimeric antigen receptor that recognizes the CD70 antigen is described in WO2012058460A2 (see also, Park et al., CD70 as a target for chimeric antigen receptor T cells in head and neck squamous cell carcinoma, Oral Oncol. 2018 Mar;78:145-150; and Jin et al., CD70, a novel target of CAR T-cell therapy for gliomas, Neuro Oncol. 2018 Jan 10;20(1):55-65). CD70 is expressed by diffuse large B-cell and follicular lymphoma and also by the malignant cells of Hodgkins lymphoma,Attorney Docket 44010.185WO-PCT / / CU24092 Waldenstrom's macroglobulinemia and multiple myeloma, and by HTLV-1- and EBV- associated malignancies. (Agathanggelou et al. Am.J.Pathol. 1995;147: 1152-1160; Hunter et al., Blood 2004; 104:4881. 26; Lens et al., J Immunol. 2005;174:6212-6219; Baba et al., J Virol. 2008;82:3843-3852.) In addition, CD70 is expressed by non-hematological malignancies such as renal cell carcinoma and glioblastoma. (Junker et al., J Urol. 2005;173:2150-2153; Chahlavi et al., Cancer Res 2005;65:5428-5438) Physiologically, CD70 expression is transient and restricted to a subset of highly activated T, B, and dendritic cells.

[0109] By means of an example and without limitation, chimeric antigen receptor that recognizes BCMA has been described (see, e.g., US20160046724A1; WO2016014789A2; WO2017211900A1; WO2015158671A1; US20180085444A1; WO2018028647A1; US20170283504A1; and WO2013154760A1).

[0110] Additional CAR T cell therapies with enhanced efficacy are further described in WO / 2017 / 049166.

[0111] In certain embodiments, the immune cell may, in addition to a CAR or exogenous TCR as described herein, further comprise a chimeric inhibitory receptor (inhibitory CAR) that specifically binds to a second target antigen and is capable of inducing an inhibitory or immunosuppressive or repressive signal to the cell upon recognition of the second target antigen. In certain embodiments, the chimeric inhibitory receptor comprises an extracellular antigen-binding element (or portion or domain) configured to specifically bind to a target antigen, a transmembrane domain, and an intracellular immunosuppressive or repressive signaling domain. In certain embodiments, the second target antigen is an antigen that is not expressed on the surface of a cancer cell or infected cell or the expression of which is downregulated on a cancer cell or an infected cell. In certain embodiments, the second target antigen is an MHC-class I molecule. In certain embodiments, the intracellular signaling domain comprises a functional signaling portion of an immune checkpoint molecule, such as for example PD-1 or CTLA4. Advantageously, the inclusion of such inhibitory CAR reduces the chance of the engineered immune cells attacking non-target (e.g., non-cancer) tissues.

[0112] Alternatively, T-cells expressing CARs may be further modified to reduce or eliminate expression of endogenous TCRs in order to reduce off-target effects. Reduction or elimination of endogenous TCRs can reduce off-target effects and increase the effectiveness of the T cells (U.S. 9,181,527). T cells stably lacking expression of a functional TCR may be produced using a variety of approaches. T cells internalize, sort, and degrade the entire T cellAttorney Docket 44010.185WO-PCT / / CU24092 receptor as a complex, with a half-life of about 10 hours in resting T cells and 3 hours in stimulated T cells (von Essen, M. et al.2004. J. Immunol.173:384-393). Proper functioning of the TCR complex requires the proper stoichiometric ratio of the proteins that compose the TCR complex. TCR function also requires two functioning TCR zeta proteins with ITAM motifs. The activation of the TCR upon engagement of its MHC-peptide ligand requires the engagement of several TCRs on the same T cell, which all must signal properly. Thus, if a TCR complex is destabilized with proteins that do not associate properly or cannot signal optimally, the T cell will not become activated sufficiently to begin a cellular response.

[0113] Accordingly, in some embodiments, TCR expression may eliminated using RNA interference (e.g., shRNA, siRNA, miRNA, etc.), CRISPR, or other methods that target the nucleic acids encoding specific TCRs (e.g., TCR-α and TCR-β) and / or CD3 chains in primary T cells. By blocking expression of one or more of these proteins, the T cell will no longer produce one or more of the key components of the TCR complex, thereby destabilizing the TCR complex and preventing cell surface expression of a functional TCR.

[0114] In some instances, CAR may also comprise a switch mechanism for controlling expression and / or activation of the CAR. For example, a CAR may comprise an extracellular, transmembrane, and intracellular domain, in which the extracellular domain comprises a target- specific binding element that comprises a label, binding domain, or tag that is specific for a molecule other than the target antigen that is expressed on or by a target cell. In such embodiments, the specificity of the CAR is provided by a second construct that comprises a target antigen binding domain (e.g., an scFv or a bispecific antibody that is specific for both the target antigen and the label or tag on the CAR) and a domain that is recognized by or binds to the label, binding domain, or tag on the CAR. See, e.g., WO 2013 / 044225, WO 2016 / 000304, WO 2015 / 057834, WO 2015 / 057852, WO 2016 / 070061, US 9,233,125, US 2016 / 0129109. In this way, a T-cell that expresses the CAR can be administered to a subject, but the CAR cannot bind its target antigen until the second composition comprising an antigen- specific binding domain is administered.

[0115] Alternative switch mechanisms include CARs that require multimerization in order to activate their signaling function (see, e.g., US 2015 / 0368342, US 2016 / 0175359, US 2015 / 0368360) and / or an exogenous signal, such as a small molecule drug (US 2016 / 0166613, Yung et al., Science, 2015), in order to elicit a T-cell response. Some CARs may also comprise a “suicide switch” to induce cell death of the CAR T-cells following treatment (Buddee et al.,Attorney Docket 44010.185WO-PCT / / CU24092 PLoS One, 2013) or to downregulate expression of the CAR following binding to the target antigen (WO 2016 / 011210).

[0116] Alternative techniques may be used to transform target immunoresponsive cells, such as protoplast fusion, lipofection, transfection or electroporation. A wide variety of vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons, such as a Sleeping Beauty transposon (see U.S. Patent Nos.6,489,458; 7,148,203; 7,160,682; 7,985,739; 8,227,432), may be used to introduce CARs, for example using 2nd generation antigen-specific CARs signaling through CD3ζ and either CD28 or CD137. Viral vectors may for example include vectors based on HIV, SV40, EBV, HSV or BPV. In certain embodiments, inducible gene switches are used to regulate expression of a CAR or TCR (see, e.g., Chakravarti, Deboki et al. “Inducible Gene Switches with Memory in Human T Cells for Cellular Immunotherapy.” ACS synthetic biology vol.8,8 (2019): 1744- 1754).

[0117] Cells that are targeted for transformation may for example include T cells, Natural Killer (NK) cells, cytotoxic T lymphocytes (CTL), regulatory T cells, human embryonic stem cells, tumor-infiltrating lymphocytes (TIL) or a pluripotent stem cell from which lymphoid cells may be differentiated. T cells expressing a desired CAR may for example be selected through co-culture with γ-irradiated activating and propagating cells (AaPC), which co-express the cancer antigen and co-stimulatory molecules. The engineered CAR T-cells may be expanded, for example by co-culture on AaPC in presence of soluble factors, such as IL-2 and IL-21. This expansion may for example be carried out so as to provide memory CAR+ T cells (which may for example be assayed by non-enzymatic digital array and / or multi-panel flow cytometry). In this way, CAR T cells may be provided that have specific cytotoxic activity against antigen-bearing tumors (optionally in conjunction with production of desired chemokines such as interferon-γ). CAR T cells of this kind may for example be used in animal models, for example to treat tumor xenografts.

[0118] In certain embodiments, ACT includes co-transferring CD4+ Th1 cells and CD8+ CTLs to induce a synergistic antitumour response (see, e.g., Li et al., Adoptive cell therapy with CD4+ T helper 1 cells and CD8+ cytotoxic T cells enhances complete rejection of an established tumour, leading to generation of endogenous memory responses to non-targeted tumour epitopes. Clin Transl Immunology. 2017 Oct; 6(10): e160).Attorney Docket 44010.185WO-PCT / / CU24092

[0119] In certain embodiments, antigen specificity can be conferred to Tregs by engineering the expression of transgenic T-cell receptor (TCR) or chimeric antigen receptor (CAR), such as to modulate immune responses in organ transplant and autoimmune diseases (see, e.g., Arjomandnejad M, Kopec AL, Keeler AM. CAR-T Regulatory (CAR-Treg) Cells: Engineering and Applications. Biomedicines. 2022;10(2):287). Regulatory T cells (Tregs) are a T-cell subset known for their immunomodulatory function. Expression of CD4, CD25, and the master transcription factor, forkhead box P3 (FOXP3), are the main characteristic markers of conventional Tregs. However, other regulatory immune cells with different properties such as CD8+ Tregs, or type 1 regulatory T cells (Tr1) have been described. Id. Tregs are divided into “natural” Tregs that develop in the thymus or “induced” Tregs that are generated in the periphery. Id. Regulatory T cells suppress immune responses through multiple mechanisms including direct interaction with other immune cells or by producing immunosuppressive cytokines such as interleukin-10 (IL-10) and Transforming growth factor beta (TGF-β). Id. Directing Tregs towards a desired antigen may boost the overall response and lower the risk of broad and systemic immunosuppression or generation of an inflammatory response. Id.

[0120] In certain embodiments, Th17 cells are transferred to a subject in need thereof. Th17 cells have been reported to directly eradicate melanoma tumors in mice to a greater extent than Th1 cells (Muranski P, et al., Tumor-specific Th17-polarized cells eradicate large established melanoma. Blood. 2008 Jul 15; 112(2):362-73; and Martin-Orozco N, et al., T helper 17 cells promote cytotoxic T cell activation in tumor immunity. Immunity. 2009 Nov 20; 31(5):787- 98). Those studies involved an adoptive T cell transfer (ACT) therapy approach, which takes advantage of CD4+T cells that express a TCR recognizing tyrosinase tumor antigen. Exploitation of the TCR leads to rapid expansion of Th17 populations to large numbers ex vivo for reinfusion into the autologous tumor-bearing hosts.

[0121] In certain embodiments, ACT may include autologous iPSC-based vaccines, such as irradiated iPSCs in autologous anti-tumor vaccines (see e.g., Kooreman, Nigel G. et al., Autologous iPSC-Based Vaccines Elicit Anti-tumor Responses In Vivo, Cell Stem Cell 22, 1– 13, 2018, doi.org / 10.1016 / j.stem.2018.01.016).

[0122] Unlike T-cell receptors (TCRs) that are MHC restricted, CARs can potentially bind any cell surface-expressed antigen and can thus be more universally used to treat patients (see Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org / 10.3389 / fimmu.2017.00267).Attorney Docket 44010.185WO-PCT / / CU24092 In certain embodiments, in the absence of endogenous T-cell infiltrate (e.g., due to aberrant antigen processing and presentation), which precludes the use of TIL therapy and immune checkpoint blockade, the transfer of CAR T-cells may be used to treat patients (see, e.g., Hinrichs CS, Rosenberg SA. Exploiting the curative potential of adoptive T-cell therapy for cancer. Immunol Rev (2014) 257(1):56–71. doi:10.1111 / imr.12132).

[0123] Approaches such as the foregoing may be adapted to provide methods of treating and / or increasing survival of a subject having a disease, such as a neoplasia, for example by administering an effective amount of an immunoresponsive cell comprising an antigen recognizing receptor that binds a selected antigen, wherein the binding activates the immunoresponsive cell, thereby treating or preventing the disease (such as a neoplasia, a pathogen infection, an autoimmune disorder, or an allogeneic transplant reaction).

[0124] In certain embodiments, the treatment can be administered after lymphodepleting pretreatment in the form of chemotherapy (typically a combination of cyclophosphamide and fludarabine) or radiation therapy. Initial studies in ACT had short lived responses and the transferred cells did not persist in vivo for very long (Houot et al., T-cell-based immunotherapy: adoptive cell transfer and checkpoint inhibition. Cancer Immunol Res (2015) 3(10):1115–22; and Kamta et al., Advancing Cancer Therapy with Present and Emerging Immuno-Oncology Approaches. Front. Oncol. (2017) 7:64). Immune suppressor cells like Tregs and MDSCs may attenuate the activity of transferred cells by outcompeting them for the necessary cytokines. Not being bound by a theory lymphodepleting pretreatment may eliminate the suppressor cells allowing the TILs to persist.

[0125] In one embodiment, the treatment can be administrated into patients undergoing an immunosuppressive treatment (e.g., glucocorticoid treatment). The cells or population of cells, may be made resistant to at least one immunosuppressive agent due to the inactivation of a gene encoding a receptor for such immunosuppressive agent. In certain embodiments, the immunosuppressive treatment provides for the selection and expansion of the immunoresponsive T cells within the patient.

[0126] In certain embodiments, the treatment can be administered before primary treatment (e.g., surgery or radiation therapy) to shrink a tumor before the primary treatment. In another embodiment, the treatment can be administered after primary treatment to remove any remaining cancer cells.Attorney Docket 44010.185WO-PCT / / CU24092

[0127] In certain embodiments, immunometabolic barriers can be targeted therapeutically prior to and / or during ACT to enhance responses to ACT or CAR T-cell therapy and to support endogenous immunity (see, e.g., Irving et al., Engineering Chimeric Antigen Receptor T-Cells for Racing in Solid Tumors: Don’t Forget the Fuel, Front. Immunol., 03 April 2017, doi.org / 10.3389 / fimmu.2017.00267).

[0128] The administration of cells or population of cells, such as immune system cells or cell populations, such as more particularly immunoresponsive cells or cell populations, as disclosed herein may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The cells or population of cells may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, intrathecally, by intravenous or intralymphatic injection, or intraperitoneally. In some embodiments, the disclosed CARs may be delivered or administered into a cavity formed by the resection of tumor tissue (i.e. intracavity delivery) or directly into a tumor prior to resection (i.e. intratumoral delivery). In one embodiment, the cell compositions of the present invention are preferably administered by intravenous injection.

[0129] The administration of the cells or population of cells can consist of the administration of 104- 109cells per kg body weight, preferably 105to 106cells / kg body weight including all integer values of cell numbers within those ranges. Dosing in CAR T cell therapies may for example involve administration of from 106to 109cells / kg, with or without a course of lymphodepletion, for example with cyclophosphamide. The cells or population of cells can be administrated in one or more doses. In another embodiment, the effective amount of cells are administrated as a single dose. In another embodiment, the effective amount of cells are administrated as more than one dose over a period time. Timing of administration is within the judgment of managing physician and depends on the clinical condition of the patient. The cells or population of cells may be obtained from any source, such as a blood bank or a donor. While individual needs vary, determination of optimal ranges of effective amounts of a given cell type for a particular disease or conditions are within the skill of one in the art. An effective amount means an amount which provides a therapeutic or prophylactic benefit. The dosage administrated will be dependent upon the age, health and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment and the nature of the effect desired.Attorney Docket 44010.185WO-PCT / / CU24092

[0130] In another embodiment, the effective amount of cells or composition comprising those cells are administrated parenterally. The administration can be an intravenous administration. The administration can be directly done by injection within a tumor.

[0131] To guard against possible adverse reactions, engineered immunoresponsive cells may be equipped with a transgenic safety switch, in the form of a transgene that renders the cells vulnerable to exposure to a specific signal. For example, the herpes simplex viral thymidine kinase (TK) gene may be used in this way, for example by introduction into allogeneic T lymphocytes used as donor lymphocyte infusions following stem cell transplantation (Greco, et al., Improving the safety of cell therapy with the TK-suicide gene. Front. Pharmacol. 2015; 6: 95). In such cells, administration of a nucleoside prodrug such as ganciclovir or acyclovir causes cell death. Alternative safety switch constructs include inducible caspase 9, for example triggered by administration of a small-molecule dimerizer that brings together two nonfunctional icasp9 molecules to form the active enzyme. A wide variety of alternative approaches to implementing cellular proliferation controls have been described (see U.S. Patent Publication No. 20130071414; PCT Patent Publication WO2011146862; PCT Patent Publication WO2014011987; PCT Patent Publication WO2013040371; Zhou et al. BLOOD, 2014, 123 / 25:3895 – 3905; Di Stasi et al., The New England Journal of Medicine 2011; 365:1673-1683; Sadelain M, The New England Journal of Medicine 2011; 365:1735-173; Ramos et al., Stem Cells 28(6):1107-15 (2010)).

[0132] In a further refinement of adoptive therapies, genome editing may be used to tailor immunoresponsive cells to alternative implementations, for example providing edited CAR T cells (see Poirot et al., 2015, Multiplex genome edited T-cell manufacturing platform for "off- the-shelf" adoptive T-cell immunotherapies, Cancer Res 75 (18): 3853; Ren et al., 2017, Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition, Clin Cancer Res.2017 May 1;23(9):2255-2266. doi: 10.1158 / 1078-0432.CCR-16-1300. Epub 2016 Nov 4; Qasim et al., 2017, Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells, Sci Transl Med. 2017 Jan 25;9(374); Legut, et al., 2018, CRISPR-mediated TCR replacement generates superior anticancer transgenic T cells. Blood, 131(3), 311-322; Georgiadis et al., Long Terminal Repeat CRISPR-CAR-Coupled “Universal” T Cells Mediate Potent Anti-leukemic Effects, Molecular Therapy, In Press, Corrected Proof, Available online 6 March 2018; Roth, T.L. Editing of Endogenous Genes in Cellular Immunotherapies. Curr Hematol Malig Rep 15, 235–240 (2020); and Webber BR, LonetreeAttorney Docket 44010.185WO-PCT / / CU24092 CL, Kluesner MG, et al. Highly efficient multiplex human T cell engineering without double- strand breaks using Cas9 base editors [published correction appears in Nat Commun.2019 Dec 6;10(1):5659. doi: 10.1038 / s41467-019-13778-y]. Nat Commun.2019;10(1):5222). Cells may be edited using any CRISPR system and method of use thereof as described herein. CRISPR systems may be delivered to an immune cell by any method described herein. In preferred embodiments, cells are edited ex vivo and transferred to a subject in need thereof. Immunoresponsive cells, CAR T cells or any cells used for adoptive cell transfer may be edited. Editing may be performed for example to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell (e.g. TRAC locus); to eliminate potential alloreactive T-cell receptors (TCR) or to prevent inappropriate pairing between endogenous and exogenous TCR chains, such as to knock-out or knock-down expression of an endogenous TCR in a cell; to disrupt the target of a chemotherapeutic agent in a cell; to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell; to knock-out or knock-down expression of other gene or genes in a cell, the reduced expression or lack of expression of which can enhance the efficacy of adoptive therapies using the cell; to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR; to knock-out or knock-down expression of one or more MHC constituent proteins in a cell; to activate a T cell; to modulate cells such that the cells are resistant to exhaustion or dysfunction; and / or increase the differentiation and / or proliferation of functionally exhausted or dysfunctional CD8+ T-cells (see PCT Patent Publications: WO2013176915, WO2014059173, WO2014172606, WO2014184744, and WO2014191128).

[0133] In certain embodiments, editing may result in inactivation of a gene. By inactivating a gene, it is intended that the gene of interest is not expressed in a functional protein form. In a particular embodiment, the CRISPR system specifically catalyzes cleavage in one targeted gene thereby inactivating said targeted gene. The nucleic acid strand breaks caused are commonly repaired through the distinct mechanisms of homologous recombination or non- homologous end joining (NHEJ). However, NHEJ is an imperfect repair process that often results in changes to the DNA sequence at the site of the cleavage. Repair via non-homologous end joining (NHEJ) often results in small insertions or deletions (Indel) and can be used for the creation of specific gene knockouts. Cells in which a cleavage induced mutagenesis event has occurred can be identified and / or selected by well-known methods in the art. In certainAttorney Docket 44010.185WO-PCT / / CU24092 embodiments, homology directed repair (HDR) is used to concurrently inactivate a gene (e.g., TRAC) and insert an endogenous TCR or CAR into the inactivated locus.

