WO2024064824A2 - Compositions et procédés d'identification de cibles membranaires pour l'amélioration d'une thérapie par cellules nk - Google Patents

Compositions et procédés d'identification de cibles membranaires pour l'amélioration d'une thérapie par cellules nk Download PDF

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WO2024064824A2
WO2024064824A2 PCT/US2023/074788 US2023074788W WO2024064824A2 WO 2024064824 A2 WO2024064824 A2 WO 2024064824A2 US 2023074788 W US2023074788 W US 2023074788W WO 2024064824 A2 WO2024064824 A2 WO 2024064824A2
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cells
cell
genetically modified
tumor
cancer
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Sidi CHEN
Lei PENG
Paul RENAUER
Ryan CHOW
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Yale University
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Definitions

  • the invention is generally related to the field of screening technology for identifying targets for enhancing natural killer (NK) cell activity, and more particularly to compositions and methods for genetic engineering in live NK cells to enhance chimeric antigen receptor (CAR)-NK cell therapy.
  • NK natural killer
  • CAR chimeric antigen receptor
  • Adoptive immunotherapy in which immune cells that are specific for tumor- associated antigens are expanded to generate large numbers of cells and transferred into tumor-bearing hosts, is a promising strategy to treat cancer.
  • Cellular immunotherapy involves the administration of “living drugs”: genetically modified immune cells that can proliferate, adapt to their environment, engage surrounding cells, and elicit dynamic responses that directly or indirectly target tumor cells for destruction (Hayes, Ir J Med Sci 190, 41-57, doi:10.1007/s11845-020-02264-w (2021)).
  • Adoptive cell transfer is one type of cellular immunotherapy which involves the transfer of cells that directly target tumor cells in the patient (Laskowski & Rezvani, J Exp Med 217, doi:10.1084/jem.20200377 (2020)).
  • Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, multiple current limitations exist that naturally hinder the successful implementation of NK cell therapy against cancer, including their effector function, persistence, and tumor infiltration.
  • regulatory NK populations can influence the functions of dendritic cells (DCs), monocytes, T cells, and B cells via cytokine production or through direct cell-cell contact in a receptor-ligand interaction-dependent manner
  • DCs dendritic cells
  • monocytes monocytes
  • T cells T cells
  • B cells B cells via cytokine production or through direct cell-cell contact in a receptor-ligand interaction-dependent manner
  • a receptor-ligand interaction-dependent manner Shiasaki, et al., Nat Rev Drug Discov 19, 200-218 (2020); Peterson, et al., Frontiers in Immunology 11 (2021); Fernandez, et al., Nature Medicine 5, 405-411 (1999); Knorr, et al., Front Physiol 5, 295 (2014); Kloss, et al., J Immunol 181, 6711-6719 (2008); Laouar, et al., Nat Immunol 6, 600-607 (2005); Krebs, et al., Blood 113,
  • CAR-NK cells can use an allogeneic NK source without concern of graft-verse-host-disease (GVHD) and recognize tumor cells through cell native NK receptors, allowing for CAR- independent cancer elimination in tumors with antigen-loss (Elahi & Esmaeilzadeh, Stem Cell Rev Rep 17, 2081-2106 (2021); Marofi, et al., Frontiers in Oncology 11 (2021); and Marofi, et al., Stem Cell Research & Therapy 12, 200 (2021)).
  • GVHD graft-verse-host-disease
  • CAR-NK therapy has shown positive clinical trial outcomes against hematological malignancies and signs of promising potential for use in solid tumors (Albinger, et al., Gene Therapy 28, 513-527 (2021); Oncology Times 42, 35 (2020); Wrona, & Potemski, Int J Mol Sci 22 (2021); and Portillo, et al. iScience 24, 102619 (2021).
  • NK cell-based immunotherapy candidates face a number of obstacles, for example, the paucity, lower proliferative capacity, and particularly decreased effectiveness, persistence or tumor infiltration (Cózar, et al., Cancer Discov 11, 34-44 (2021); and Ge, et al., Immunopharmacol Immunotoxicol 42, 187-198 (2020)).