[0134] Hence, in certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to insert or knock-in an exogenous gene, such as an exogenous gene encoding a CAR or a TCR, at a preselected locus in a cell. Conventionally, nucleic acid molecules encoding CARs or TCRs are transfected or transduced to cells using randomly integrating vectors, which, depending on the site of integration, may lead to clonal expansion, oncogenic transformation, variegated transgene expression and / or transcriptional silencing of the transgene. Directing of transgene(s) to a specific locus in a cell can minimize or avoid such risks and advantageously provide for uniform expression of the transgene(s) by the cells. Without limitation, suitable ‘safe harbor’ loci for directed transgene integration include CCR5 or AAVS1. Homology-directed repair (HDR) strategies are known and described elsewhere in this specification allowing to insert transgenes into desired loci (e.g., TRAC locus).

[0135] Further suitable loci for insertion of transgenes, in particular CAR or exogenous TCR transgenes, include without limitation loci comprising genes coding for constituents of endogenous T-cell receptor, such as T-cell receptor alpha locus (TRA) or T-cell receptor beta locus (TRB), for example T-cell receptor alpha constant (TRAC) locus, T-cell receptor beta constant 1 (TRBC1) locus or T-cell receptor beta constant 2 (TRBC1) locus. Advantageously, insertion of a transgene into such locus can simultaneously achieve expression of the transgene, potentially controlled by the endogenous promoter, and knock-out expression of the endogenous TCR. This approach has been exemplified in Eyquem et al., (2017) Nature 543: 113-117, wherein the authors used CRISPR / Cas9 gene editing to knock-in a DNA molecule encoding a CD19-specific CAR into the TRAC locus downstream of the endogenous promoter; the CAR-T cells obtained by CRISPR were significantly superior in terms of reduced tonic CAR signaling and exhaustion.

[0136] T cell receptors (TCR) are cell surface receptors that participate in the activation of T cells in response to the presentation of antigen. The TCR is generally made from two chains, α and β, which assemble to form a heterodimer and associates with the CD3-transducing subunits to form the T cell receptor complex present on the cell surface. Each α and β chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region,Attorney Docket 44010.185WO-PCT / / CU24092 a hydrophobic transmembrane domain, and a short cytoplasmic region. As for immunoglobulin molecules, the variable region of the α and β chains are generated by V(D)J recombination, creating a large diversity of antigen specificities within the population of T cells. However, in contrast to immunoglobulins that recognize intact antigen, T cells are activated by processed peptide fragments in association with an MHC molecule, introducing an extra dimension to antigen recognition by T cells, known as MHC restriction. Recognition of MHC disparities between the donor and recipient through the T cell receptor leads to T cell proliferation and the potential development of graft versus host disease (GVHD). The inactivation of TCRα or TCRβ can result in the elimination of the TCR from the surface of T cells preventing recognition of alloantigen and thus GVHD. However, TCR disruption generally results in the elimination of the CD3 signaling component and alters the means of further T cell expansion.

[0137] Hence, in certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of an endogenous TCR in a cell. For example, NHEJ-based or HDR-based gene editing approaches can be employed to disrupt the endogenous TCR alpha and / or beta chain genes. For example, gene editing system or systems, such as CRISPR / Cas system or systems, can be designed to target a sequence found within the TCR beta chain conserved between the beta 1 and beta 2 constant region genes (TRBC1 and TRBC2) and / or to target the constant region of the TCR alpha chain (TRAC) gene.

[0138] Allogeneic cells are rapidly rejected by the host immune system. It has been demonstrated that, allogeneic leukocytes present in non-irradiated blood products will persist for no more than 5 to 6 days (Boni, Muranski et al. 2008 Blood 1;112(12):4746-54). Thus, to prevent rejection of allogeneic cells, the host's immune system usually has to be suppressed to some extent. However, in the case of adoptive cell transfer the use of immunosuppressive drugs also have a detrimental effect on the introduced therapeutic T cells. Therefore, to effectively use an adoptive immunotherapy approach in these conditions, the introduced cells would need to be resistant to the immunosuppressive treatment. Thus, in a particular embodiment, the present invention further comprises a step of modifying T cells to make them resistant to an immunosuppressive agent, preferably by inactivating at least one gene encoding a target for an immunosuppressive agent. An immunosuppressive agent is an agent that suppresses immune function by one of several mechanisms of action. An immunosuppressive agent can be, but isAttorney Docket 44010.185WO-PCT / / CU24092 not limited to a calcineurin inhibitor, a target of rapamycin, an interleukin-2 receptor α-chain blocker, an inhibitor of inosine monophosphate dehydrogenase, an inhibitor of dihydrofolic acid reductase, a corticosteroid or an immunosuppressive antimetabolite. The present invention allows conferring immunosuppressive resistance to T cells for immunotherapy by inactivating the target of the immunosuppressive agent in T cells. As non-limiting examples, targets for an immunosuppressive agent can be a receptor for an immunosuppressive agent such as: CD52, glucocorticoid receptor (GR), a FKBP family gene member and a cyclophilin family gene member.

[0139] In certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to block an immune checkpoint, such as to knock-out or knock-down expression of an immune checkpoint protein or receptor in a cell. Immune checkpoints are inhibitory pathways that slow down or stop immune reactions and prevent excessive tissue damage from uncontrolled activity of immune cells. In certain embodiments, the immune checkpoint targeted is the programmed death-1 (PD-1 or CD279) gene (PDCD1) (see, e.g., Rupp LJ, Schumann K, Roybal KT, et al. CRISPR / Cas9-mediated PD-1 disruption enhances anti-tumor efficacy of human chimeric antigen receptor T cells. Sci Rep. 2017;7(1):737). In other embodiments, the immune checkpoint targeted is cytotoxic T-lymphocyte-associated antigen (CTLA-4). In additional embodiments, the immune checkpoint targeted is another member of the CD28 and CTLA4 Ig superfamily such as BTLA, LAG3, ICOS, PDL1 or KIR. In further additional embodiments, the immune checkpoint targeted is a member of the TNFR superfamily such as CD40, OX40, CD137, GITR, CD27 or TIM-3.

[0140] Additional immune checkpoints include Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) (Watson HA, et al., SHP-1: the next checkpoint target for cancer immunotherapy? Biochem Soc Trans. 2016 Apr 15;44(2):356-62). SHP-1 is a widely expressed inhibitory protein tyrosine phosphatase (PTP). In T-cells, it is a negative regulator of antigen-dependent activation and proliferation. It is a cytosolic protein, and therefore not amenable to antibody-mediated therapies, but its role in activation and proliferation makes it an attractive target for genetic manipulation in adoptive transfer strategies, such as chimeric antigen receptor (CAR) T cells. Immune checkpoints may also include T cell immunoreceptor with Ig and ITIM domains (TIGIT / Vstm3 / WUCAM / VSIG9) and VISTA (Le Mercier I, et al.,Attorney Docket 44010.185WO-PCT / / CU24092 (2015) Beyond CTLA-4 and PD-1, the generation Z of negative checkpoint regulators. Front. Immunol. 6:418).

[0141] WO2014172606 relates to the use of MT1 and / or MT2 inhibitors to increase proliferation and / or activity of exhausted CD8+ T-cells and to decrease CD8+ T-cell exhaustion (e.g., decrease functionally exhausted or unresponsive CD8+ immune cells). In certain embodiments, metallothioneins are targeted by gene editing in adoptively transferred T cells.

[0142] In certain embodiments, targets of gene editing may be at least one targeted locus involved in the expression of an immune checkpoint protein. Such targets may include, but are not limited to CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, ICOS (CD278), PDL1, KIR, LAG3, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, LAIR1, SIGLEC7, SIGLEC9, CD244 (2B4), TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT1, FOXP3, PRDM1, BATF, VISTA, GUCY1A2, GUCY1A3, GUCY1B2, GUCY1B3, MT1, MT2, CD40, OX40, CD137, GITR, CD27, SHP-1, TIM-3, CEACAM-1, CEACAM-3, or CEACAM-5. In preferred embodiments, the gene locus involved in the expression of PD-1 or CTLA-4 genes is targeted. In other preferred embodiments, combinations of genes are targeted, such as but not limited to PD-1 and TIGIT.

[0143] By means of an example and without limitation, WO2016196388 concerns an engineered T cell comprising (a) a genetically engineered antigen receptor that specifically binds to an antigen, which receptor may be a CAR; and (b) a disrupted gene encoding a PD- L1, an agent for disruption of a gene encoding a PD- L1, and / or disruption of a gene encoding PD-L1, wherein the disruption of the gene may be mediated by a gene editing nuclease, a zinc finger nuclease (ZFN), CRISPR / Cas9 and / or TALEN. WO2015142675 relates to immune effector cells comprising a CAR in combination with an agent (such as CRISPR, TALEN or ZFN) that increases the efficacy of the immune effector cells in the treatment of cancer, wherein the agent may inhibit an immune inhibitory molecule, such as PD1, PD-L1, CTLA-4, TIM-3, LAG-3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, TGFR beta, CEACAM-1, CEACAM- 3, or CEACAM-5. Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR,Attorney Docket 44010.185WO-PCT / / CU24092 β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.

[0144] In certain embodiments, cells may be engineered to express a CAR, wherein expression and / or function of methylcytosine dioxygenase genes (TET1, TET2 and / or TET3) in the cells has been reduced or eliminated, such as by CRISPR, ZNF or TALEN (for example, as described in WO201704916).

[0145] In certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of an endogenous gene in a cell, said endogenous gene encoding an antigen targeted by an exogenous CAR or TCR, thereby reducing the likelihood of targeting of the engineered cells. In certain embodiments, the targeted antigen may be one or more antigen selected from the group consisting of CD38, CD138, CS-1, CD33, CD26, CD30, CD53, CD92, CD100, CD148, CD150, CD200, CD261, CD262, CD362, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P4501B1 (CYP1B), HER2 / neu, Wilms’ tumor gene 1 (WT1), livin, alphafetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate-specific membrane antigen (PSMA), p53, cyclin (D1), B cell maturation antigen (BCMA), transmembrane activator and CAML Interactor (TACI), and B-cell activating factor receptor (BAFF-R) (for example, as described in WO2016011210 and WO2017011804).

[0146] In certain embodiments, editing of cells (such as by CRISPR / Cas), particularly cells intended for adoptive cell therapies, more particularly immunoresponsive cells such as T cells, may be performed to knock-out or knock-down expression of one or more MHC constituent proteins, such as one or more HLA proteins and / or beta-2 microglobulin (B2M), in a cell, whereby rejection of non-autologous (e.g., allogeneic) cells by the recipient’s immune system can be reduced or avoided. In preferred embodiments, one or more HLA class I proteins, such as HLA-A, B and / or C, and / or B2M may be knocked-out or knocked-down. Preferably, B2M may be knocked-out or knocked-down. By means of an example, Ren et al., (2017) Clin Cancer Res 23 (9) 2255-2266 performed lentiviral delivery of CAR and electro-transfer of Cas9 mRNA and gRNAs targeting endogenous TCR, β-2 microglobulin (B2M) and PD1 simultaneously, to generate gene-disrupted allogeneic CAR T cells deficient of TCR, HLA class I molecule and PD1.Attorney Docket 44010.185WO-PCT / / CU24092

[0147] In other embodiments, at least two genes are edited. Pairs of genes may include, but are not limited to PD1 and TCRα, PD1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, Tim3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, B2M and TCRα, B2M and TCRβ.

[0148] In certain embodiments, a cell may be multiply edited (multiplex genome editing) as taught herein to (1) knock-out or knock-down expression of an endogenous TCR (for example, TRBC1, TRBC2 and / or TRAC), (2) knock-out or knock-down expression of an immune checkpoint protein or receptor (for example PD1, PD-L1 and / or CTLA4); and (3) knock-out or knock-down expression of one or more MHC constituent proteins (for example, HLA-A, B and / or C, and / or B2M, preferably B2M).

[0149] Whether prior to or after genetic modification of the T cells, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Patents 6,352,694; 6,534,055; 6,905,680; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and 7,572,631. T cells can be expanded in vitro or in vivo.

[0150] Immune cells may be obtained using any method known in the art. In one embodiment, allogenic T cells may be obtained from healthy subjects. In one embodiment T cells that have infiltrated a tumor are isolated. T cells may be removed during surgery. T cells may be isolated after removal of tumor tissue by biopsy. T cells may be isolated by any means known in the art. In one embodiment, T cells are obtained by apheresis. In one embodiment, the method may comprise obtaining a bulk population of T cells from a tumor sample by any suitable method known in the art. For example, a bulk population of T cells can be obtained from a tumor sample by dissociating the tumor sample into a cell suspension from which specific cell populations can be selected. Suitable methods of obtaining a bulk population of T cells may include, but are not limited to, any one or more of mechanically dissociating (e.g., mincing) the tumor, enzymatically dissociating (e.g., digesting) the tumor, and aspiration (e.g., as with a needle).Attorney Docket 44010.185WO-PCT / / CU24092

[0151] The bulk population of T cells obtained from a tumor sample may comprise any suitable type of T cell. Preferably, the bulk population of T cells obtained from a tumor sample comprises tumor infiltrating lymphocytes (TILs).

[0152] The tumor sample may be obtained from any mammal. Unless stated otherwise, as used herein, the term "mammal" refers to any mammal including, but not limited to, mammals of the order Logomorpha, such as rabbits; the order Carnivora, including Felines (cats) and Canines (dogs); the order Artiodactyla, including Bovines (cows) and Swines (pigs); or of the order Perssodactyla, including Equines (horses). The mammals may be non-human primates, e.g., of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some embodiments, the mammal may be a mammal of the order Rodentia, such as mice and hamsters. Preferably, the mammal is a non-human primate or a human. An especially preferred mammal is the human.

[0153] T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, spleen tissue, and tumors. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor) according to the manufacturer's instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.Attorney Docket 44010.185WO-PCT / / CU24092

[0154] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. A specific subpopulation of T cells, such as CD28+, CD4+, CDC, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one preferred embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3×28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, or XCYTE DYNABEADS™ for a time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells.

[0155] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and / or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0156] Further, monocyte populations (i.e., CD14+ cells) may be depleted from blood preparations by a variety of methodologies, including anti-CD14 coated beads or columns, or utilization of the phagocytotic activity of these cells to facilitate removal. Accordingly, in one embodiment, the invention uses paramagnetic particles of a size sufficient to be engulfed by phagocytotic monocytes. In certain embodiments, the paramagnetic particles are commercially available beads, for example, those produced by Life Technologies under the trade name Dynabeads™. In one embodiment, other non-specific cells are removed by coating the paramagnetic particles with “irrelevant” proteins (e.g., serum proteins or antibodies). IrrelevantAttorney Docket 44010.185WO-PCT / / CU24092 proteins and antibodies include those proteins and antibodies or fragments thereof that do not specifically target the T cells to be isolated. In certain embodiments, the irrelevant beads include beads coated with sheep anti-mouse antibodies, goat anti-mouse antibodies, and human serum albumin.

[0157] In brief, such depletion of monocytes is performed by preincubating T cells isolated from whole blood, apheresed peripheral blood, or tumors with one or more varieties of irrelevant or non-antibody coupled paramagnetic particles at any amount that allows for removal of monocytes (approximately a 20:1 bead:cell ratio) for about 30 minutes to 2 hours at 22 to 37 degrees C., followed by magnetic removal of cells which have attached to or engulfed the paramagnetic particles. Such separation can be performed using standard methods available in the art. For example, any magnetic separation methodology may be used including a variety of which are commercially available, (e.g., DYNAL® Magnetic Particle Concentrator (DYNAL MPC®)). Assurance of requisite depletion can be monitored by a variety of methodologies known to those of ordinary skill in the art, including flow cytometric analysis of CD14 positive cells, before and after depletion.

[0158] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In a further embodiment, greater than 100 million cells / ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / ml is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells / ml is used. In further embodiments, concentrations of 125 or 150 million cells / ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.Attorney Docket 44010.185WO-PCT / / CU24092

[0159] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4+ T cells express higher levels of CD28 and are more efficiently captured than CD8+ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5×106 / ml. In other embodiments, the concentration used can be from about 1×105 / ml to 1×106 / ml, and any integer value in between.

[0160] T cells can also be frozen. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After a washing step to remove plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media, the cells then are frozen to −80° C at a rate of 1° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at −20° C. or in liquid nitrogen.

[0161] T cells for use in the present invention may also be antigen-specific T cells. For example, tumor-specific T cells can be used. In certain embodiments, antigen-specific T cells can be isolated from a patient of interest, such as a patient afflicted with a cancer or an infectious disease. In one embodiment, neoepitopes are determined for a subject and T cells specific to these antigens are isolated. Antigen-specific cells for use in expansion may also be generated in vitro using any number of methods known in the art, for example, as described in U.S. Patent Publication No. US 20040224402 entitled, Generation and Isolation of Antigen- Specific T Cells, or in U.S. Pat. Nos. 6,040,177. Antigen-specific cells for use in the present invention may also be generated using any number of methods known in the art, for example, as described in Current Protocols in Immunology, or Current Protocols in Cell Biology, both published by John Wiley & Sons, Inc., Boston, Mass.

[0162] In a related embodiment, it may be desirable to sort or otherwise positively select (e.g. via magnetic selection) the antigen specific cells prior to or following one or two rounds of expansion. Sorting or positively selecting antigen-specific cells can be carried out using peptide-MHC tetramers (Altman, et al., Science. 1996 Oct. 4; 274(5284):94-6). In anotherAttorney Docket 44010.185WO-PCT / / CU24092 embodiment, the adaptable tetramer technology approach is used (Andersen et al., 2012 Nat Protoc. 7:891-902). Tetramers are limited by the need to utilize predicted binding peptides based on prior hypotheses, and the restriction to specific HLAs. Peptide-MHC tetramers can be generated using techniques known in the art and can be made with any MHC molecule of interest and any antigen of interest as described herein. Specific epitopes to be used in this context can be identified using numerous assays known in the art. For example, the ability of a polypeptide to bind to MHC class I may be evaluated indirectly by monitoring the ability to promote incorporation of125I labeled β2-microglobulin (β2m) into MHC class I / β2m / peptide heterotrimeric complexes (see Parker et al., J. Immunol. 152:163, 1994).

[0163] In one embodiment cells are directly labeled with an epitope-specific reagent for isolation by flow cytometry followed by characterization of phenotype and TCRs. In one embodiment, T cells are isolated by contacting with T cell specific antibodies. Sorting of antigen-specific T cells, or generally any cells of the present invention, can be carried out using any of a variety of commercially available cell sorters, including, but not limited to, MoFlo sorter (DakoCytomation, Fort Collins, Colo.), FACSAria™, FACSArray™, FACSVantage™, BD™ LSR II, and FACSCalibur™ (BD Biosciences, San Jose, Calif.).