  • Various methods have been utilized to improve anti-tumor efficacy of NK cells, including ex vivo activation, expansion, and genetic modifications (Chu, J. et al. Journal of Translational Medicine 20, 240 (2022)).
  • NK cells encode the same collection of ⁇ 20,000 protein coding genes in their genome, many of which might play critical roles in regulating or limiting the anti-tumor function of NK cells.
  • perturbation mapping technologies of the NK genome to guide the identification of new genes that can be targeted to enhance NK function, such as activation, proliferation, repression of inhibitory signals or exhaustion, persistence, or tumor infiltration.
  • compositions and methods for highly efficient screening of genetically engineered NK cells are provided.
  • the disclosed compositions and methods are especially applicable to development of enhanced chimeric antigen receptor engineered NK cell therapy (CAR-NK).
  • Genetically modified Natural Killer (NK) cells are provided.
  • the modified Natural Killer (NK) cells include at least one modified gene targeted by one or more of the sgRNA including a nucleotide sequence selected from the group including SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (“mSurfeome2”).
  • the modified Natural Killer (NK) cells are modified by mutation of at least one gene selected from the group including tga1/Cd49a, Itga2/Cd49b, Itga3/Cd49c, Spn/Cd43Klrk1/NKG2D, CD27, Tigit, Pdcd1/PD-1, Lag3 Vnn3, Ccr2, Slc2a8, Prnp, Cd59b, Ceacam14, and Calhm2 .
  • the genetically modified NK cell includes a mutation that causes lack or reduction of the expression of one or more of the genes and/or the protein(s) (e.g., full-length protein(s)) encoded by the gene(s).
  • the modified NK cell includes a mutation that causes lack or reduction of the expression of one or more of the genes Vnn3, Ccr2, Slc2a8, Prnp, Cd59b, Ceacam14, and Calhm2, and/or the protein(s) encoded by the foregoing genes in the cell.
  • the mutation enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified NK cell.
  • the mutation causes lack or reduction of the expression of Calhm2, and/or protein encoded by Calhm2.
  • NK cells lacking expression of one or more of the genes Vnn3, Ccr2, Slc2a8, Prnp, Cd59b, Ceacam14, and Calhm2, and at least one additional gene are also provided.
  • the modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR).
  • the modified NK cell expresses or encodes a CAR that targets a cancer antigen.
  • Exemplary cancer antigen are selected from the group including a neoantigen derived from the subject, 41BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD 152, CD19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, Ll CAM, IL 13,
  • the NK cell is derived from a subject diagnosed as having, or who is identified as being at increased risk of having a disease or disorder.
  • the subject is diagnosed as having, or is identified as being at increased risk of having cancer.
  • the NK is derived from a healthy subject.
  • Populations of NK cells derived by expanding the genetically modified NK cells are also described.
  • Pharmaceutical composition including the populations of NK cells and a pharmaceutically acceptable buffer, carrier, diluent or excipient for administration in vivo are also provided. Methods of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of the pharmaceutical composition including the populations of NK cells have also been developed.
  • Methods of treating a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen are also provided.
  • the methods include administering to the subject an effective amount of a population of genetically modified NK cells, wherein the NK cells include a CAR that targets the antigen.
  • Methods of treating cancer in a subject in need thereof include administering to the subject an effective amount of a population of genetically modified NK cells, wherein at least one gene selected from the group including Vnn3, Ccr2, Slc2a8, Prnp, Cd59b, Ceacam14, and Calhm2 has been mutated in the NK cell.
  • the NK cell has been mutated to reduce or knock out the expression of Calhm2, and/or protein encoded by Calhm2.
  • the NK cell expresses or encodes a Chimeric Antigen Receptor (CAR).
  • CAR targets an antigen expressed by the cancer.
  • the methods can be used to treat cancers selected from the group including leukemia, vascular cancer such as multiple myeloma, adenocarcinomas and bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine cancer.
  • the methods treat triple negative breast cancer or glioblastoma.
  • the NK cells are derived from the subject prior to genetic modification.
  • the administration includes injection of the composition of cells into or directly adjacent to a tumor, or into the blood stream, or into the brain or into a ventricle of the heart of the subject.
  • the methods can further include administering to the subject one or more additional therapeutic agents and/or procedures.
  • the additional treatment is selected from the group including a chemotherapeutic agent, and antimicrobial agent, an immune checkpoint inhibitor, a PD-I inhibitor, a CTLA-4 inhibitor, radiation treatment and surgery.
  • a non-naturally occurring or engineered nucleic acid library including a plurality of nucleic acids including at least one nucleotide sequence selected from the group including SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2) is also provided.
  • the library includes a plurality of sgRNA nucleic acids targeting membrane-bound molecules of Natural Killer (NK) cells, wherein each of the sgRNAs include (i) a guide sequence; and (ii) a tracrRNA including a nucleic acid sequence selected from SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • NK Natural Killer
  • a vector library including the sgRNA library has also been developed.
  • the vector library includes a plurality of vectors, where each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from the group including SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the vector is an Adeno-associated virus vector (AAV).
  • AAV Adeno-associated virus vector
  • the sgRNA(s) are encapsulated within the vectors.
  • CRISPR- based editing vector libraries for CRISPR-mediated editing of NK cells have also been developed.
  • the CRISPR-based editing vector libraries include a plurality of vectors that each include a first ITR, a second ITR, an antibiotic resistance sequence, two sleeping beauty (SB) IR/DR repeats, a first promoter, a second promoter, a Thy 1.1 selection marker, an SB I 00x transposase, and a poly A sequence in addition to an sgRNA including a nucleotide sequence selected from the group including SEQ ID NOs: 1- 69,747 of the sequence listing of WO 2020/028533 (mSurfeome2). Methods of performing genome perturbation screening of a Natural Killer (NK) cell are also provided.
  • NK Natural Killer
  • the methods include the steps of (i) contacting an NK cell with Cas9 and an AAV library, wherein the AAV library includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from the group including SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2); (ii) causing the NK cell to be genetically modified by CRISPR-mediated genome editing of a gene targeted by the sgRNA including any one of SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2); and (iii) screening the NK cell for a mutation.
  • the AAV library includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from the group including SEQ ID NOs: 1-69,747 of the sequence listing of WO 2020/028533 (m
  • the screening is carried out in vitro, or in vivo.
  • the in vivo screening is carried out using a tumor-bearing animal model, wherein the screening includes selecting genetically modified NK cells from animals with enhanced survival/reduced tumor burden as compared to control animals that did not receive the same genetically modified NK cells.
  • the methods further include characterizing the mutant NK cells by single cell transcriptome analysis and/or by sequence analysis to identify mutated genes.
  • the methods include repeating the screening steps using a selected pool of sgRNAs, for one or more additional rounds. Genetically modified NK cells created according to the methods are also described.
  • FIG. 1A is a schematic representation of the AAV-SB barcoded vector maps and the detailed barcode tracking experiment design.
  • Fig.1B is a bar plot of the barcode library coverage for all samples. Barcode detection is represented by bar height.
  • Fig.1C is a box-whisker plot of barcode detection rates, displayed with the percent of all possible barcode permutations that were detected for each barcode length.
  • Figures 2A-2E show functional genetic screens in four in vivo tumor models identifies candidate genes that enhance NK cell tumor infiltration.
  • Fig.2A is a schematic representation of the described system for in vivo AAV-SB-Surf-v2 CRISPR KO screens for NK cell tumor infiltration performed in four independent syngeneic tumor models, B16F10, E0771, GL261 and Pan02 in C57BL/6J mice.
  • Figs.2B-2E are volcano plot graphs of the CRISPR-KO screen results for in vivo NK tumor-infiltration, showing Adj. p value (-log10) over gene score for each of the indicated genes in each of the four tumor models, B16F10 ( Figure 2B); E0771 (Figure 2C); GL261 ( Figure 2D); and Pan02 ( Figure 2E), respectively.