[0164] In a preferred embodiment, the method comprises selecting cells that also express CD3. The method may comprise specifically selecting the cells in any suitable manner. Preferably, the selecting is carried out using flow cytometry. The flow cytometry may be carried out using any suitable method known in the art. The flow cytometry may employ any suitable antibodies and stains. Preferably, the antibody is chosen such that it specifically recognizes and binds to the particular biomarker being selected. For example, the specific selection of CD3, CD8, TIM-3, LAG-3, 4-1BB, or PD-1 may be carried out using anti-CD3, anti-CD8, anti-TIM-3, anti-LAG-3, anti-4-lBB, or anti-PD-1 antibodies, respectively. The antibody or antibodies may be conjugated to a bead (e.g., a magnetic bead) or to a fluorochrome. Preferably, the flow cytometry is fluorescence-activated cell sorting (FACS). TCRs expressed on T cells can be selected based on reactivity to autologous tumors. Additionally, T cells that are reactive to tumors can be selected for based on markers using the methods described in patent publication Nos. WO2014133567 and WO2014133568, herein incorporated by reference in their entirety. Additionally, activated T cells can be selected for based on surface expression of CD107a.Attorney Docket 44010.185WO-PCT / / CU24092

[0165] In one embodiment of the invention, the method further comprises expanding the numbers of T cells in the enriched cell population. Such methods are described in U.S. Patent No. 8,637,307 and is herein incorporated by reference in its entirety. The numbers of T cells may be increased at least about 3-fold (or 4-, 5-, 6-, 7-, 8-, or 9-fold), more preferably at least about 10-fold (or 20-, 30-, 40-, 50-, 60-, 70-, 80-, or 90-fold), more preferably at least about 100-fold, more preferably at least about 1,000 fold, or most preferably at least about 100,000- fold. The numbers of T cells may be expanded using any suitable method known in the art. Exemplary methods of expanding the numbers of cells are described in patent publication No. WO 2003057171, U.S. Patent No. 8,034,334, and U.S. Patent Application Publication No. 2012 / 0244133, each of which is incorporated herein by reference.

[0166] In one embodiment, ex vivo T cell expansion can be performed by isolation of T cells and subsequent stimulation or activation followed by further expansion. In one embodiment of the invention, the T cells may be stimulated or activated by a single agent. In another embodiment, T cells are stimulated or activated with two agents, one that induces a primary signal and a second that is a co-stimulatory signal. Ligands useful for stimulating a single signal or stimulating a primary signal and an accessory molecule that stimulates a second signal may be used in soluble form. Ligands may be attached to the surface of a cell, to an Engineered Multivalent Signaling Platform (EMSP), or immobilized on a surface. In a preferred embodiment both primary and secondary agents are co-immobilized on a surface, for example a bead or a cell. In one embodiment, the molecule providing the primary activation signal may be a CD3 ligand, and the co-stimulatory molecule may be a CD28 ligand or 4-1BB ligand.

[0167] In certain embodiments, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in WO2015120096, by a method comprising: enriching a population of lymphocytes obtained from a donor subject; stimulating the population of lymphocytes with one or more T-cell stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using a single cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells for a predetermined time to produce a population of engineered T cells, wherein the expansionAttorney Docket 44010.185WO-PCT / / CU24092 is performed in a closed system using serum-free culture medium. In certain embodiments, T cells comprising a CAR or an exogenous TCR, may be manufactured as described in WO2015120096, by a method comprising: obtaining a population of lymphocytes; stimulating the population of lymphocytes with one or more stimulating agents to produce a population of activated T cells, wherein the stimulation is performed in a closed system using serum-free culture medium; transducing the population of activated T cells with a viral vector comprising a nucleic acid molecule which encodes the CAR or TCR, using at least one cycle transduction to produce a population of transduced T cells, wherein the transduction is performed in a closed system using serum-free culture medium; and expanding the population of transduced T cells to produce a population of engineered T cells, wherein the expansion is performed in a closed system using serum-free culture medium. The predetermined time for expanding the population of transduced T cells may be 3 days. The time from enriching the population of lymphocytes to producing the engineered T cells may be 6 days. The closed system may be a closed bag system. Further provided is population of T cells comprising a CAR or an exogenous TCR obtainable or obtained by said method, and a pharmaceutical composition comprising such cells.

[0168] In certain embodiments, T cell maturation or differentiation in vitro may be delayed or inhibited by the method as described in WO2017070395, comprising contacting one or more T cells from a subject in need of a T cell therapy with an AKT inhibitor (such as, e.g., one or a combination of two or more AKT inhibitors disclosed in claim 8 of WO2017070395) and at least one of exogenous Interleukin-7 (IL-7) and exogenous Interleukin-15 (IL-15), wherein the resulting T cells exhibit delayed maturation or differentiation, and / or wherein the resulting T cells exhibit improved T cell function (such as, e.g., increased T cell proliferation; increased cytokine production; and / or increased cytolytic activity) relative to a T cell function of a T cell cultured in the absence of an AKT inhibitor.

[0169] In certain embodiments, a patient in need of a T cell therapy may be conditioned by a method as described in WO2016191756 comprising administering to the patient a dose of cyclophosphamide between 200 mg / m2 / day and 2000 mg / m2 / day and a dose of fludarabine between 20 mg / m2 / day and 900 mg / m2 / day.

[0170] In certain embodiments, a patient in need of adoptive cell transfer may be administered a TLR agonist to enhance anti-tumor immunity (see, e.g., Urban-Wojciuk, et al., The Role of TLRs in Anti-cancer Immunity and Tumor Rejection, Front Immunol. 2019; 10:Attorney Docket 44010.185WO-PCT / / CU24092 2388; and Kaczanowska et al., TLR agonists: the best frenemy in cancer immunotherapy, J Leukoc Biol. 2013 Jun; 93(6): 847–863). In certain embodiments, TLR agonists are delivered in a nanoparticle system (see, e.g., Buss and Bhatia, Nanoparticle delivery of immunostimulatory oligonucleotides enhances response to checkpoint inhibitor therapeutics, Proc Natl Acad Sci USA. 2020 Jun 3;202001569). In certain embodiments, the agonist is a TLR9 agonist. Id. Editing T cells to include Gain-of-function and Loss-of-function mutations

[0171] In example embodiments, the mutations described herein can be introduced to T cells using any genome engineering method known in the art. In preferred embodiments, T cells are edited using a CRISPR system. As used herein generating a mutation in a cell can be referred to as a perturbation. As used herein a genetic modification agent can be used to generate the mutations as disclosed herein. In example embodiments, T cells for modification are obtained from a subject. In example, embodiments, modified cells are expanded and administered to the subject. Genetic Modification Systems

[0172] In one example embodiment, the genetic modifying agent may comprise a programmable nuclease, such as, a CRISPR system, a zinc finger nuclease system, a TALEN, or a meganuclease, or an OMEGA system. In addition, a number of alternate gene modification systems have been developed by modifying Cas nuclease so that they are catalytically inactive (“dead Cas” or “dCas”) or cut only a single strand of DNA (“nickase”) and then coupling these modified Cas nucleases with a further functional domain such as base editors, reverse transcriptases, recombinases, transposases and retrotransposases. For sake of convenience these alternative systems (e.g., Base Editors, Prime Editors, CAST, Non-LTR Retrotransposon Systems) are described further below in the context of use with a modified Cas. However, it is further contemplated that the modified Cas could be substituted with another similarly modified programmable nuclease like Zinc Finger nucleases, TALENs, Omega nucleases (e.g., Iscb, Isrb, TnpB, Fanzor), or a meganuclease. In example embodiments, the genetic modifying agent is administered using a vector, such as a viral vector or liposome. In example embodiments, the genetic modifying agent is administered as mRNA (e.g., by electroporation). Programmable nucleases may use two different cell repair pathways to effectuate edits to one or more target sequences, non-homologous end joining (NHEJ) or homology-directed repair (HDR).Attorney Docket 44010.185WO-PCT / / CU24092

[0173] In one example embodiment, a donor template is provided along with a programmable nuclease to facilitate homology direct repair (HDR) which results insertion of a donor sequence comprising one or more insertions, deletions, or substitutions relative to the target sequence it replaces. A donor template may comprise an insertion sequence flanked by two homology regions. The insertion sequence comprises an edited sequence to be inserted in place of the target sequence (e.g., a portion of genomic DNA to be edited). The homology regions comprise sequences that are homologous to the genomic DNA strands at the site of the CRISPR-Cas induced double-strand break. Cellular HDR mechanisms then facilitate insertion of the insertion sequence at the site of the DSB. The donor template comprises a sequence to be integrated (e.g., a mutated gene). Example Programmable Nucleases

[0174] The following provides further details and nuclease specific considerations for example programmable nucleases that may be used to make the NHEJ-mediated and HDR- mediated modifications described above. CRISPR-Cas

[0175] In one example embodiment, the genetic modifying agent is a CRISPR-Cas system. CRISPR-Cas systems comprise a Cas polypeptide and a guide sequence, wherein the guide sequence is capable of forming a CRISPR-Cas complex with the Cas polypeptide and directing site-specific binding of the CRISPR-Cas sequence to a target sequence in one or more of the target genes. The Cas polypeptide may induce a double- or single-stranded break at a designated site in the target sequence. The site of CRISPR-Cas cleavage, for most CRISPR- Cas systems, is dictated by distance from a protospacer-adjacent motif (PAM), discussed in further detail below. Accordingly, a guide sequence may be selected to direct the CRISPR-Cas system to a desired target site at or near the one or more target genes. Additionally, CRISPR systems can be used in vivo (see, e.g., Chen H, Shi M, Gilam A, et al. Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII. Sci Rep. 2019;9(1):16838; Hana S, Peterson M, McLaughlin H, et al. Highly efficient neuronal gene knockout in vivo by CRISPR-Cas9 via neonatal intracerebroventricular injection of AAV in mice. Gene Ther. 2021;28(10-11):646-658; and Rosenblum D, Gutkin A, Kedmi R, et al. CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy. Sci Adv. 2020;6(47):eabc9450).Attorney Docket 44010.185WO-PCT / / CU24092

[0176] In general, a CRISPR-Cas or CRISPR system as used in herein and in documents, such as International Patent Publication No. WO 2014 / 093622 (PCT / US2013 / 074667), refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr- mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or “RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas, such as Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). See, e.g., Shmakov et al. (2015) “Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems”, Molecular Cell, DOI: dx.doi.org / 10.1016 / j.molcel.2015.10.008.

[0177] In example embodiments, a gRNA is made up of two parts: crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA (also referred to as a “spacer” in the context of an endogenous CRISPR system), and a tracr RNA, which serves as a binding scaffold for the Cas nuclease. In example embodiments, a sgRNA is composed of four different components named as target sequence (crRNA sequence or spacer), crRNA repeat sequence, tetra loop sequence and tracrRNA sequence and has six secondary structural modules (spacer, lower stem, bulge, upper stem, nexus, and hairpins). The loop sequence joins the crRNA and tracRNA.

[0178] CRISPR-Cas systems can generally fall into two classes based on their architectures of their effector molecules, which are each further subdivided by type and subtype. The two class are Class 1 and Class 2. Class 1 CRISPR-Cas systems have effector modules composed of multiple Cas proteins, some of which form crRNA-binding complexes, while Class 2 CRISPR-Cas systems include a single, multi-domain crRNA-binding protein.

[0179] In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system. In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 2 CRISPR-Cas system.Attorney Docket 44010.185WO-PCT / / CU24092 Class 1 CRISPR-Cas Systems

[0180] In some embodiments, the CRISPR-Cas system that can be used to modify a polynucleotide of the present invention described herein can be a Class 1 CRISPR-Cas system. Class 1 CRISPR-Cas systems are divided into types I, II, and IV. Makarova et al. 2020. Nat. Rev.18: 67-83., particularly as described in Figure 1. Type I CRISPR-Cas systems are divided into 9 subtypes (I-A, I-B, I-C, I-D, I-E, I-F1, I-F2, I-F3, and IG). Makarova et al., 2020. Class 1, Type I CRISPR-Cas systems can contain a Cas3 protein that can have helicase activity. Type III CRISPR-Cas systems are divided into 6 subtypes (III-A, III-B, III-C, III-D, III-E, and III- F). Type III CRISPR-Cas systems can contain a Cas10 that can include an RNA recognition motif called Palm and a cyclase domain that can cleave polynucleotides. Makarova et al., 2020. Type IV CRISPR-Cas systems are divided into 3 subtypes. (IV-A, IV-B, and IV-C)..Makarova et al., 2020. Class 1 systems also include CRISPR-Cas variants, including Type I-A, I-B, I-E, I-F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I-B systems. Peters et al., PNAS 114 (35) (2017); DOI: 10.1073 / pnas.1709035114; see also, Makarova et al. 2018. The CRISPR Journal, v. 1 , n5, Figure 5.

[0181] The Class 1 systems typically comprise a multi-protein effector complex, which can, in some embodiments, include ancillary proteins, such as one or more proteins in a complex referred to as a CRISPR-associated complex for antiviral defense (Cascade), one or more adaptation proteins (e.g., Cas1, Cas2, RNA nuclease), and / or one or more accessory proteins (e.g., Cas 4, DNA nuclease), CRISPR associated Rossman fold (CARF) domain containing proteins, and / or RNA transcriptase.

[0182] The backbone of the Class 1 CRISPR-Cas system effector complexes can be formed by RNA recognition motif domain-containing protein(s) of the repeat-associated mysterious proteins (RAMPs) family subunits (e.g., Cas 5, Cas6, and / or Cas7). RAMP proteins are characterized by having one or more RNA recognition motif domains. In some embodiments, multiple copies of RAMPs can be present. In some embodiments, the Class I CRISPR-Cas system can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more Cas5, Cas6, and / or Cas 7 proteins. In some embodiments, the Cas6 protein is an RNAse, which can be responsible for pre-crRNA processing. When present in a Class 1 CRISPR-Cas system, Cas6 can be optionally physically associated with the effector complex.Attorney Docket 44010.185WO-PCT / / CU24092

[0183] Class 1 CRISPR-Cas system effector complexes can, in some embodiments, also include a large subunit. The large subunit can be composed of or include a Cas8 and / or Cas10 protein. See, e.g., Figures 1 and 2. Koonin EV, Makarova KS.2019. Phil. Trans. R. Soc. B 374: 20180087, DOI: 10.1098 / rstb.2018.0087 and Makarova et al. 2020.

[0184] Class 1 CRISPR-Cas system effector complexes can, in some embodiments, include a small subunit (for example, Cas11). See, e.g., Figures 1 and 2. Koonin EV, Makarova KS. 2019 Origins and Evolution of CRISPR-Cas systems. Phil. Trans. R. Soc. B 374: 20180087, DOI: 10.1098 / rstb.2018.0087.

[0185] In some embodiments, the Class 1 CRISPR-Cas system can be a Type I CRISPR- Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-A CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-B CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-C CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-D CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-E CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F1 CRISPR-Cas system. In some embodiments, the Type I CRISPR- Cas system can be a subtype I-F2 CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-F3 CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a subtype I-G CRISPR-Cas system. In some embodiments, the Type I CRISPR-Cas system can be a CRISPR Cas variant, such as a Type I-A, I-B, I-E, I- F and I-U variants, which can include variants carried by transposons and plasmids, including versions of subtype I-F encoded by a large family of Tn7-like transposon and smaller groups of Tn7-like transposons that encode similarly degraded subtype I-B systems as previously described.

[0186] In some embodiments, the Class 1 CRISPR-Cas system can be a Type III CRISPR- Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-A CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-B CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-C CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-D CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-E CRISPR-Cas system. In some embodiments, the Type III CRISPR-Cas system can be a subtype III-F CRISPR-Cas system.Attorney Docket 44010.185WO-PCT / / CU24092

[0187] In some embodiments, the Class 1 CRISPR-Cas system can be a Type IV CRISPR- Cas-system. In some embodiments, the Type IV CRISPR-Cas system can be a subtype IV-A CRISPR-Cas system. In some embodiments, the Type IV CRISPR-Cas system can be a subtype IV-B CRISPR-Cas system. In some embodiments, the Type IV CRISPR-Cas system can be a subtype IV-C CRISPR-Cas system.

[0188] The effector complex of a Class 1 CRISPR-Cas system can, in some embodiments, include a Cas3 protein that is optionally fused to a Cas2 protein, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas10, a Cas11, or a combination thereof. In some embodiments, the effector complex of a Class 1 CRISPR-Cas system can have multiple copies, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14, of any one or more Cas proteins. Class 2 CRISPR-Cas Systems

[0189] The compositions, systems, and methods described in greater detail elsewhere herein can be designed and adapted for use with Class 2 CRISPR-Cas systems. Thus, in some embodiments, the CRISPR-Cas system is a Class 2 CRISPR-Cas system. Class 2 systems are distinguished from Class 1 systems in that they have a single, large, multi-domain effector protein. In certain example embodiments, the Class 2 system can be a Type II, Type V, or Type VI system, which are described in Makarova et al. “Evolutionary classification of CRISPR- Cas systems: a burst of class 2 and derived variants” Nature Reviews Microbiology, 18:67-81 (Feb 2020), incorporated herein by reference. Each type of Class 2 system is further divided into subtypes. See Markova et al. 2020, particularly at Figure. 2. Class 2, Type II systems can be divided into 4 subtypes: II-A, II-B, II-C1, and II-C2. Class 2, Type V systems can be divided into 17 subtypes: V-A, V-B1, V-B2, V-C, V-D, V-E, V-F1, V-F1(V-U3), V-F2, V-F3, V-G, V-H, V-I, V-K (V-U5), V-U1, V-U2, and V-U4. Class 2, Type IV systems can be divided into 5 subtypes: VI-A, VI-B1, VI-B2, VI-C, and VI-D.

[0190] The distinguishing feature of these types is that their effector complexes consist of a single, large, multi-domain protein. Type V systems differ from Type II effectors (e.g., Cas9), which contain two nuclear domains that are each responsible for the cleavage of one strand of the target DNA, with the HNH nuclease inserted inside the Ruv-C like nuclease domain sequence. The Type V systems (e.g., Cas12) only contain a RuvC-like nuclease domain that cleaves both strands. Type VI (Cas13) are unrelated to the effectors of Type II and V systems and contain two HEPN domains and target RNA. Cas13 proteins also display collateral activityAttorney Docket 44010.185WO-PCT / / CU24092 that is triggered by target recognition. Some Type V systems have also been found to possess this collateral activity with two single-stranded DNA in in vitro contexts.

[0191] In some embodiments, the Class 2 system is a Type II system. In some embodiments, the Type II CRISPR-Cas system is a II-A CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-B CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-C1 CRISPR-Cas system. In some embodiments, the Type II CRISPR-Cas system is a II-C2 CRISPR-Cas system. In some embodiments, the Type II system is a Cas9 system. In some embodiments, the Type II system includes a Cas9.

[0192] In some embodiments, the Class 2 system is a Type V system. In some embodiments, the Type V CRISPR-Cas system is a V-A CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-B1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-B2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-C CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-D CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-E CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F1 (V-U3) CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-F3 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-G CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-H CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-I CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-K (V-U5) CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-U1 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-U2 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system is a V-U4 CRISPR-Cas system. In some embodiments, the Type V CRISPR-Cas system includes a Cas12a (Cpf1), Cas12b (C2c1), Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas14, and / or CasΦ.

[0193] In some embodiments the Class 2 system is a Type VI system. In some embodiments, the Type VI CRISPR-Cas system is a VI-A CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-B1 CRISPR-Cas system. In someAttorney Docket 44010.185WO-PCT / / CU24092 embodiments, the Type VI CRISPR-Cas system is a VI-B2 CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-C CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system is a VI-D CRISPR-Cas system. In some embodiments, the Type VI CRISPR-Cas system includes a Cas13a (C2c2), Cas13b (Group 29 / 30), Cas13c, and / or Cas13d. Guide Molecules

[0194] The following include general design principles that may be applied to the guide molecule. The terms guide molecule, guide sequence and guide polynucleotide refer to polynucleotides capable of guiding Cas to a target genomic locus and are used interchangeably as in foregoing cited documents such as International Patent Publication No. WO 2014 / 093622 (PCT / US2013 / 074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. The guide molecule can be a polynucleotide.

[0195] The ability of a guide sequence (within a nucleic acid-targeting guide RNA) to direct sequence-specific binding of a nucleic acid-targeting complex to a target nucleic acid sequence may be assessed by any suitable assay. For example, the components of a nucleic acid-targeting CRISPR system sufficient to form a nucleic acid-targeting complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target nucleic acid sequence, such as by transfection with vectors encoding the components of the nucleic acid-targeting complex, followed by an assessment of preferential targeting (e.g., cleavage) within the target nucleic acid sequence, such as by Surveyor assay (Qui et al. 2004. BioTechniques. 36(4)702-707). Similarly, cleavage of a target nucleic acid sequence may be evaluated in a test tube by providing the target nucleic acid sequence, components of a nucleic acid-targeting complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible and will occur to those skilled in the art.

[0196] In some embodiments, the guide molecule is an RNA. The guide molecule(s) (also referred to interchangeably herein as guide polynucleotide and guide sequence) that are included in the CRISPR-Cas or Cas based system can be any polynucleotide sequence having sufficient complementarity with a target nucleic acid sequence to hybridize with the targetAttorney Docket 44010.185WO-PCT / / CU24092 nucleic acid sequence and direct sequence-specific binding of a nucleic acid-targeting complex to the target nucleic acid sequence. In some embodiments, the degree of complementarity, when optimally aligned using a suitable alignment algorithm, can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0197] A guide sequence, and hence a nucleic acid-targeting guide, may be selected to target any target nucleic acid sequence. The target sequence may be DNA. The target sequence may be any RNA sequence. In some embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of messenger RNA (mRNA), pre- mRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), micro-RNA (miRNA), small interfering RNA (siRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), double stranded RNA (dsRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), and small cytoplasmatic RNA (scRNA). In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of mRNA, pre- mRNA, and rRNA. In some preferred embodiments, the target sequence may be a sequence within an RNA molecule selected from the group consisting of ncRNA, and lncRNA. In some more preferred embodiments, the target sequence may be a sequence within an mRNA molecule or a pre-mRNA molecule.

[0198] In some embodiments, a nucleic acid-targeting guide is selected to reduce the degree secondary structure within the nucleic acid-targeting guide. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the nucleic acid-targeting guide participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res.9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroidAttorney Docket 44010.185WO-PCT / / CU24092 structure prediction algorithm (see e.g., A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).

[0199] In one example embodiment, a guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat (DR) sequence and a guide sequence or spacer sequence. In another example embodiment, the guide RNA or crRNA may comprise, consist essentially of, or consist of a direct repeat sequence fused or linked to a guide sequence or spacer sequence. In another example embodiment, the direct repeat sequence may be located upstream (i.e., 5’) from the guide sequence or spacer sequence. In other embodiments, the direct repeat sequence may be located downstream (i.e., 3’) from the guide sequence or spacer sequence.

[0200] In one example embodiment, the crRNA comprises a stem loop, preferably a single stem loop. In one example embodiment, the direct repeat sequence forms a stem loop, preferably a single stem loop.

[0201] In one example embodiment, the spacer length of the guide RNA is from 15 to 35 nt. In another example embodiment, the spacer length of the guide RNA is at least 15 nucleotides. In another example embodiment, the spacer length is from 15 to 17 nt, e.g., 15, 16, or 17 nt, from 17 to 20 nt, e.g., 17, 18, 19, or 20 nt, from 20 to 24 nt, e.g., 20, 21, 22, 23, or 24 nt, from 23 to 25 nt, e.g., 23, 24, or 25 nt, from 24 to 27 nt, e.g., 24, 25, 26, or 27 nt, from 27 to 30 nt, e.g., 27, 28, 29, or 30 nt, from 30 to 35 nt, e.g., 30, 31, 32, 33, 34, or 35 nt, or 35 nt or longer.

[0202] The “tracrRNA” sequence or analogous terms includes any polynucleotide sequence that has sufficient complementarity with a crRNA sequence to hybridize. In some embodiments, the degree of complementarity between the tracrRNA sequence and crRNA sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and crRNA sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.

[0203] In general, degree of complementarity is with reference to the optimal alignment of the spacer sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm and may furtherAttorney Docket 44010.185WO-PCT / / CU24092 account for secondary structures, such as self-complementarity within either the spacer sequence or tracr sequence. In some embodiments, the degree of complementarity between the tracr sequence and spacer sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.

[0204] In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence can be about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%; a guide or RNA or sgRNA can be about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length; or guide or RNA or sgRNA can be less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length; and tracr RNA can be 30 or 50 nucleotides in length. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5% or 95% or 95.5% or 96% or 96.5% or 97% or 97.5% or 98% or 98.5% or 99% or 99.5% or 99.9%, or 100%. Off target is less than 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% or 94% or 93% or 92% or 91% or 90% or 89% or 88% or 87% or 86% or 85% or 84% or 83% or 82% or 81% or 80% complementarity between the sequence and the guide, with it being advantageous that off target is 100% or 99.9% or 99.5% or 99% or 99% or 98.5% or 98% or 97.5% or 97% or 96.5% or 96% or 95.5% or 95% or 94.5% complementarity between the sequence and the guide.

[0205] In some embodiments according to the invention, the guide RNA (capable of guiding Cas to a target locus) may comprise (1) a guide sequence capable of hybridizing to a genomic target locus in the eukaryotic cell; (2) a tracr sequence; and (3) a tracr mate sequence. All of (1) to (3) may reside in a single RNA, i.e., an sgRNA (arranged in a 5’ to 3’ orientation), or the tracr RNA may be a different RNA than the RNA containing the guide and tracr sequence. The tracr hybridizes to the tracr mate sequence and directs the CRISPR / Cas complex to the target sequence. Where the tracr RNA is on a different RNA than the RNA containing the guide and tracr sequence, the length of each RNA may be optimized to be shortened from their respective native lengths, and each may be independently chemically modified to protect from degradation by cellular RNase or otherwise increase stability.

[0206] Many modifications to guide sequences are known in the art and are further contemplated within the context of this invention. Various modifications may be used toAttorney Docket 44010.185WO-PCT / / CU24092 increase the specificity of binding to the target sequence and / or increase the activity of the Cas protein and / or reduce off-target effects. Example guide sequence modifications are described in International Patent Application No. PCT US2019 / 045582, specifically paragraphs

[0178] -

[0333] . which is incorporated herein by reference. Target Sequences, PAMs, and PFSs

[0207] In the context of formation of a CRISPR complex, “target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. In other words, the target polynucleotide can be a polynucleotide or a part of a polynucleotide to which a part of the guide sequence is designed to have complementarity with and to which the effector function mediated by the complex comprising the CRISPR effector protein and a guide molecule is to be directed. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0208] PAM elements are sequences that can be recognized and bound by Cas proteins. Cas proteins / effector complexes can then unwind the dsDNA at a position adjacent to the PAM element. It will be appreciated that Cas proteins and systems target RNA do not require PAM sequences (Marraffini et al. 2010. Nature. 463:568-571). Instead, many rely on PFSs, which are discussed elsewhere herein. In one example embodiment, the target sequence should be associated with a PAM (protospacer adjacent motif) or PFS (protospacer flanking sequence or site), that is, a short sequence recognized by the CRISPR complex. Depending on the nature of the CRISPR-Cas protein, the target sequence should be selected, such that its complementary sequence in the DNA duplex (also referred to herein as the non-target sequence) is upstream or downstream of the PAM. In the embodiments, the complementary sequence of the target sequence is downstream or 3’ of the PAM or upstream or 5’ of the PAM. The precise sequence and length requirements for the PAM differ depending on the Cas protein used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Examples of the natural PAM sequences for different Cas proteins are provided herein below and the skilled person will be able to identify further PAM sequences for use with a given Cas protein.

[0209] The ability to recognize different PAM sequences depends on the Cas polypeptide(s) included in the system. See e.g., Gleditzsch et al. 2019. RNA Biology. 16(4):504-517. In a preferred embodiment, the CRISPR effector protein may recognize a 3’Attorney Docket 44010.185WO-PCT / / CU24092 PAM. In one example embodiment, the CRISPR effector protein may recognize a 3’ PAM which is 5’H, wherein H is A, C or U.

[0210] Further, engineering of the PAM Interacting (PI) domain on the Cas protein may allow programing of PAM specificity, improve target site recognition fidelity, and increase the versatility of the CRISPR-Cas protein, for example as described for Cas9 in Kleinstiver BP et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature. 2015 Jul 23;523(7561):481-5. doi: 10.1038 / nature14592. As further detailed herein, the skilled person will understand that Cas13 proteins may be modified analogously. Gao et al, “Engineered Cpf1 Enzymes with Altered PAM Specificities,” bioRxiv 091611; doi: dx.doi.org / 10.1101 / 091611 (Dec.4, 2016). Doench et al. created a pool of sgRNAs, tiling across all possible target sites of a panel of six endogenous mouse and three endogenous human genes and quantitatively assessed their ability to produce null alleles of their target gene by antibody staining and flow cytometry. The authors showed that optimization of the PAM improved activity and also provided an on-line tool for designing sgRNAs.

[0211] PAM sequences can be identified in a polynucleotide using an appropriate design tool, which are commercially available as well as online. Such freely available tools include, but are not limited to, CRISPRFinder and CRISPRTarget. Mojica et al. 2009. Microbiol. 155(Pt. 3):733-740; Atschul et al. 1990. J. Mol. Biol. 215:403-410; Biswass et al. 2013 RNA Biol. 10:817-827; and Grissa et al. 2007. Nucleic Acid Res. 35:W52-57. Experimental approaches to PAM identification can include, but are not limited to, plasmid depletion assays (Jiang et al. 2013. Nat. Biotechnol. 31:233-239; Esvelt et al. 2013. Nat. Methods. 10:1116- 1121; Kleinstiver et al. 2015. Nature. 523:481-485), screened by a high-throughput in vivo model called PAM-SCNAR (Pattanayak et al. 2013. Nat. Biotechnol. 31:839-843 and Leenay et al.2016.Mol. Cell.16:253), and negative screening (Zetsche et al.2015. Cell.163:759-771). OMEGA systems

[0212] In one example embodiment, the programmable nuclease to modify the one or more target genes is a transposon-encoded RNA-guided nuclease system, referred to herein as OMEGA (obligate mobile element–guided activity). See, e.g., Altae-Tran H, Kannan S, Demircioglu FE, et al. The widespread IS200 / IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science. 2021;374(6563):57-65. OMEGA systems include, but are not limited to IscB, IsrB, TnpB systems.Attorney Docket 44010.185WO-PCT / / CU24092

[0213] In some embodiments, the nucleic acid-guided nucleases herein may be an IscB protein (see, e.g., International patent application publication No. WO2022087494A1; and Altae-Tran H, et al. 2021). An IscB protein may comprise an X domain and a Y domain as described herein. In some examples, the IscB proteins may form a complex with one or more guide molecules. In some cases, the IscB proteins may form a complex with one or more hRNA molecules which serve as a scaffold molecule and comprise guide sequences. In some examples, the IscB proteins are CRISPR-associated proteins, e.g., the loci of the nucleases are associated with an CRISPR array. In some examples, the IscB proteins are not CRISPR- associated. In some examples, the IscB protein may be homolog or ortholog of IscB proteins described in Kapitonov VV et al., ISC, a Novel Group of Bacterial and Archaeal DNA Transposons That Encode Cas9 Homologs, J Bacteriol. 2015 Dec 28;198(5):797-807. doi: 10.1128 / JB.00783-15, which is incorporated by reference herein in its entirety.

[0214] In some embodiments, the nucleic acid-guided nucleases herein may be an IsrB (Insertion sequence RuvC-like OrfB) protein (see, e.g., International patent application publication No. WO2022087494A1; and Altae-Tran H, et al. 2021). IsrB refers to a group of shorter, ~350 aa IscB homologs that are also encoded in IS200 / 605 superfamily transposons. These proteins contain a PLMP domain and split RuvC but lack the HNH domain.

[0215] In some embodiments, the nucleic acid-guided nucleases herein may be a TnpB protein (see, e.g., International patent application publication No. WO2022159892A1; and Altae-Tran H, et al. 2021). TnpB is a putative endonuclease distantly related to IscB and thought to be the ancestor of Cas12, the type V CRISPR effector. The TnpB system comprises a TnpB polypeptide and a nucleic acid component capable of forming a complex with the TnpB polypeptide and directing the complex to a target polynucleotide. The TnpB systems and TnpB / nucleic acid component complexes may also be referred to herein as OMEGA (Obligate Mobile Element Guided Activity) systems or complexes, or Ω systems or complexes for short. TnpB systems are a distinct type of Ω system, which further include IscB, IsrB, and IshB systems. The nucleic acid component of Ω systems is structurally distinct from other RNA- guided nucleases, such as CRISPR-Cas systems, and may also be referred to as a ωRNA. In certain example embodiments, the TnpB systems are RNA-predominate, that is the nucleic acid component makes a larger contribution to the overall size of the TnpB complex relative to other RNA-guided nuclease systems such as CRISPR-Cas. Also, given the more minimal structural features of TnpB relative other known programmable nucleases such as CRISPR-Cas, theAttorney Docket 44010.185WO-PCT / / CU24092 polynucleotide binding pocket is open and more accessible, which can facilitate greater access to and ability to manipulate, modify, edit, remove, or delete nucleotides at a target region on the bound polynucleotide.

[0216] Accordingly, it is contemplated within the scope of the present invention that OMEGA systems may be used in place of CRISPR-Cas systems due to their reprogrammable nature. These embodiments include further modified versions of CRISPR-Cas systems such as base editing systems, prime editing systems, CAST systems, and non-LTR retrotransposons, as discussed below. Other Genetic Modification Systems

[0217] A number of alternative gene modification systems have been developed that utilize the target specificity of a programmable nuclease but that modify or replace that nuclease activity with another functional activity. For example, programmable nucleases may be modified such that they cleave only a single-strand as opposed to both strands of a target polynucleotide. Such “nickases” may then be paired with other functional domains such as reverse transcriptases, recombinases and non-LTR retrotransposon polypeptides to make genetic modifications that do not rely on creating double strand breaks. Similarly, programmable nucleases may also be modified to eliminate the nuclease activity altogether. These catalytically inactive or “dead” nucleases may then be combined with other functional domains like nucleotide deaminases, transposases, non-LTR retrotransposon polypeptides, methylases, deactylases, and acetylases, among other domains. The following provides further examples of gene modification systems that may be used in the context of the present invention. For ease of reference the gene modifications systems that follow will be discussed in the context of using CRISPR-Cas as the programmable nuclease system, but it is contemplated within the scope of this invention that the nickase or dead Cas versions described below could be replaced by a comparable nickase or dead nuclease variant of other programmable nucleases / systems such as OMEGA systems, Zn finger nucleases, TALE nucleases, and meganucleases. DNA and RNA Base Editing

[0218] In one example embodiment, the genetic modifying agent comprises a DNA base editing system to introduce gain-of-function or loss-of-function mutations to one or more genes. In one example embodiment, a catalytically inactive Cas protein is connected or fused to a nucleotide deaminase. As used herein, “base editing” refers generally to the process ofAttorney Docket 44010.185WO-PCT / / CU24092 polynucleotide modification via a CRISPR-Cas-based or Cas-based system that does not include excising nucleotides to make the modification. Base editing can convert base pairs at precise locations without generating excess undesired editing byproducts that can be made using traditional CRISPR-Cas systems. Accordingly, in one example embodiment, the base editing system edits the target gene to reduce or eliminate its expression or to increase its expression.

[0219] In one example embodiment, the nucleotide deaminase may be a DNA base editor used in combination with a DNA binding Cas protein such as, but not limited to, Class 2 Type II and Type V systems. Two classes of DNA base editors are generally known: cytosine base editors (CBEs) and adenine base editors (ABEs). CBEs convert a C•G base pair into a T•A base pair (Komor et al. 2016. Nature. 533:420-424; Nishida et al. 2016. Science. 353; and Li et al. Nat. Biotech. 36:324-327) and ABEs convert an A•T base pair to a G•C base pair. Collectively, CBEs and ABEs can mediate all four possible transition mutations (C to T, A to G, T to C, and G to A). Rees and Liu. 2018.Nat. Rev. Genet. 19(12): 770-788, particularly at Figures 1b, 2a-2c, 3a-3f, and Table 1. In some embodiments, the base editing system includes a CBE and / or an ABE. In some embodiments, a polynucleotide of the present invention described elsewhere herein can be modified using a base editing system. Rees and Liu. 2018. Nat. Rev. Gent.19(12):770-788. Base editors also generally do not need a DNA donor template and / or rely on homology-directed repair. Komor et al. 2016. Nature. 533:420-424; Nishida et al.2016. Science.353; and Gaudeli et al.2017. Nature.551:464-471. Upon binding to a target locus in the DNA, base pairing between the guide RNA of the system and the target DNA strand leads to displacement of a small segment of ssDNA in an “R-loop”. Nishimasu et al. Cell. 156:935-949. DNA bases within the ssDNA bubble are modified by the enzyme component, such as a deaminase. In some systems, the catalytically disabled Cas protein can be a variant or modified Cas can have nickase functionality and can generate a nick in the non- edited DNA strand to induce cells to repair the non-edited strand using the edited strand as a template. Komor et al. 2016. Nature. 533:420-424; Nishida et al. 2016. Science. 353; and Gaudeli et al. 2017. Nature. 551:464-471.

[0220] Other Example Type V base editing systems are described in International Patent Publication Nos. WO 2018 / 213708, WO 2018 / 213726, and International Patent Applications No. PCT / US2018 / 067207, PCT / US2018 / 067225, and PCT / US2018 / 067307, each of which is incorporated herein by reference.Attorney Docket 44010.185WO-PCT / / CU24092

[0221] An example method for delivery of base-editing systems, including use of a split- intein approach to divide CBE and ABE into reconstitutable halves, is described in Levy et al. Nature Biomedical Engineering doi.org / 10.1038 / s41441-019-0505-5 (2019), which is incorporated herein by reference.

[0222] Other example base editing systems have been described (see, e.g., WO / 2021 / 025750; WO / 2023 / 024089; US20220177877; US20220127622; Davis JR, Wang X, Witte IP, et al. Efficient in vivo base editing via single adeno-associated viruses with size- optimized genomes encoding compact adenine base editors [published correction appears in Nat Biomed Eng. 2022 Nov;6(11):1317; Gaudelli NM, Lam DK, Rees HA, et al. Directed evolution of adenine base editors with increased activity and therapeutic application. Nat Biotechnol. 2020;38(7):892-900; Lee RG, Mazzola AM, Braun MC, et al. Efficacy and Safety of an Investigational Single-Course CRISPR Base-Editing Therapy Targeting PCSK9 in Nonhuman Primate and Mouse Models. Circulation. 2023;147(3):242-253; and Neugebauer ME, Hsu A, Arbab M, et al. Evolution of an adenine base editor into a small, efficient cytosine base editor with low off-target activity. Nat Biotechnol. 2023;41(5):673-685). Prime Editors

[0223] In one example embodiment, the perturbation comprises administering a prime editing system to either decrease expression of one or more genes or increase the expression of one or more genes. Prime editing systems comprise a programable nuclease (e.g. Cas), most often a nickase, linked to a reverse transcriptase domain and a guide molecule (prime editing guide pegRNA), which comprises a target-specific spacer, a primer binding site, and RT template. See e.g., Anzalone et al. 2019. Nature. 576: 149-157; and International Patent Application Publication No. WO2022150790A2. In some embodiments, the prime editing guide molecule can specify both the target polynucleotide information (e.g., sequence) and contain a new polynucleotide cargo that replaces target polynucleotides. To initiate transfer from the guide molecule to the target polynucleotide, the PE system can nick the target polynucleotide at a target side to expose a 3’hydroxyl group, which can prime reverse transcription of an edit-encoding extension region of the guide molecule (e.g., a prime editing guide molecule or peg guide molecule) directly into the target site in the target polynucleotide. See e.g., Anzalone et al. 2019. Nature. 576: 149-157, particularly at Figures 1b, 1c, related discussion, and Supplementary discussion.Attorney Docket 44010.185WO-PCT / / CU24092

[0224] Prime editing systems can also be used in tandem such that, the two pegRNAs template the synthesis of complementary DNA flaps on opposing strands of genomic DNA, which replace the endogenous DNA sequence between the PE-induced nick sites. See, e.g., Anzalone AV, Gao XD, Podracky CJ, et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat Biotechnol. 2022;40(5):731-740. Thus, use of two pegRNAs allows for larger insertions or deletions because of the two overlapping 3’ flaps created by the two nicked sites. In one example embodiment, the system can be used to insert or replace a sequence into one or more target genes. In example embodiments, the insertion or replacement results in an inactive target gene or less active form of the target gene. In one example embodiment, the system is used to replace all or a portion of the entire target gene. In one example embodiment, the system is used to replace all or a portion of an enhancer controlling the target gene expression.