  • Figures 3A-3F are bar graphs of cancer cell killing capability of mouse primary NK cells with top hit genes knocked out showing % lysis for knockouts (KO) over time (hr) for each of the genes Ccr2, Vnn3, Slc2a8, and Prnp in B16F10-PL cancer cells (Figure 3A) and E0yy1-mCh-OVA-GL cancer cells ( Figure 3B); Ceacam14 and Cd59b in B16F10-PL cancer cells ( Figure 3C) and E0yy1-mCh-OVA-GL cancer cells ( Figure 3D); and Vector Control and Calhm2 in B16F10-PL cancer cells ( Figure 3E) and E0yy1-mCh-OVA-GL cancer cells ( Figure 3F), respectively.
  • Figures 4A-4D are circular bar plots of meta-pathway analysis results for enriched genes of each in vivo NK CRISPR screen...Meta-pathways are shown for relevant immune-related categories (bar), and pathway significance is represented by bar height in each of B16F10-PL cancer cells (Figure 4A); E0771 cancer cells (Figure 4B); GL261 cancer cells ( Figure 4C); and Pan02 cancer cells (Figure 4D), respectively.
  • Figure 5 is a Venn diagram of the overlap between screen result genes and select pathways including Regulation of Cell adhesion; Leukocyte proliferation; Positive regulation of apoptotic processes and Screen hits.
  • Figures 6A-6C are heatmaps of the CRISPR-KO screen enrichment (z-score) in genes of tumor infiltration-related pathways including Regulation of Cell adhesion (Figure 6A); Leukocyte proliferation (Figure 6B); and Positive regulation of apoptotic processes (Figure 6C).
  • z-score CRISPR-KO screen enrichment
  • FIG. 7 is a schematic representation of the described system for the single-cell transcriptomic exploration of NK cells within the tumor and spleen in two different in vivo cancer models, B16F10, and E0771, in C57BL/6J mice, across multiple time points (Day -7 to 15 relative to i.v. injection of NK cells).
  • Figures 8A-8I are violin plots showing expression data.
  • Figs 8A-8C are violin plots of the expression of select NK phenotype genes Itgam, CD27 and Cd3e (Fig.8A); Gzmb and Ifng (Fig.8B); and Lag3*, Ctla4* and Tox (Fig.8C), compared across different NK subset populations, with the distribution of log-scaled expression data shown for each NK subset, and a dashed line represents a log-scale expression threshold of 1.
  • Figs 8D-8E are violin plots of the expression of select immune marker genes, compared across different cell subset populations. The distribution of log-scaled expression data is shown for each cell subset, and a dashed line represents a log-scale expression threshold of 2.
  • Figs 8F-8I are violin plots of the expression of select immune marker genes, grouped as Activating receptors (Fig.8F); Inhibitory receptors or Miscellaneous (Fig.8G); Chemokine receptors (Fig.8H), and Adhesion Receptors (Fig. 8I), respectively, compared across different cell subset populations.
  • the distribution of log-scaled expression data is shown for each cell subset, and a dashed line represents a log-scale expression threshold of 2.
  • Genes with low detection rates have been imputed using the ALRA R package with default settings (Linderman, et al., Nat Commun 13, 192 (2022)), and imputed genes are depicted with an asterisk.
  • Figures 9A-9C are bar graphs of NK subset percentages, compared across each of tumor-model (Control, B16F10, and E0771; Fig.9A); time (0 days post infection (dpi), 7 dpi and 15 dpi, respectively; Fig.9B); and the tissue type (Culture, Spleen, and Tumor; Fig.9C).
  • FIG. 10 is a Uniform Manifold Approximation and Projection (UMAP) graph which visually shows how separable the different NK subset populations (Cell types) are with respect to the selected group of features.
  • UMAP Uniform Manifold Approximation and Projection
  • Figure 11 is a Volcano plot of differential expression (DE) analyses for tumor infiltration of NK single-cell data showing Adj. p value (-log10) over Log-FC for each of the indicated genes.