[0225] The peg guide molecule can be about 10 to about 200 or more nucleotides in length, such as 10 to / or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or 200 or more nucleotides in length. Optimization of the peg guide molecule can be accomplished as described in Anzalone et al.2019. Nature.576: 149-157, particularly at pg.3, Fig. 2a-2b, and Extended Data Figs. 5a-c.

[0226] Prime Editing systems may be used to introduce insertions, deletions, or substitutions (modifications) in a gene. As a threshold matter, Primer Editing systems are capable of making all 4 base edits (A, T, C, G) and thus can be used to make all of the same DNA base edits described above in the Base Editor section. Cancer

[0227] In example embodiments, cancer can be treated using the CD8+ T cells modified to include mutations that enhance anti-tumor immunity as described herein. CD8+ T cells specificAttorney Docket 44010.185WO-PCT / / CU24092 to any cancer can be obtained. For example, CD8+ T cells can be engineered to express a CAR or TCR specific for a cancer or T cells specific for a cancer can be obtained from a subject and modified to include a mutation described herein. Example cancers are provided below.

[0228] The cancer may include, without limitation, liquid tumors such as leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin’s disease, non- Hodgkin’s disease), Waldenstrom’s macroglobulinemia, heavy chain disease, or multiple myeloma.

[0229] The cancer may include, without limitation, solid tumors such as sarcomas and carcinomas. Examples of solid tumors include, but are not limited to fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, epithelial carcinoma, bronchogenic carcinoma, hepatoma, colorectal cancer (e.g., colon cancer, rectal cancer), anal cancer, pancreatic cancer (e.g., pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors), breast cancer (e.g., ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma), ovarian carcinoma (e.g., ovarian epithelial carcinoma or surface epithelial-stromal tumour including serous tumour, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor), prostate cancer, liver and bile duct carcinoma (e.g., hepatocelluar carcinoma, cholangiocarcinoma, hemangioma), choriocarcinoma, seminoma, embryonal carcinoma, kidney cancer (e.g., renal cell carcinoma, clear cell carcinoma, Wilm's tumor, nephroblastoma), cervical cancer, uterine cancer (e.g., endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas and leiomyosarcomas, mixed mullerian tumors), testicular cancer, germ cell tumor, lung cancer (e.g., lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma), bladder carcinoma, signet ring cell carcinoma, cancer of the head and neck (e.g., squamous cell carcinomas), esophagealAttorney Docket 44010.185WO-PCT / / CU24092 carcinoma (e.g., esophageal adenocarcinoma), tumors of the brain (e.g., glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma), neuroblastoma, retinoblastoma, neuroendocrine tumor, melanoma, cancer of the stomach (e.g., stomach adenocarcinoma, gastrointestinal stromal tumor), or carcinoids. Lymphoproliferative disorders are also considered to be proliferative diseases. Autoimmune diseases

[0230] In example embodiments, autoimmune diseases are treated with CD8+ T cells that have decreased immune activity. In example embodiments, the modified CD8+ T cells suppress autoimmunity of other endogenous T cells not modified. Autoimmune conditions that may benefit from treatment using the compositions and methods described herein include, but are not limited to, for example, multiple sclerosis (MS), Addison's Disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune oophoritis, Bechet's disease, bullous pemphigoid, celiac disease, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, cold agglutinin disease, CREST Syndrome, Crohn’s disease, diabetes (e.g., type I), dysautonomia, endometriosis, eosinophilia-myalgia syndrome, essential mixed cryoglobulinemia, fibromyalgia, syndrome / fibromyositis, Graves’ disease, Guillain Barré syndrome, Hashimoto’s thyroiditis, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), inflammatory bowel disease (IBD), lichen planus, lupus, Ménière's disease, mixed connective tissue disease (MCTD), multiple sclerosis, myasthenia gravis, pemphigus, pernicious anemia, polyarteritis nodosa, polychondritis, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, Raynaud's phenomenon, Reiter’s syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy (spondyloarthritides), stiff-man syndrome, Takayasu arteritis, temporal arteritis / giant cell arteritis, autoimmune thyroid disease, ulcerative colitis, autoimmune uveitis, autoimmune vasculitis, vitiligo, and Wegener's granulomatosis. In some embodiments, the autoimmune disease is MS, IBD, Crohn’s disease, spondyloarthritides, Systemic Lupus Erythematosus, Vitiligo, rheumatoid arthritis, psoriasis, Sjögren’s syndrome, or diabetes, e.g., Type I diabetes.Attorney Docket 44010.185WO-PCT / / CU24092 SCREENING METHODS

[0231] In example embodiments, pooled screening is used to identify mutations that modulate T cell Function. In example embodiments, primary cells are screened. In example embodiments, primary cells are human primary cells. In example embodiments, the primary cells are human cells. In example embodiments, the primary cells screened are obtained from an animal model. In example embodiments, the animal model expresses a CRISPR system, such as a base editing system described herein. Such CRISPR mouse models and genome editing of primary cells obtained from the mouse is known in the art (see, e.g., US Patent US11197467B2). In example embodiments, CD8+ T cell function is screened. In example embodiments, the methods described herein can be applied to any immune cells and immune cell function described herein. In example embodiments, the T cell function screened for includes changes in activation, short-term and long-term proliferation, and cytokine production.

[0232] In example embodiments, a library of guide RNAs is introduced to a population of primary cells. In example embodiments, a guide RNA directed base editor as described herein is introduced to the primary cells. In example embodiments, the base editor is produced entirely using in vitro transcription, resulting in a single encoding RNA transcript, delivered as a single RNA molecule translated by the host cell (rather than transduction with a virus), and enables timed delivery of single-guide RNAs for individual gene edits. In example embodiments, the edited cells are screened for an altered function (e.g., altered immune cell function) and guide RNAs linked to the altered function are identified by sequencing. Immune Cell Functions

[0233] In example embodiments, any immune cell function can be screened with a library of guide RNAs and base editors, such as described in the present invention. In example embodiments, functions specific to the immune cell type being screened are used. In example embodiments, the present invention provides for screening modifications that alter immune function (e.g., immune response).

[0234] The term “immune cell” as used throughout this specification generally encompasses any cell derived from a hematopoietic stem cell that plays a role in the immune response. The term is intended to encompass immune cells both of the innate or adaptive immune system. The immune cell as referred to herein may be a leukocyte, at any stage of differentiation (e.g., a stem cell, a progenitor cell, a mature cell) or any activation stage.Attorney Docket 44010.185WO-PCT / / CU24092 Immune cells include lymphocytes (such as natural killer cells, T-cells (including, e.g., thymocytes, Th or Tc; Th1, Th2, Th17, Thαβ, CD4+, CD8+, effector Th, memory Th, regulatory Th, CD4+ / CD8+thymocytes, CD4– / CD8– thymocytes, γδ T cells, etc.) or B-cells (including, e.g., pro-B cells, early pro-B cells, late pro-B cells, pre-B cells, large pre-B cells, small pre-B cells, immature or mature B-cells, producing antibodies of any isotype, T1 B-cells, T2, B-cells, naïve B-cells, GC B-cells, plasmablasts, memory B-cells, plasma cells, follicular B-cells, marginal zone B-cells, B-1 cells, B-2 cells, regulatory B cells, etc.), such as for instance, monocytes (including, e.g., classical, non-classical, or intermediate monocytes), (segmented or banded) neutrophils, eosinophils, basophils, mast cells, histiocytes, microglia, including various subtypes, maturation, differentiation, or activation stages, such as for instance hematopoietic stem cells, myeloid progenitors, lymphoid progenitors, myeloblasts, promyelocytes, myelocytes, metamyelocytes, monoblasts, promonocytes, lymphoblasts, prolymphocytes, small lymphocytes, macrophages (including, e.g., Kupffer cells, stellate macrophages, M1 or M2 macrophages), (myeloid or lymphoid) dendritic cells (including, e.g., Langerhans cells, conventional or myeloid dendritic cells, plasmacytoid dendritic cells, mDC- 1, mDC-2, Mo-DC, HP-DC, veiled cells), granulocytes, polymorphonuclear cells, antigen- presenting cells (APC), etc.

[0235] As used throughout this specification, “immune response” or “immune function” refers to a response by a cell of the immune system, such as a B cell, T cell (CD4+or CD8+), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil, to a stimulus. In some embodiments, the response is specific for a particular antigen (an “antigen-specific response”) and refers to a response by a CD4 T cell, CD8 T cell, or B cell via their antigen-specific receptor. In some embodiments, an immune response is a T cell response, such as a CD4+response or a CD8+response. Such responses by these cells can include, for example, cytotoxicity, proliferation, cytokine or chemokine production, trafficking, or phagocytosis, and can be dependent on the nature of the immune cell undergoing the response.

[0236] T cell response refers more specifically to an immune response in which T cells directly or indirectly mediate or otherwise contribute to an immune response in a subject. T cell-mediated response may be associated with cell mediated effects, cytokine mediated effects, and even effects associated with B cells if the B cells are stimulated, for example, by cytokines secreted by T cells. By means of an example but without limitation, effector functions of MHCAttorney Docket 44010.185WO-PCT / / CU24092 class I restricted Cytotoxic T lymphocytes (CTLs), may include cytokine and / or cytolytic capabilities, such as lysis of target cells presenting an antigen peptide recognized by the T cell receptor (naturally-occurring TCR or genetically engineered TCR, e.g., chimeric antigen receptor, CAR), secretion of cytokines, preferably IFN gamma, TNF alpha and / or or more immunostimulatory cytokines, such as IL-2, and / or antigen peptide-induced secretion of cytotoxic effector molecules, such as granzymes, perforins or granulysin. By means of example but without limitation, for MHC class II restricted T helper (Th) cells, effector functions may be antigen peptide-induced secretion of cytokines, preferably, IFN gamma, TNF alpha, IL-4, IL5, IL-10, and / or IL-2. By means of example but without limitation, for T regulatory (Treg) cells, effector functions may be antigen peptide-induced secretion of cytokines, preferably, IL- 10, IL-35, and / or TGF-beta. B cell response refers more specifically to an immune response in which B cells directly or indirectly mediate or otherwise contribute to an immune response in a subject. Effector functions of B cells may include in particular production and secretion of antigen-specific antibodies by B cells (e.g., polyclonal B cell response to a plurality of the epitopes of an antigen (antigen-specific antibody response)), antigen presentation, and / or cytokine secretion.

[0237] During persistent immune activation, such as during uncontrolled tumor growth or chronic infections, subpopulations of immune cells, particularly of CD8+ or CD4+ T cells, become compromised to different extents with respect to their cytokine and / or cytolytic capabilities. Such immune cells, particularly CD8+ or CD4+ T cells, are commonly referred to as “dysfunctional” or as “functionally exhausted” or “exhausted”. As used herein, the term “dysfunctional” or “functional exhaustion” refer to a state of a cell where the cell does not perform its usual function or activity in response to normal input signals, and includes refractivity of immune cells to stimulation, such as stimulation via an activating receptor or a cytokine. Such a function or activity includes, but is not limited to, proliferation (e.g., in response to a cytokine, such as IFN-gamma) or cell division, entrance into the cell cycle, cytokine production, cytotoxicity, migration and trafficking, phagocytotic activity, or any combination thereof. Normal input signals can include, but are not limited to, stimulation via a receptor (e.g., T cell receptor, B cell receptor, co-stimulatory receptor). Unresponsive immune cells can have a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or even 100% in cytotoxic activity, cytokine production, proliferation, trafficking, phagocytotic activity, or any combination thereof, relative to a corresponding control immuneAttorney Docket 44010.185WO-PCT / / CU24092 cell of the same type. In some particular embodiments of the aspects described herein, a cell that is dysfunctional is a CD8+ T cell that expresses the CD8+ cell surface marker. Such CD8+ cells normally proliferate and produce cell killing enzymes, e.g., they can release the cytotoxins perforin, granzymes, and granulysin. However, exhausted / dysfunctional T cells do not respond adequately to TCR stimulation, and display poor effector function, sustained expression of inhibitory receptors and a transcriptional state distinct from that of functional effector or memory T cells. Dysfunction / exhaustion of T cells thus prevents optimal control of infection and tumors. Exhausted / dysfunctional immune cells, such as T cells, such as CD8+ T cells, may produce reduced amounts of IFN-gamma, TNF-alpha and / or one or more immunostimulatory cytokines, such as IL-2, compared to functional immune cells. Exhausted / dysfunctional immune cells, such as T cells, such as CD8+ T cells, may further produce (increased amounts of) one or more immunosuppressive transcription factors or cytokines, such as IL-10 and / or Foxp3, compared to functional immune cells, thereby contributing to local immunosuppression. Dysfunctional CD8+ T cells can be both protective and detrimental against disease control. As used herein, a “dysfunctional immune state” refers to an overall suppressive immune state in a subject or microenvironment of the subject (e.g., tumor microenvironment). For example, increased IL-10 production leads to suppression of other immune cells in a population of immune cells.

[0238] CD8+ T cell function is associated with their cytokine profiles. It has been reported that effector CD8+ T cells with the ability to simultaneously produce multiple cytokines (polyfunctional CD8+ T cells) are associated with protective immunity in patients with controlled chronic viral infections as well as cancer patients responsive to immune therapy (Spranger et al., 2014, J. Immunother. Cancer, vol. 2, 3). In the presence of persistent antigen CD8+ T cells were found to have lost cytolytic activity completely over time (Moskophidis et al., 1993, Nature, vol.362, 758–761). It was subsequently found that dysfunctional T cells can differentially produce IL-2, TNFa and IFNg in a hierarchical order (Wherry et al., 2003, J. Virol., vol.77, 4911–4927). Decoupled dysfunctional and activated CD8+ cell states have also been described (see, e.g., Singer, et al. (2016). A Distinct Gene Module for Dysfunction Uncoupled from Activation in Tumor-Infiltrating T Cells. Cell 166, 1500-1511 e1509; WO / 2017 / 075478; and WO / 2018 / 049025).

[0239] In example embodiments, cell sorting is used to obtain populations of cells having an altered T cell function phenotype. In example embodiments, single cell sequencing is usedAttorney Docket 44010.185WO-PCT / / CU24092 to identify single cells having an altered T cell function phenotype. In example embodiments, to assess edits affecting short-term proliferation capacity, cells can be stained with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE), which enables tracking of cell proliferation by dye dilution. To assess edits impacting T cell activation status, sorted T cells with high or low expression of a marker of activation can be used. For example, the IL2 receptor alpha chain (CD25) can be detected. To determine variant effects on cytokine production, cells can be sorted based on the intracellular presence of inflammatory or suppressive cytokines. For example, IFNɣ and / or TNF⍺ can be detected. In example embodiments, the distribution of sgRNAs in each condition can be read out using next- generation sequencing. Sequencing

[0240] In example embodiments, sequencing is used to identify guide RNAs (i.e., base edit targets) that alter T cell function in either a population of sorted cells or in single cells. In example embodiments, next generation sequencing is used to identify guide RNAs. In example embodiments, Sanger sequencing can be used to confirm that a mutation is introduced to a cell.

[0241] In example embodiments, sequencing comprises high-throughput (formerly "next- generation") technologies to generate sequencing reads. In DNA sequencing, a read is an inferred sequence of base pairs (or base pair probabilities) corresponding to all or part of a single DNA fragment. A typical sequencing experiment involves fragmentation of the genome into millions of molecules or generating complementary DNA (cDNA) fragments, which are size-selected and ligated to adapters. The set of fragments is referred to as a sequencing library, which is sequenced to produce a set of reads. Methods for constructing sequencing libraries are known in the art (see, e.g., Head et al., Library construction for next-generation sequencing: Overviews and challenges. Biotechniques. 2014; 56(2): 61–77; and Trombetta, J. J., Gennert, D., Lu, D., Satija, R., Shalek, A. K. & Regev, A. Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing. Curr Protoc Mol Biol. 107, 4 22 21-24 22 17, doi:10.1002 / 0471142727.mb0422s107 (2014). PMCID:4338574). A “library” or “fragment library” may be a collection of nucleic acid molecules derived from one or more nucleic acid samples, in which fragments of nucleic acid have been modified, generally by incorporating terminal adapter sequences comprising one or more primer binding sites and identifiable sequence tags. In example embodiments, the library members (e.g., genomic DNA, cDNA) may include sequencing adaptors that are compatible with use in, e.g., Illumina's reversibleAttorney Docket 44010.185WO-PCT / / CU24092 terminator method, long read nanopore sequencing, Roche's pyrosequencing method (454), Life Technologies' sequencing by ligation (the SOLiD platform), PacBio long read sequencing, or Life Technologies' Ion Torrent platform. Recent advances in long-read sequencing have enabled sequencing full-length transcripts; Pacific Biosciences (PacBio) single-molecule real- time (SMRT) sequencing and Oxford Nanopore Technologies (ONT) nanopore sequencing can generate reads >10 Kb (see, e.g., Amarasinghe SL, Su S, Dong X, Zappia L, Ritchie ME, Gouil Q. Opportunities and challenges in long-read sequencing data analysis. Genome Biol. 2020;21(1):30). Examples of such methods are described in the following references: Margulies et al (Nature 2005437: 376-80); Schneider and Dekker (Nat Biotechnol. 2012 Apr 10;30(4):326-8); Ronaghi et al (Analytical Biochemistry 1996 242: 84-9); Shendure et al (Science 2005 309: 1728-32); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al (Methods Mol. Biol.2009; 553:79-108); Appleby et al (Methods Mol. Biol.2009; 513:19-39); Wenger, A. M., et al. (2019) Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome. Nature Biotechnology, 37, 1155–1162, Leung SK, Jeffries AR, Castanho I, et al. Full-length transcript sequencing of human and mouse cerebral cortex identifies widespread isoform diversity and alternative splicing. Cell Rep. 2021;37(7):110022, Gordon SP, Tseng E, Salamov A, et al. Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing. PLoS One. 2015;10(7):e0132628, and Morozova et al (Genomics. 2008 92:255-64), which are incorporated by reference for the general descriptions of the methods and the particular steps of the methods, including all starting products, reagents, and final products for each of the steps.

[0242] In example embodiments, the invention involves single cell RNA sequencing (see, e.g., Qi Z, Barrett T, Parikh AS, Tirosh I, Puram SV. Single-cell sequencing and its applications in head and neck cancer. Oral Oncol.2019;99:104441; Kalisky, T., Blainey, P. & Quake, S. R. Genomic Analysis at the Single-Cell Level. Annual review of genetics 45, 431-445, (2011); Kalisky, T. & Quake, S. R. Single-cell genomics. Nature Methods 8, 311-314 (2011); Islam, S. et al. Characterization of the single-cell transcriptional landscape by highly multiplex RNA- seq. Genome Research, (2011); Tang, F. et al. RNA-Seq analysis to capture the transcriptome landscape of a single cell. Nature Protocols 5, 516-535, (2010); Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nature Methods 6, 377-382, (2009); Ramskold, D. et al. Full-length mRNA-Seq from single-cell levels of RNA and individual circulatingAttorney Docket 44010.185WO-PCT / / CU24092 tumor cells. Nature Biotechnology 30, 777-782, (2012); and Hashimshony, T., Wagner, F., Sher, N. & Yanai, I. CEL-Seq: Single-Cell RNA-Seq by Multiplexed Linear Amplification. Cell Reports, Cell Reports, Volume 2, Issue 3, p666–673, 2012).

[0243] In example embodiments, the invention involves plate based single cell RNA sequencing (see, e.g., Picelli, S. et al., 2014, “Full-length RNA-seq from single cells using Smart-seq2” Nature protocols 9, 171-181, doi:10.1038 / nprot.2014.006).

[0244] In example embodiments, the invention involves high-throughput single-cell RNA- seq. In this regard reference is made to Macosko et al., 2015, “Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets” Cell 161, 1202–1214; International patent application number PCT / US2015 / 049178, published as WO2016 / 040476 on March 17, 2016; Klein et al., 2015, “Droplet Barcoding for Single-Cell Transcriptomics Applied to Embryonic Stem Cells” Cell 161, 1187–1201; International patent application number PCT / US2016 / 027734, published as WO2016168584A1 on October 20, 2016; Zheng, et al., 2016, “Haplotyping germline and cancer genomes with high-throughput linked-read sequencing” Nature Biotechnology 34, 303–311; Zheng, et al., 2017, “Massively parallel digital transcriptional profiling of single cells” Nat. Commun. 8, 14049 doi: 10.1038 / ncomms14049; International patent publication number WO2014210353A2; Zilionis, et al., 2017, “Single-cell barcoding and sequencing using droplet microfluidics” Nat Protoc. Jan;12(1):44-73; Cao et al., 2017, “Comprehensive single cell transcriptional profiling of a multicellular organism by combinatorial indexing” bioRxiv preprint first posted online Feb.2, 2017, doi: dx.doi.org / 10.1101 / 104844; Rosenberg et al., 2017, “Scaling single cell transcriptomics through split pool barcoding” bioRxiv preprint first posted online Feb.2, 2017, doi: dx.doi.org / 10.1101 / 105163; Rosenberg et al., “Single-cell profiling of the developing mouse brain and spinal cord with split-pool barcoding” Science 15 Mar 2018; Vitak, et al., “Sequencing thousands of single-cell genomes with combinatorial indexing” Nature Methods, 14(3):302–308, 2017; Cao, et al., Comprehensive single-cell transcriptional profiling of a multicellular organism. Science, 357(6352):661–667, 2017; Gierahn et al., “Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput” Nature Methods 14, 395–398 (2017); and Hughes, et al., “Highly Efficient, Massively-Parallel Single-Cell RNA- Seq Reveals Cellular States and Molecular Features of Human Skin Pathology” bioRxiv 689273; doi: doi.org / 10.1101 / 689273, all the contents and disclosure of each of which are herein incorporated by reference in their entirety.Attorney Docket 44010.185WO-PCT / / CU24092

[0245] In example embodiments, CITE-seq (Stoeckius, M. et al. Simultaneous epitope and transcriptome measurement in single cells. Nat. Methods 14, 865–868 (2017)) (cellular proteins) is used to generate single cell RNA-seq and proteomics data.

[0246] In example embodiments, guide RNAs are delivered to primary immune cells using a vector library. In example embodiments, perturb-seq vectors are used. Perturb-seq vectors are compatible with scRNA-seq because they allow pol II expression of sequences (e.g., barcodes) that can be used to identify the guide sequences in single cells. Example perturb-seq vectors have been described, such as the CROP-seq vector used in the examples herein. Any vector is applicable to the present invention (see e.g., Dixit et al., “Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens” 2016, Cell 167, 1853–1866; Adamson et al., “A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response” 2016, Cell 167, 1867–1882; Jaitin DA, Weiner A, Yofe I, et al. Dissecting Immune Circuits by Linking CRISPR-Pooled Screens with Single-Cell RNA-Seq. Cell. 2016;167(7):1883-1896.e15; Feldman et al., Lentiviral co-packaging mitigates the effects of intermolecular recombination and multiple integrations in pooled genetic screens, bioRxiv 262121, doi: doi.org / 10.1101 / 262121; Datlinger, et al., 2017, Pooled CRISPR screening with single-cell transcriptome readout. Nature Methods. Vol.14 No.3 DOI: 10.1038 / nmeth.4177; Hill et al., On the design of CRISPR- based single cell molecular screens, Nat Methods. 2018 Apr; 15(4): 271–274; Replogle, et al., “Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing” Nat Biotechnol (2020). doi.org / 10.1038 / s41587-020-0470-y; Schraivogel D, Gschwind AR, Milbank JH, et al. "Targeted Perturb-seq enables genome-scale genetic screens in single cells". Nat Methods. 2020;17(6):629-635; Frangieh CJ, Melms JC, Thakore PI, et al. Multimodal pooled Perturb-CITE-seq screens in patient models define mechanisms of cancer immune evasion. Nat Genet. 2021;53(3):332-341; US patent application publication number US20200283843A1; and US Patent number US11214797B2).

[0247] Further embodiments are illustrated in the following Examples which are given for illustrative purposes only and are not intended to limit the scope of the invention.Attorney Docket 44010.185WO-PCT / / CU24092 EXAMPLES Example 1 – Massively parallel base editing screens to map variant effects on anti-tumor hallmarks of primary human T cells

[0248] Here, Applicants overcome existing challenges through improved production, delivery, and scalability of BE and achieve unprecedented single and multiplex editing efficiency in primary human T cells, while finding optimal balances between genome editing and maintaining cell viability. Applicants performed several large-scale BE screens in multiple donors under acute and chronic T cell activation conditions and found novel variants that improve T cell polyfunctionality across multiple donors. Through completely virus-free production and protein-free base editing of specific epitope-reactive human T cells that harbor base edits nominated from these screens, Applicants engineered T cell products with superior, on-target tumor-lytic ability. Thus, this study paves the way for rapid, efficient, and safe application of base editing to improve existing and future cellular immunotherapies.

[0249] The Tables described herein can also be viewed in Walsh ZH, Shah P, Kothapalli N, et al. Mapping variant effects on anti-tumor hallmarks of primary human T cells with base- editing screens. Nat Biotechnol. Published online May 23, 2024. RESULTS High-efficiency base editing using different delivery methods in primary human T cells.

[0250] Applicants first sought to establish and test methods for delivering adenine- and cytosine base editors (ABE and CBE, respectively) and single-guide RNAs (sgRNAs) to primary human T cells that result in high editing efficiency while maintaining cell viability, two major determinants in the successful development of cell-based therapies (Fig. 1a)1. First, Applicants devised a strategy using in vitro transcription (IVT) to generate high-quality, stable mRNA molecules with reduced immunogenicity encoding for the ABE or CBE (Fig.6a,b) and deliver these to T cells via optimized electroporation with an sgRNA targeting either a splice donor or acceptor site or a start codon mutation in CD2 or B2M, respectively (Fig. 1b,c). The base editor mRNA is translated by the endogenous T cell ribosomal machinery to a fully functional synthetic protein. With this approach, Applicants achieved high efficiency with all tested sgRNAs resulting in protein loss as measured by flow cytometry, ranging up to 99.5% with ABE (Fig. 1d). Applicants also show that multiple base edits can be performed through simultaneous delivery of two sgRNAs targeting CD2 and B2M, without loss of editing efficiency for either target (Fig. 6c). Similarly, Applicants achieved up to 85.9% editing efficiency using a CBE (Fig. 1e).Attorney Docket 44010.185WO-PCT / / CU24092

[0251] To prepare for massively parallel BE screening, Applicants tested another strategy: Applicants delivered sgRNAs cloned into the CROP-seq (containing a mTurquoise fluorescent reporter) vector using lentiviral transduction followed by electroporation of BE mRNA (Fig. 1f). Applicants first validated the feasibility of this approach with CRISPR-Cas9 knockout, with lentiviral delivery of a previously validated sgRNA targeting CD2 followed by nucleofection of SpCas9 mRNA (Fig. 6d). Applicants then extended this approach to both ABE and CBE delivery and found unprecedented high efficiency in introducing start site mutations in B2M in two independent donors using ABE (90.6% and 85.3%) (Fig. 1g). The editing efficiency was essentially equal in both CD4+ and CD8+ T cells (Fig.6e). Furthermore, using a CBE Applicants achieved high editing efficiency (56.5%) of the TRBC locus (Fig.6f). Importantly, Applicants determined dosing of BE mRNA delivered to produce an optimal balance between high editing efficiency and cell viability (Fig.1h), which is a prerequisite for performing large-scale screens in primary human T cells. In summary, Applicants established a scalable strategy for high-efficiency base editing which enables base editor screening in primary human T cells. Massively parallel base editing to map variants to hallmarks of T cell function.

[0252] To characterize variants broadly and deeply across a wide range of genes implicated in T cell function Applicants designed two ABE sgRNA libraries. In the first library ("ClinVar library"), which included 8,142 sgRNAs, Applicants mutagenized 102 genes involved in all major functions of T cells (Fig.2a, Fig.7a, Table 1), including known variants associated with clinical immune syndromes, variants of unknown significance (VUS), and other uncharacterized mutations that Applicants generated at these defined loci. In the second library, Applicants systematically tiled 7,815 sgRNAs to introduce every possible ABE-mediated mutation across the entire coding sequence and exon-intron boundaries of twelve genes with central functions in T cell activity, including T cell receptor signaling (ZAP70, CD3Z, LCK, LAT), costimulatory signaling (CD2, CD28), cytokine receptor signaling (IL2RG, IL7RA, JAK1, STAT3, STAT5B), and stemness (TCF7) (Fig. 2b, Fig. 7b). Each library was designed to include multiple subtypes of control sgRNAs: Negative controls included empty-window sgRNAs (i.e. sgRNAs in which the defined base-editing window does not contain a targetable base), sgRNAs predicted to produce only silent mutations in the target gene set, and sgRNAs tiling a gene not involved in T cell function (PPP1R12C). Positive control sgRNAs were designed to generate an array of presumed-deleterious mutations, such as splice site mutationsAttorney Docket 44010.185WO-PCT / / CU24092 and missense mutations resulting in substitutions to proline, across 50 essential genes encoding factors critical for cell survival, including DNA polymerases (e.g. POLR3C, POLR2E), RNA splicing (e.g. SF3B3), and cell-division (e.g. KIF11) (Fig. 7a,b, Table 2). In all experiments, Applicants used a highly active adenine base editor (ABE8e) with a relaxed protospacer- adjacent motif requirement (NG rather than NGG)17, thus, maximizing the number of mutations that can be introduced. Table 1. Genes selected for ClinVar screen, with STRING clustering by functional interactions.Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Attorney Docket 44010.185WO-PCT / / CU24092Table 2. List of essential genes used for positive controls in screens.

[0253] Following lentiviral transductions of these sgRNAs (cloned into the CROP-seq vector using optimized Golden Gate assembly, Methods) (Fig. 7c-d) into pre-stimulated primary human CD3+ T cells isolated from two healthy donors, Applicants electroporated ABE mRNA and allowed cells to edit and then proliferate for at least 7 days (Fig. 7e). The T cells from each screen were then probed with various assays to capture multiple critical axes of T cell function (Fig. 2c), including activation, short- and long-term proliferation, and cytokine production. To approximate acute and chronic T cell receptor (TCR) engagement as seen in infections or cancer, respectively, base edited T cells were stimulated either through a single-Attorney Docket 44010.185WO-PCT / / CU24092 time activation or with repetitive stimulations using CD3 / CD28 microbeads (Fig. 2d). To assess edits affecting short-term proliferation capacity, cells were stained with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE), which enables tracking of cell proliferation by dye dilution, bead-stimulated, and allowed to proliferate for 4 days followed by flow sorting (CFSElowvs. CFSEhigh) (Fig. 2e). To determine variant effects on cytokine production, cells were briefly stimulated and sorted on intracellular presence of the proinflammatory cytokines IFNɣ and TNF⍺ (Fig. 2f). Finally, to assess edits impacting T cell activation status, Applicants sorted T cells with high or low expression of the IL2 receptor alpha chain (CD25) (Fig.7f). The distribution of sgRNAs in each condition was read out using next-generation sequencing (Methods). Base editor screens recover known variants critical for T cell survival and proliferation.

[0254] Applicants first analyzed several built-in controls across all experiments. In the ClinVar screen, Applicants determined the distribution of sgRNAs targeting the gene set with either empty editing windows or generating silent mutations only, which showed no significant change in distribution at late timepoints in the long-term proliferation arm (Fig.3a, Fig.8a,b), thus suggesting relatively low off-target base edit events. In contrast, sgRNAs disrupting essential genes (Table 2) through either splice donor or splice acceptor splice site edits or, base edits resulting in amino acid changes to a proline (thus altering the structural organization of resulting gene products) were strongly depleted (Fig. 3a,b, Fig. 8a). Applicants noted that across both donors, sgRNAs generating splice donor disruption in essential genes had greater dropout than those generating splice acceptor disruption, consistent with previous reports indicating that splice donor mutations more reliably impact gene function18. sgRNAs generating deleterious mutations in CD3 complex genes (CD3D, CD3E, CD3G, and CD3Z), which are essential for T cell activation, were significantly depleted across both donors (Fig. 3a,b, Fig.8a). Finally, when assessing gene-wise dropout in the CD25hi(activated) and CFSElo(highly proliferative) flow-sorted populations of the ClinVar screen, Applicants observed depletion of variants in expected targets, including multiple members of the CD3 complex and genes essential for cell division (Fig. 8c,d).

[0255] IL2RG encodes for the gamma chain of the IL2 receptor complex, whose ligand interleukin-2 (IL2) is required for T cell proliferation. In the 12-gene tiling screen, sgRNAs generating mutations at IL2RG splice-sites or introducing proline conversions (e.g. L16P, L14P, S10P) in the signal peptide, which is responsible for plasma membrane trafficking, wereAttorney Docket 44010.185WO-PCT / / CU24092 strongly depleted following multiple rounds of T cell restimulation (Fig. 3c, Fig. 8e). Applicants also observed strong depletion of known IL2RG mutations (e.g. C62R, Y89C, L230P) that have been documented in patients with X-linked severe combined immunodeficiency (X-SCID), suggesting that base editor screens can recover and characterize variants of clinical significance19. These results were highly consistent between both donors, indicating reproducibility of these screens (Fig. 3c, Fig. 8e).

[0256] Conversely, Applicants also identified rational enrichment of sgRNAs in the screens reflecting mutations with known clinical, biological, and structural significance. In the long-term proliferation arm of the ClinVar screen, Applicants observed enrichment of an sgRNA generating a mutation in the inhibitory histone domain interface of SOS1 (W85R), a guanine exchange factor which positively regulates Ras pathway signaling (Fig. 8f). W85R is a pathogenic mutation associated with Noonan Syndrome20, a RASopathy driven by hyperactive Ras pathway signaling. Preclinical structural and functional studies have demonstrated that mutating this residue facilitates a partial release of SOS1 auto-inhibition, thus, increasing SOS1 signaling. Additionally, Applicants found enrichment of a sgRNA predicted to generate a D323G mutation in AKT1, a likely-pathogenic mutation associated with familial breast cancer (Fig. 3d). Clinical and preclinical studies have shown that another missense mutation in the same residue – D323H – is a gain-of-function mutation driving increased downstream signaling, resistance to pharmacological allosteric inhibition21. Moreover, the same preclinical studies identified a similar AKT1 variant (W80R) of unknown significance with, which enriched in the long-term proliferation screen (see below) (Fig. 3d).

[0257] Together, these data suggest that Applicants achieved excellent on-target base editing recovering enrichment and depletion of several expected gain- and loss-of-function mutations, respectively, while synonymous or empty-window base edits showed no differential abundance, suggesting low off-target base edits. Variants modulating T cell proliferation, activation, and effector function.

[0258] Having established the robustness and reproducibility of the screens, Applicants next quantified sgRNA abundance across multiple functional read-outs and identified variants modulating T cell proliferation, activation, and effector function (Figure 3d-f, Fig.8f-l, Fig 9- 10, Extended Data Table 3-4 (Walsh, et al. 2024)). In the long-term proliferation assay, Applicants found significant enrichment of sgRNAs introducing an array of base edits in genes encoding protein kinases (PIK3CD, PIK3R1, PIK3R3, AKT1, LCK), phosphatases (PTPRC,Attorney Docket 44010.185WO-PCT / / CU24092 which encodes CD45), and immune checkpoints (CTLA4), among others, as well as depletion of perturbations in genes encoding GTP binding proteins (RHOA) (Fig. 3d, Fig. 8f-j). Applicants discovered clusters of highly enriched PIK3CD variants in the C2 domain (C416R, S444P) and helical domain (Y524H, Y524C, E525G, E527G, D529G) (Fig. 3d). Other highly enriched variants involved the pleckstrin homology and protein kinase domains (W80R and I165V, respectively) of AKT1, the C-terminal tyrosine residue of LCK (Y505C), and the tyrosine phosphatase 1 domain of CD45 (N760S) (Fig. 3d, Fig. 8f). SgRNAs generating variants of RHOA in the effector domain (Y42H, Y42C) and switch II domain22(Y66H, R70G) were among the most significantly depleted across multiple donors (Fig. 3d, Fig. 8g,h). For many of these variants, Applicants observed concordant enrichment / depletion patterns in the CFSE-proliferation assay (Fig. 3e, Fig. 8i). This suggests that these variants, particularly involving PIK3CD, have the potential not only for short-term, but sustained proliferative capacity, which is a prerequisite for robust T cell antitumor activity. Applicants next identified putative variants associated with increased cytokine production, including in several genes with central functions in TCR signaling, co-stimulation, signal transduction and transcription regulation, such as ITK (Y588C, Y578H), ZAP70 (I203V), NFKB1 (E439G, N846D, E63G), CARD11 (I1076T), CTLA4 (D153G), and CD40LG (Q220R, Y172H, among others (Extended Data Table 3 (Walsh, et al. 2024)). In the same readout, Applicants identified dozens of known or likely pathogenic variants in several genes resulting in a range of immunodeficiency disorders (Extended Data Table 3 (Walsh, et al. 2024)). Additionally, variants in genes identified in other readouts (e.g. PIK3CD and CTLA4) were also enriched in the cytokine assay (Extended Data Table 3 (Walsh, et al. 2024)). Together, these results suggested that putative and known gain- and loss-of-function variants produced in these base editing screens may modulate one or multiple hallmarks of T cell function. Identification and structural modeling of PIK3CD variant hotspots associated with altering T cell polyfunctionality.

[0259] Applicants next characterized a group of PIK3CD-targeting sgRNAs, which enriched across multiple screen readouts and in multiple human donors, thus suggesting that these mutations broadly influence T cell activity (Fig 3d,e, Fig. 8i). Class I PI3Ks are obligatory heterodimers composed of a catalytic subunit (p110⍺, p110β or p110δ) and a regulatory subunit (summarized as p85, with p85a being the most common)23. PI3Kδ (encoded by PIK3CD which forms a heterodimer with p85, encoded by PIK3R1, 2 or 3) is predominantlyAttorney Docket 44010.185WO-PCT / / CU24092 expressed in immune cells and regulates central cell functions such as cell survival and proliferation in response to receptor-tyrosine kinase phosphorylation. Physiologically, p85 binds to p110δ to fulfill three major functions: (i) stabilization of the catalytic subunit, (ii) inhibition of catalytic activity, and (iii) physical localization to phosphorylated RTKs, and local activation in the cell membrane following disruption of the p110δ-p85 interaction and release of the p110 kinase catalytic activity24. Activating mutations in the kinase domain of PIK3CD (e.g., E1021K) are associated with an immunodeficiency syndrome (Activated PI3K Delta Syndrome, APDS25,26, which is surprising, given that the screen identified variants that resulted in improved polyfunctionality of T cells.

[0260] Applicants noted a significant enrichment “hotspot” of multiple base edits in the helical domain of PIK3CD resulting in missense mutations in codons 524 (two independent sgRNAs producing Y524C), 525 (two independent sgRNAs producing E525G), 527 (two independent sgRNAs producing E527G), and N529G; additionally, two independent sgRNAs resulting in C416R located in the C2 domain (Fig. 4a, Fig. 11a-c; Extended Data Table 3 (Walsh, et al. 2024)). Since the helical domain interacts with the N-terminal SH2 domains (nSH2) and the C2 domain interacts with both nSH2 and the coiled-coil domain (iSH2 domain) of p85, Applicants reasoned that mutations in codons 416 and 524-529 disrupt inhibitory functions of p85, and thereby enable increased catalytic activity of p110δ. In line with this, Applicants also identified putative loss-of-function variants of PIK3R1 (L570P) and PIK3R3 (H440R) located in SH2 domains predicted to be important for interacting with the p110 subunits (Fig. 3d,e, Fig. 4b). PIK3CD mutations in residues Y524, E525, D527 and D529 likely interfere with interactions with the opposing Lys / Arg-rich surface of p85, thereby disrupting the electrostatic interactions at this interface (Fig. 4b). Similarly, the identified mutations in the C416 residue are also adjacent to a Lys (L567 of p85). Thus, mutation to a positive charged amino acid (e.g. C416R) is likely responsible for the functional effect. The L570P mutation identified in PIK3R1 is likely incompatible with the coiled coil region due to the dihedral angle allowed by the proline substitution, thus disrupting the physiologic interface with p110δ (Fig. 4b). Functional validation of the effect of PIK3CD variants on multiple hallmarks of T cell function contrasted with other variants affecting predominantly T cell proliferation.

[0261] Applicants next sought to functionally characterize several highly enriched PIK3CD variants and associated gene variants which were also expected to impact the PI3KAttorney Docket 44010.185WO-PCT / / CU24092 axis, including PIK3R1 L570P as described above, and AKT1 W80R, an activating mutation downstream of PI3Kδ (Fig. 11d-h)21. Applicants reasoned that to determine the specific role of these variants in enhancing multiple axes of T cell function, it would be useful to contrast them with variants that apparently only impacted one T cell hallmark in the screens (e.g. proliferation). From both the ClinVar and 12-gene tiling screens, Applicants identified variants of LCK (lymphocyte-specific protein tyrosine kinase) a central regulator of TCR signaling, including Y505C, Y505H and Q506R, all located in the distal C-terminal domain of LCK, and scored strongly in only proliferation assays (Fig.11i,j). Y505C is a ClinVar variant of unknown significance. The tyrosine residue (Y505) is a phosphorylation site that interacts with its own SH2 domain, resulting in an inactive or closed confirmation27,28(Extended Data Fig. 6k). Furthermore, as loss-of-function mutation (i.e., negative control for T cell stimulation experiments), Applicants also included a CD3D M92T mutation, a ClinVar VUS which was significantly depleted in multiple readouts in the screens (Fig 3e, Fig.8k,l). To control for non- specific effects of base editor electroporation, Applicants used a sgRNA which only generates a silent mutation in PPP1R12C (P405P).

[0262] Using the same sgRNA sequences from the screening libraries, Applicants next generated the following single base edits (through simultaneous delivery of BE mRNA and sgRNA via electroporation, see Fig. 1a) in three independent healthy donors: putative PIK3CD-activating mutations C416R, Y524C, Y524H, E525G and E527G, and a putative loss- of-function mutation S281P which was depleted in multiple screens (Fig.3d-e, Fig 8l), PIK3R1 L570P, LCK Y505C and AKT1 W80R. Applicants confirmed on-target base editing for these by Sanger sequencing and achieved an estimated average efficiency of 80.25% (ranging from 58 to 93.5%) across all edits and donors (Fig. 4c,d).

[0263] Applicants performed a short-term CD3 / CD28 stimulation (using crosslinked soluble antibodies) of cells harboring these variants and measured the frequency of S6 and AKT phosphorylation (which are downstream surrogates for PI3K activity). Consistent with the screen readouts, Applicants found increased pS6 (pS235 / pS236) and pAKT (pS473) in activating PI3KCD variants and PIK3R1 L570P, and decreased levels in the putative loss-of- function variants CD3D M92T and PIK3CD S281P (Fig. 4e). Next, Applicants stimulated edited T cells with immobilized OKT3 and soluble CD28, followed by intracellular measurement of cytokines associated with activation (IL2) and effector cytokines (TNF⍺) using flow cytometry. This resulted in a significantly higher fraction of IL2-positive cells withAttorney Docket 44010.185WO-PCT / / CU24092 PIK3CD variants C416R and E525G and PIK3R1 L570P (Fig.4f) and increased production of TNF⍺ (Fig.4g) in T cells harboring PIK3CD Y524C, E525G and E527G and PIK3R1 L570P, but not PIK3CD S281P or mutations in AKT1, LCK or CD3D. Lastly, Applicants tested the impact of these variants on proliferation rates using the CFSE assay. In all donors, Applicants found increased proliferation in PIK3CD C416R, E525G, E527G and Y524C and at a slightly lower rate, PIK3R1 L570P. As predicted from the screens, AKT1 W80R and LCK Y505C, but not PI3KCD S281P or CD3D W92T, also resulted in increased proliferation (Fig. 4h,i).

[0264] Together, these results allow three important conclusions: first, Applicants validated several variants across different functional axes reflecting the phenotypic readouts used in the screens. Second, Applicants demonstrate that some variants affect multiple T cell functions, while others predominantly influence proliferation. Thus, proliferation as sole readout is insufficient to capture or infer T cell functions critical for effective tumor lysis and emphasizes the importance of the multi-dimensional screen readouts and validation pipelines used in this study. Finally, Applicants also note a spectrum of effect sizes in the signaling, cytokine, and proliferation readouts across the landscape of putative PI3K pathway-activating mutations Applicants validated. This suggests that inferring the effect of a gain-of-function variant based solely on its presence in a signaling cascade is not sufficient to capture the nuance of its influence on tuning signal strength and quality. Improving cellular cancer immunotherapies informed by hits nominated in base editing screens.

[0265] Applicants reasoned that variants that improve multiple axes of T cell activity, such as the selected PIK3CD mutations Applicants validated, may be leveraged to improve existing and emerging cell immunotherapies derived from CD8+T cells. To examine this in an epitope- specific manner, Applicants used a previously established co-culture model of the melanoma cell line A3754, which expresses a common cancer-testis antigen (NY-ESO-1) and human T cells in which Applicants knocked out the endogenous T cell receptor (TCR) and knocked in the cognate NY-ESO-1 T cell receptor (NY-ESO-1 TCR T cells) (Fig. 5a, Fig. 12a-c). First, Applicants confirmed that Applicants were able to achieve high efficiency base editing in these antigen-specific cells (Fig. 12d). Next, Applicants generated several NY-ESO-1 TCR T cells variants, including PIK3CD S281P, C416R, and E252G, AKT1 W80R, LCK Y505C, and a silent control mutation in PPP1R12C (P405P). A375 cells were plated and co-cultured withAttorney Docket 44010.185WO-PCT / / CU24092 NY-ESO-1 TCR T cells harboring the mutants listed above with or without addition of anti- MHC Class I antibody, which blocks TCR-MHC interactions (Fig. 5a, Fig. 12c).

[0266] Following co-culture experiments with or without addition of MHC class I blockade, Applicants isolated T cells and performed intracellular flow cytometry for IL2, TNF⍺ and Granzyme B (GrzB). Applicants found that compared to the silent mutation, the fraction TNF⍺-producing cells was significantly increased in NY-ESO-1-TCR T cells with PIK3CD C416R and E525G, and AKT1 W80R, and significantly decreased with PIK3CD S281P and LCK Y505C (Fig. 5b,c, Fig. 12e). The intensity of TNF⍺ production in PIK3CD C416R and E525G T cells was also enhanced. Importantly, TNF⍺-positive cells was also elevated in. MHC class I blocking antibody entirely abrogated TNF⍺ production. Similarly, the PIK3CD C416R and E525G variants also resulted in increased fractions of both IL2- and GrzB-producing cells (Fig. 5d,e, Fig. 12f), as well as higher intensity of GrzB (Fig. 12g,h). Integrated multi-dimensional analyses across measurements confirmed co-expression of all cytokines in cells with PIK3CD C416R and E525G, confirming their polyfunctional state (Fig. 5f,g, Fig. 12i).

[0267] Lastly, Applicants tested the impact of these variants on tumor lysis. Applicants co- cultured NY-ESO-1 TCR T cells with dsRed-expressing A375-melanoma (A375-dsRed, target) cells at 1:1 or 0.5:1 ratio and determined the number of viable tumor cells over time. Compared to the silent mutation, T cells with PIK3CD C416R and E525G demonstrated significantly improved tumor killing, while PIK3CD S281P or LCK Y505C both demonstrated significantly diminished tumor killing (Fig. 5h,i, Fig. 12j,k). Interestingly, T cells with AKT1 W80R also demonstrated improved tumor lysis. Applicants also noted that across co-culture conditions, the C416R variant showed superior polyfunctionality and tumor-lytic activity compared the other PIK3CD variant E525G, suggesting that polyfunctional T cell effects can be tuned through specific base edits. Together, these results demonstrate that synthetic variants detected in base editing screens inform engineering of primary human T cells to make improved cell-based cancer immunotherapies. DISCUSSION

[0268] The potential clinical efficacy of cell-based cancer immunotherapies, such as TIL transfer products or CAR-T cells, are heavily impacted by the precursor T cell state and function5–7. Recent studies suggest that point mutations (either germline or engineered) may significantly alter the function of T cells and ensuing cell products. For example, a phospho-Attorney Docket 44010.185WO-PCT / / CU24092 silencing mutation in TSC2 results in antigen-specific, conditional activation and persistence of T cells, and improved anti-tumor activity following ACT8. Similarly, point mutations in genes encoding for co-stimulatory proteins (e.g., CD28) and other signaling molecules (e.g., PTPN22 or STAT3) significantly affect T cell receptor signaling strength, effector and memory phenotype, proliferation, and long-term persistence9,10, thus, may engender a potentially favorable cell state for production of more efficient cell therapies.

[0269] Inspired by such sporadic or naturally occurring examples, Applicants sought to systematically identify variants that may improve the polyfunctionality T cell, thus, enhance the therapeutic activity of products generated from these. While a series of CRISPR-Cas9 studies demonstrated how loss of function screens may identify perturbations that enhance or decrease T cell functions29–31, these approaches cannot produce specific mutations at sufficient accuracy or scale, and may have several undesirable off-target activities, such as development of aneuploidy15, a hallmark of cancer. Furthermore, complete gene knockout may result in unknown compensatory upregulation of inhibitory immune checkpoints through loss of steric protein interactions or other mechanisms, and therefore have unexpected immune suppressive consequences4,13.

[0270] In contrast, base editors enable generation of site-specific mutations at single- nucleotide resolution16however, application of these methods has been limited due to editing efficiency in primary human T cells and scalability for large-scale discovery16. Here, Applicants overcame several of these barriers and established methods that enable massively parallel, high efficiency base editing screens in primary human T cells. Additionally, Applicants established optimized procedures for virus- and protein-free single base edits in primary human T cells and engineered T cells that may serve as cell-based immunotherapies.

[0271] In multiple base editing screens across different human T cell donors, Applicants simulated acute and tonic T cell receptor engagement, and read out a range of T cell hallmarks that are critical for effective anti-tumor immunity. This multi-modal phenotypic evaluation associated with variants generated in these screens was critical for rational selection of mutations driving a favorable T cell state characterized by activation, proliferation, effector cytokine function and subsequently enhanced tumor lysis. For example, Applicants identified several activating PIK3CD mutations that were nominated through the screen and later identified in a series of experiments to confer improved T cell polyfunctionality, proliferation, and improved tumor-lytic capacity, thus, may represent candidate variants that may alsoAttorney Docket 44010.185WO-PCT / / CU24092 improve cell-based immunotherapies. In contrast, other mutations, such as LCK Y505C predominantly enhanced cell proliferation, but not cytokine production and ultimately tumor- lytic capacity. Thus, proliferation (or survival) assays alone, which are frequently the readout for large-scale perturbation screens30,32, are insufficient to nominate potentially useful variants of broad T cell function. Notably, while Applicants identified several activating PIK3CD mutations, there were qualitative differences in their phenotypic outputs. For example, C416R consistently produced superior improvement of T cell hallmarks compared to E525G or other activating mutations. Furthermore, mutations in negative regulators of PIK3CD gene product p110δ or activating mutations of key nodes downstream of PI3K activity, such as AKT1 W80R had a significantly lower magnitude in altering T cell polyfunctionality. This suggests that seemingly redundant mutants within the same protein or signaling nodes in the same pathway can have meaningful differential impact on resulting T cell phenotypes. Thus, screens such as the one presented here, identify opportunities to calibrate and tune T cell functions at a previously underappreciated granularity.

[0272] The discovery of activating mutations of PIK3CD (and mutations in PIK3R1 / 3) as a means of improving cell-based immunotherapies seems unexpected. Germline mutations in PIK3CD have been described to cause a rare syndrome known as activated PI3K delta syndrome (APDS), which is characterized by immunodeficiency and a predisposition for recurrent respiratory infections, yet in a subset associated with auto-immunity26. This apparent discrepancy – immunodeficiency when occurring in germline and enhanced anti-tumor immunity when introduced in differentiated T cells in the experiments – likely has several explanations. The germline mutations affect immune cells beyond CD8+ T cells, including immunosuppressive T regulatory T cells, and, due to early hyperactivating of CD8+ T cells may be eliminated early during thymic selection, leaving the host deficient for effective cell- mediated immunity. Irrespective, this example highlights an important lesson from these unbiased screens: it is unlikely that one would pick these mutations a priori to improve T cell function given the clinical association with immunodeficiency. This emphasizes the power of unbiased discovery coupled with multi-modal functional readouts.

[0273] In summary, in this first massively parallel base editing screen in primary human T cells, Applicants overcome multiple technical and design barriers, and identified several unexpected mutations that confer enhanced T cell polyfunctionality and antigen-specific tumor killing, thus providing an important blueprint for unbiased discovery to improve existing andAttorney Docket 44010.185WO-PCT / / CU24092 future cellular immunotherapies. Applicants also present virus- and protein-free approach for highly efficient base editing, which has several advantages: it is rapid, cost-efficient, and compared to existing approaches for cell-engineering leaves no foreign material after desired base edits. Coupled with the high precision and reduced off-target activities (compared to CRISPR-Cas9 approaches) this approach is safer and will likely also reduce regulatory barriers for such novel cell products to enter clinical testing. METHODS

[0274] Primary T cell isolation. Buffy coats from deidentified healthy human donors were obtained from the New York Blood Center. Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats using SepMate 50 ml conical tubes (StemCell, #85450) containing 15mL of Ficoll-Paque Plus media (1.077g / mL, Cytiva, #17144002) per manufacturer’s protocol. PBMCs were then resuspended in ACK buffer (Gibco, #A1049201) for 5 minutes for red blood cell lysis. CD3+ T cells were isolated from PBMCs via negative selection using the EasySep human T cell isolation kit (StemCell, #17951). All T cell cultures were performed with OpTmizer SFM (Gibco #A1048501) supplemented with 1:40 OpTmizer Supplement (Gibco #A1048501) and 1:100 GlutaMAX (Gibco #35050061) using a humidified incubator at 37 °C with 5% CO2atmosphere.

[0275] Lentiviral production and concentration. HEK-293T cells were passaged at least two times in DMEM (Gibco #11965092) + 10% FBS (Gibco #A5670701), splitting at ~80% confluence, and then plated at 825,000 cells / well in 6-well plates. 24 hours later, at ~70-80% confluence, cells were transfected using the TransIT-LT1 system (MirusBio, #MIR2304). Per well, cells were transfected with 500 ng psPAX2 (a gift from Didier Trono, Addgene plasmid #12260), 250 ng VSV-G (a gift from Didier Trono, Addgene plasmid #12259), and 500 ng lentiviral transfer plasmid.18 hours after transfection, media was replaced with DMEM + 20% FBS. 24 hours after the media change, lentivirus-containing supernatant was harvested from cells and clarified by filtration with a 0.45 uM PES filter. Clarified lentiviral supernatant was concentrated (to 1 / 10ththe original volume) using Lenti-X concentrator (Alstem Bio, #631232) according to the manufacturer’s instructions. Concentrated lentivirus was resuspended in sterile PBS (Gibco, #10010023) and either stored at –80 °C or used immediately for transductions.

[0276] General culture of cell lines. HEK293T and A375 cell lines were cultured in DMEM (Gibco, #11965092) supplemented with 10% FBS (Gibco, #A5670701). All cell linesAttorney Docket 44010.185WO-PCT / / CU24092 were cultured at 37 °C in a 5% CO2 humidified incubator. Cells were kept at low passage number and routinely tested for mycoplasma using PlasmoTest (Invivogen, #rep-pt1).

[0277] Lentiviral transduction of primary T cells. Primary human T cells were pre- activated with DynabeadsTMhuman T-Activator CD3 / CD28 beads (Gibco, #11131D) at a 1:1 ratio of cells:beads with 100 IU / ml IL-2 (Chiron #53905-991-01) for 48 hours prior to transduction. T cells were then plated in flat-bottom 96-well plates at 100,000 T cells per well with 1:100 v / v LentiBOOST transduction enhancer (Mayflower Biosciences, # SBPLV10112) and 1:10 v / v of 10x concentrated lentivirus. Plates were centrifuged at 800 x G for 90 minutes at 32 °C in a pre-warmed centrifuge and then cultured overnight at 37 °C. 24 hours after centrifugation, beads were removed using a magnetic rack (Stemcell, #18103), and T cells were expanded in G-Rex 6-well plates (Wilson Wolf, #80240M) with 300 IU / ml IL-2, keeping the density between 0.5-2e6 cells / ml.

[0278] In vitro transcription (IVT) of Base Editor mRNA. Constructs encoding base editors NG-CBE3.9MAX (gift from John Doench and David Root, Addgene #179095) and NG-ABE8e (gift from David Liu, Addgene #138491), and an in vitro transcrition (IVT) template vector (gift from David Liu, Addgene #193843) were obtained from Addgene. Base editors were cloned into the IVT template vector using NEBuilder®HiFi DNA Assembly Cloning Kit (New England Biolabs, #E5520S) (Table 5) and validated by whole-plasmid sequencing (Plasmidsaurus). The in vitro transcription protocol was adapted from Neugebauer et al33. The base editor IVT template was simultaneously amplified and poly-T tailed by PCR using primers IVT-F and IVT-R (Table 5). PCR was performed with Q5®Hot Start High- Fidelity DNA Polymerase with 25 total cycles (New England Biosciences, # M0493S). The PCR amplicon was purified using a QIAquick PCR Purification Kit (Qiagen #28104), eluted in nuclease-free water (Invitrogen, #10977015), and purity of the amplicon was confirmed by gel electrophoresis prior to IVT. IVT was done using the HiScribe®T7 High-Yield RNA Synthesis Kit (New England Biolabs, #E2040S), with substitution of N-1-methyl- pseudouridine-5'-triphosphate (TriLink Biotechnologies, #N-1081-1) for UTP and co- transcriptional capping with CleanCap®Reagent AG (TriLink Biotechnologies, # N-7113-1). For large-scale mRNA production, 320ul of IVT reaction was performed for each editor construct. IVT mRNA was then precipitated by mixing with 0.5 v / v Lithium Chloride (Invitrogen, #AM9480) and incubating at -20 °C for 30 minutes. mRNA was pelleted by centrifugation at 15,000xG for 20 minutes at 4 °C, washed once with ice-cold 70% ethanol,Attorney Docket 44010.185WO-PCT / / CU24092 and then supernatant was removed and pellet air-dried at room temperature for 5 minutes. The mRNA pellet was resuspended by gentle pipetting in 500 µl Ambion THE RNA Storage Solution (Thermo Fisher, #AM7000) per 320 µl of initial IVT reaction. mRNA concentration was quantified using a Nanodrop Spectrophotometer (Thermo Fisher), and mRNA was further diluted to a final concentration of 1.5 µg / µl. Appropriate purity and size of the mRNA products was confirmed using an RNA tapestation kit (Agilent, #5067-5579). Prior to use in screens or validation experiments, editing activity of the BE encoded by the mRNA was confirmed by knockout of a constitutively expressed T cell protein with a validated high efficiency sgRNA. Table 5. All PCR oligos used in this study.

[0279] Design of base editor sgRNAs for targeted gene disruption experiments. For targeted gene knockout experiments to validate base editor efficiency, sgRNAs creating mutations in CD2 splice sites were generated using SpliceR (github.com / MoriarityLab / SpliceR)18. sgRNAs creating premature stop codons in B2M were generated using iSTOP (github.com / CicciaLab / iSTOP)34. sgRNAs ablating the start codon of B2M were generated using the Base Editor Design Tool (github.com / mhegde / base-editor- design-tool). All sgRNAs were ordered as full-length synthetic sgRNAs with modified bases (2’ O-methyl analog on first and last 3 bases; 3’ phosphorothioate between first 3 and last 2Attorney Docket 44010.185WO-PCT / / CU24092 bases) (Synthego). A full list of sgRNAs used for single-guide perturbation experiments are included in Table 6. Table 6. All sgRNA sequences used in this study.

[0280] Base editing of primary T cells. For single and multi-plex (non-library) base- editing, T cells were activated for 48h in 100 IU / ml IL2 with a 1:1 ratio of CD3 / CD28 activator beads, as indicated. T cells were then washed 1x with PBS, resuspended at 1e6 / 20 µl in P3 nucleofection buffer (Lonza, #V4XP-3032), and 1e6 cells were combined with 4.5 µg BEAttorney Docket 44010.185WO-PCT / / CU24092 mRNA and 100 pmol sgRNA (Synthego). T cells were electroporated at 1e6 per cuvette well in a Lonza 4D nucleofector using electroporation program EO-115, and then 100 µl pre- warmed media was immediately added to each cuvette well. Cells were recovered in the cuvette for 15 minutes at 37 °C, and then cultured in OpTmizer SFM at 1e6 / ml with 300 IU / ml IL2. Editing efficiency was assessed at least 4 days after nucleofection by flow cytometry or Sanger sequencing (Azenta).

[0281] Lentiviral-based single-guide editing of primary T cells. For single-guide perturbation pilot experiments using lentiviral guide integration, sgRNAs were cloned into a modified CROPseq-mTurquoise-PuroR backbone (CropSeq-mTurq) using the GeCKO single- guide cloning protocol. Briefly, for each sgRNA, two oligos were ordered, Oligo1: 5’- CACCG[20nt guide]-3’, Oligo2: 3’-C[revcom of 20nt guide]CAAA-5'. The oligos were annealed and phosphorylated, and then ligated into BsmBI-digested, dephosphorylated CROPseq backbone with T4 ligase. A full list of cloning oligos is included in Table 7. Ligation products were transformed into Stbl3 bacteria (Thermo Fisher, #C737303), DNA was purified from bacterial cultures using a HiSpeed Plasmid MidiPrep Kit (Qiagen, #12643), and cloned products were validated with whole-plasmid sequencing or Sanger sequencing of the insert. CropSeq lentivirus was generated, and primary T cells were transduced with lentivirus as described above. After lentiviral transduction cells were left to recover for 72 hours prior to nucleofection. For Cas9-editing proof-of-concept experiments, Cas9 was then introduced using nucleofection of CleanCap 5moU Cas9 mRNA (Trilink, #L-7206). For base-editing experiments, base editors were introduced using IVT mRNA encoding either NG-ABE8e or NG-CBE3.9MAX. To this end T cells were washed once in PBS and resuspended in complete P3 nucleofection buffer (Lonza, #V4XP-3032). 1e6 cells were then mixed with either Cas9 or base editor mRNA (4 - 4.5 µg) and nucleofected with program EO-115 using a 4D Nucleofector (Lonza). Immediately after nucleofection, 100 µl complete T cell media was added, and cells were left to recover for 15 min at 37 °C before transfer to culture plates at 1e6 / ml with T cell media supplemented with 300 IU / ml IL2. Editing efficiency was assessed 4-5 days after nucleofection using a Cytek Aurora flow cytometer (Cytek). Transduced cells were identified by gating on viable, mTurqoise positive cells and target gene KO was assessed by comparing target gene expression in mTurqoise positive vs. mTurquoise negative cells and cells which had not received Cas9 mRNA or Protein. Table 7. All cloning oligos used in this study.Attorney Docket 44010.185WO-PCT / / CU24092Flow cytometry and flow cytometry assisted cell sorting (FACS)

[0282] Surface staining. For all surface staining experiments, cells were collected, washed 1x with ice-cold FACS buffer, and stained on ice for 20 minutes in FACS buffer with surface antibodies at dilutions pre-determined by titration experiments. When multiple brilliant dyes were used together, Brilliant Stain Buffer Plus (BD, #566385) was added according to the manufacturer’s instructions. Cells were then stained with Sytox Green Ready Flow Reagent (Invitrogen, #R37168) according to the manufacturer’s protocol, and analyzed on a Cytek Aurora flow cytometer (Cytek). In cases where staining with Sytox Green was not feasible (fixed samples, fluorophore overlap), cells were instead stained with a 1:1000 dilution of Zombie NIR Viability Dye (Biolegend, #423105) in PBS on ice for 10 minutes prior to surface antibody staining.

[0283] Intracellular cytokine staining. For intracellular cytokine staining experiments, cells were collected and stained on ice for 10 minutes with a 1:1000 dilution of Zombie NIR in PBS. Cells were then stained for surface markers of interest for 20 minutes on ice. Surface- stained cells were fixed and permeabilized using the eBioscience FoxP3 / Transcription Factor Staining Buffer Set (Invitrogen, #00-5523-00), stained for intracellular cytokines of interest and analyzed on a Cytek Aurora flow cytometer. When multiple brilliant dyes were usedAttorney Docket 44010.185WO-PCT / / CU24092 together, Brilliant Stain Buffer Plus (BD, #566385) was added according to the manufacturer’s instructions.

[0284] Intracellular phosphoprotein staining. For all phospho-flow cytometry experiments, cells were stimulated at 37 °C for a pre-determined time period (additional information in “Phospho-flow cytometry of T cell variants” section) Following stimulation at 37 °C, cells were immediately fixed by adding an equal volume of pre-warmed BD Cytofix Buffer (BD, #554655), permeabilized with BD Phosflow Perm Buffer III (BD, #558050), and then stained with antibodies against intracellular phospho-targets for 30 minutes at room temperature in the dark. Samples were run on a Cytek Aurora cytometer and analyzed using Flowjo v10.

[0285] High-dimensional visualization of flow cytometry data. For high-dimensional visualization of intracellular T cell cytokine production, samples were indexed based on T cell variant, concatenated, and visualized using PaCMAP dimensionality reduction using all default parameters35. Dimensionality reduction was performed based on four intracellular proteins: IFNɣ, IL2, TNF⍺, GrzB. All analysis was and visualizations were performed in FlowJo V10.8.1 with the PaCMAP plugin.

[0286] Design of base editor libraries. For the ClinVar library, the KEGG gene set “T CELL RECEPTOR SIGNALING PATHWAY” (hsa04660) was used to generate a starting list of 108 target genes. Genes encoding secreted proteins (interleukins, IFNG) were excluded, as the impact of their perturbation on the cell of origin is difficult to determine. Using the final list of 102 genes (Table 1), Applicants used the Base Editor Design Tool (github.com / mhegde / base-editor-design-tool) to design a library tiling each gene. The experimental perturbation library was filtered to include only sgRNAs introducing missense mutations in amino acids with known variants classified as “variant of unknown significance,” “likely pathogenic,” or “pathogenic.” To broaden the mutation library, Applicants did not filter sgRNAs making non-identical missense mutations at these loci (e.g. an sgRNA that makes a Y100H missense mutation at Tyr100, when the known ClinVar variant is Y100C, would not be filtered). sgRNAs containing a “TTTT” sequence or BsmBI cut site were also excluded. The negative control library consisted of 300 randomly-selected guides predicted to make only silent mutations in these genes, and 300 guides with no target base in the 5-nucleotide editing window (“empty window”). 200 additional control guides were selected which tiled the PPP1R12C gene, which is not expected to play a role in T cell function. For positive controlsAttorney Docket 44010.185WO-PCT / / CU24092 (i.e. expected dropouts) 50 genes classified as pan-species essential by the Bayesian Analysis of Gene Essentiality 2 (BAGEL2)36were selected as targets. Applicants generated a library of 600 sgRNAs predicted to perturb these genes: 300 sgRNAs introducing splice site mutations, including both splice acceptor and splice donor sites, and 300 sgRNAs introducing proline mutations (i.e. mutation of any non-proline amino acid to proline), which are expected to disrupt protein structure.

[0287] For the 12-Gene tiling library, target genes were selected based on their central roles in orchestrating T cell responses to extracellular cues. Each gene was tiled with sgRNAs using the Base Editor Design Tool as above. Filtration was performed to remove sgRNAs containing a BsmBI cut site. For negative controls, 300 guides introducing silent mutations and 300 empty-window guides targeting these genes were included, in addition to 200 PPP1R12C tiling guides, as described above. For positive controls, the identical library of 600 sgRNAs was used as described in the ClinVar library.

[0288] Visualization of targeted geneset for ClinVar base editor library. To visualize known functional protein interactions and groups of targets included in the ClinVar library the gene list was uploaded to the STRING database (https: / / string-db.org / ) and interactions were visualized using the full STRING network including confident interactions based on textmining, experiments, databases, co-expresison, neighborhood, gene fusion and co- occurrence using the highest confidence setting (0.9)37. The protein interactions were then clustered using STRING and k-means clustering with the number of clusters set to 8. The resulting clusters were annotated based on biological function.

[0289] Base editor library cloning. Custom oligonucleotide pools (TWIST Bioscience) encoding the individual sgRNA sequences and flanking regions for targeted subpool amplification and cloning were used for pooled golden-gate cloning of sgRNA gene editing libraries into the CropSeq vector as previously described38. The following sequence was used for all oligonucleotide pools: 5′-[Forward Primer] CGTCTCACACCG (SEQ ID NO: 48) [sgRNA, 20 nt] GTTTCGAGACG (SEQ ID NO: 49) [Reverse Primer]-3’. The individual library pools were amplified using subpool specific primers and KAPA HiFi HotStart ReadyMix (Roche, #KK2601). After PCR, the reactions were cleaned using the QIAquick PCR purification kit (Qiagen, #28104) according to manufacturer instruction and elute in 50 μL TE buffer (Invitrogen, #12090015). To prepare the CropSeq-mTurq backbone for pooled golden- gate cloning, 5 µg of sequencing verified plasmid were restriction digested using BsmBI V2Attorney Docket 44010.185WO-PCT / / CU24092 (NEB, #R0739S) in a 50 µl reaction with NEB3.1 buffer (NEB, #B7203S) for 2 hours at 55 °C. After digestion the linearized backbone was dephosphorylated by addition of 2 µl rSAP (NEB, #M0371S) and incubation at 37 °C for 1 hour followed by heat inactivation of the enzymes at 80 °C for 20 minutes. The reaction was then loaded on a 1% agarose gel confirming the release of a ~1.8 kb fragment. The linearized backbone was extracted from the gel using the Zymogen Gel DNA recovery Kit (#D4001) followed by additional clean up using 1x SPRI bead selection (Beckman Coulter, #B23318) and eluted in TE buffer. Next, the linearized backbone and amplified oligonucleotide pool were mixed at equimolar ratio and assembled using a 50 µl golden-gate reaction containing 1x Tango Buffer (Thermo Fisher, #BY5) , 1 mM DTT (Thermo Fisher, #R0861), 1 mM ATP (NEB, # P0756S), 1 µl Esp3l (Thermo Scientific, #ER0451), T7 ligase (NEB, #M0318S) and nuclease free water. The reaction was conducted in a thermocycler using incubation at 37 °C for 5 minutes followed by 20for 5 minutes for a total of 99 cycles (final holding stage at 4 °C). After the reaction had been completed the final product was cleaned using 1x SPRI beads and eluted for 1 minute at room temperature in 11 µl TE. Next, 10 µl of the reaction were electroporated in 50 µl Lucigen Endura electro competent cells (Lucigen, #602421) using 1 mm electroporation cuvettes (Biorad, #1652089) and a BioRad Gene Pulser Xcell (Biorad, #1652660) set to 10 µF, 600 Ohms, 1.8 kV. Immediately after electroporation 1 ml of prewarmed recovery medium (Lucigen, #800261 was added to the bacteria and the cells were incubated for 1 hour at 37 °C shaking at 250 rpm in a bacterial incubator. ...

Claims

Attorney Docket 44010.185WO-PCT / / CU24092 CLAIMS What is claimed:

1. An isolated CD8+ T cell genetically modified in vitro to comprise one or more gain- of-function (GOF) or loss-of-function (LOF) mutations in one or more genes selected from the group consisting of PIK3CD, PIK3R1, AKT1, RHOA, LCK, RHOA, CTLA4, ITK, ZAP70, NFKB1, CD40LG, and LAT, wherein the one or more GOF mutations are in PIK3CD, PIK3R1, AKT1, LCK, CTLA4, ITK, NFKB1, ZAP70, or CD40LG; or wherein the one or more LOF mutations are in PIK3CD, RHOA, ZAP70, or LAT.

2. The isolated CD8+ T cell of claim 1, wherein the one or more GOF mutations in PIK3CD is a mutation generating a conversion in residue Cys416 to a positive charged amino acid; or wherein the one or more GOF mutations in PIK3CD is a mutation generating a conversion in residues Tyr524, Glu525, Glu527, or Asp529.

3. The isolated CD8+ T cell of claim 1, wherein the one or more GOF mutations is selected from the group consisting of PIK3CD Cys416Arg, PIK3CD Glu525Gly_His526Arg, PIK3CD Glu525Gly, PIK3CD Ser444Pro, PIK3CD Tyr524His, PIK3CD Tyr524Cys, PIK3CD Glu527Gly_Lys528Glu, PIK3CD Asp529Gly, PIK3CD Tyr524His_Leu523Pro, PIK3CD His526Arg_Glu527Gly, PIK3CD Glu527Gly_Lys528Glu, PIK3R1 Leu570Pro, AKT1 Trp80Arg, AKT1 Ile165Val, AKT1 Glu322Gly, AKT1 Asp323Gly, LCK Tyr505Cys_Gln506Arg, LCK Tyr505His, LCK Tyr505Cys, LCK Gln506Arg, CTLA4 Leu47Pro, CTLA4 Val46Ala, CTLA4 Ile102Thr CTLA4 Asp153Gly, ITK Tyr588Cys, ITK Tyr578His, ZAP70 Ile203Val, NFKB1 Glu439Gly, NFKB1 Asn846Asp, NFKB1 Glu63Gly, CD40LG Gln220Arg, and CD40LG Tyr172His; and wherein the one or more LOF mutations is selected from the group consisting of PIK3CD Ser281Pro, PIK3CD Tyr440His_Leu439Pro, PIK3CD Ser318Pro, PIK3CD Ile899Val_Met900Val, PIK3CD Lys922Gly, RHOA Tyr42His_Val43Ala, RHOA Tyr42Cys, RHOA Tyr66His, RHOA Arg70Gly, RHOA Asp65Asp, RHOA Val43Ala, ZAP70 Cys39Arg, ZAP70 Met558Val, ZAP70 Val438Ala, ZAP70 Val589Ala, ZAP70 Asp574Gly, ZAP70 Trp576Arg, ZAP70 Cys575Arg, LAT Leu19Pro, LAT Ile18Thr, LAT Asp125Gly, and LAT Asp126Gly.Attorney Docket 44010.185WO-PCT / / CU24092 4. The isolated CD8+ T cell of any of claims 1 to 3, wherein the isolated CD8+ T cell was obtained from a subject and genetically modified in vitro, whereby the genetically modified CD8+ T cell is autologous to the subject.

5. The isolated CD8+ T cell of any of claims 1 to 4, wherein the isolated CD8+ T cell is derived from a subject having a tumor; or wherein the isolated CD8+ T cell is derived from a subject having an autoimmune disease; or wherein the isolated CD8+ T cell is tumor specific; or wherein the isolated CD8+ T cell is specific to an autoantigen; or wherein the isolated CD8+ T cell expresses a chimeric antigen receptor (CAR); or wherein the isolated CD8+ T cell expresses an exogenous T cell receptor (TCR).

6. The isolated CD8+ T cell of claim 5, wherein the CAR or TCR is specific to a tumor antigen or an autoantigen.

7. A population of CD8+ T cells comprising a plurality of CD8+ T cells expanded from the isolated CD8+ T cell of any of claims 1 to 6.

8. A method of treating cancer in a subject in need thereof comprising administering the population of CD8+ T cells of claim 7 having a GOF mutation to the subject.

9. The method of claim 8, wherein the CD8+ T cells are autologous to the subject; and / or wherein the subject has a loss of function of CD58 expression or activity or wherein the subject is resistant to checkpoint blockade therapy.

10. A method of treating an autoimmune disease in a subject in need thereof comprising administering the population of CD8+ T cells of claim 8 having a LOF mutation to the subject.

11. The method of claim 10, wherein the CD8+ T cells are autologous to the subject.

12. A method of pooled screening of primary CD8+ T cells for GOF or LOF mutations that modulate T cell function comprising: a) delivering to a population of primary CD8+ T cells a library of guide RNAs targeting one or more genes selected from Table 1;Attorney Docket 44010.185WO-PCT / / CU24092 b) expressing an mRNA encoding a guide RNA directed base editor in the CD8+ T cells having a guide RNA, wherein the library of guide RNAs target the base editor to generate a plurality of variants in the one or more genes selected from Table 1; and c) determining by sequencing, guide RNAs in cells having an altered T cell function, whereby the guide RNAs identify the GOF or LOF mutations.

13. The method of claim 12, wherein the guide RNAs are delivered by using a lentiviral vector encoding each guide RNA; and / or wherein the mRNA encoding a guide RNA directed base editor is delivered by electroporation.

14. The method of claim 12 or 13, wherein the primary CD8+ T cells are human primary CD8+ T cells.

15. The method of claim 12 or 13, wherein the primary CD8+ T cells are obtained from a mouse capable of expressing mRNA encoding a guide RNA directed base editor.

16. The method of any of claims 12 to 15, wherein primary CD8+ T cells receiving a guide RNA are selected followed by delivering the mRNA encoding a guide RNA directed base editor to the selected cells.

17. The method of any of claims 12 to 16, further comprising sorting cells of interest based on altered T cell function and identifying enriched and / or depleted guide RNAs in the sorted cells.

18. The method of any of claims 12 to 16, wherein single cell RNA-seq is used to identify both the guide sequence in each single cell and gene expression in each single cell, whereby altered T cell function in single cells can be associated to a guide sequence.

19. The method of any of claims 12 to 18, wherein the T cell function is selected from the group consisting of activation, short-term proliferation, long-term proliferation, and cytokine production.

20. The method of any of claims 12 to 19, further comprising assaying CD8+ T cells comprising the GOF or LOF mutations in tumor T cell killing assays.

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