  • DE analyses were performed using single-cell expression data fitted to Gamma-Poisson generalized linear models (shown above plots), and quasi-likelihood F tests that assessed the effect of “tumor-infiltration” as a coefficient. Upregulated and downregulated genes are shown by respective dots (q ⁇ 0.01, absolute log-FC > 2), and the top 5 gene names are presented for each.
  • Figure 12 is a circular display of bar plots of meta-pathway analysis results for DE genes of the tumor infiltration analysis of NK single-cell expression data (top and bottom panels, respectively).
  • Meta-pathways are shown for relevant immune-related categories, and pathway significance is represented by bar height. Meta-pathways from the analyses of upregulated and downregulated DE genes are indicated by shaded bars, respectively.
  • Figures 13A-13N show the results of single-cell DE analyses of tumor- infiltration revealed NK subset-specific pathway signatures.
  • Fig.13A is a heatmap of select meta-pathways from the enrichment analysis of NK tumor infiltration DE genes ranked according to Tissue (Spleen, Tumor or Culture, respectively) and by Pathway (Positive regulation of cell death; Chemotaxis; Leukocyte Differentiation; and Regulation of Cell adhesion proliferation), respectively. For each DE gene, population- averaged scaled-normalized gene expression is presented.
  • Fig.13B is a Venn diagram showing identification of Calhm2 as a convergent target from in vivo screen, single cell RNAseq, and numbers of pathways of interest indicated in each segment, accordingly.
  • Fig.13C is a micrograph of a Western blot gel loaded with Calhm2 knock out (KO) and Vector control (Ctrl), showing the presence of gel bands corresponding to Calhm2 and Vinculin, with relative intensity (Rel. intensity 0.29, 1.0) indicated above.
  • Fig.13E is a Schematic of in vivo tumor infiltration assay of Calhm2 KO mouse NK cells, with protocol events and time course in days (D0-D13) indicated.
  • Figs.13F-13H are bar graphs showing tumor volume measured at the time point of NK cells injection (Fig.13F), weight of spleen (Fig.13G) and weight of tumor (Fig.13H) in tumor-burden mice observed in each of Calhm2 KO and Vector Ctrl groups, respectively.
  • Figs.13I-13J are bar graphs showing quantification (%) of NK cells (NKp46+, CD3-), in each of spleen (Fig.13I) and tumor of mice treated with Vector Ctrl NK cells and Calhm2 KO NK cells (Fig.13J), respectively.
  • Figures 14A-14B are Volcano plots of differential expression (DE) analyses for tumor infiltration (TI) of immature (iNK; Fig.14A) and mature NK (mNK; Fig.14B) single-cell data, respectively showing Adj. p value (-log10) over Log-FC for each of the indicated genes.
  • DE differential expression
  • FIG. 15A-15B show differential expression analyses of tumor infiltration in transitional NK and the immature NK 4 subset single-cell data.
  • Figs 15A-15B Circular bar plots of meta-pathway analysis results for DE genes related to TI of immature (iNK; Fig.15A) and mature NK (mNK; Fig.15B) single-cell data in iNK and mNK single-cell expression data, respectively.
  • Figs 15C-15D are Volcano plots of differential expression (DE) analyses for tumor infiltration (TI) of transitional (tNK; Fig.15C) and immature NK (iNK; Fig. 15D) single-cell data, respectively, showing Adj. p value (-log10) over Log-FC for each of the indicated genes.
  • DE differential expression
  • Figs 15E-15F are circular bar plots of meta-pathway analysis results for DE genes related to TI of transitional (TI-tNK; Fig.15E) and immature NK (iNK; Fig.15F) single-cell expression data, respectively.
  • Figures 16A-16E are data showing the effects of CALHM2-knockout in CAR- NK92 cells on anti-tumor efficacy in vitro and in vivo.
  • Fig.16A is an image of a gel showing Gene editing of CALHM2 in NK92 cells, as measured by T7E1 assay with bands visible in each of AAVS1 and CALHM2 lanes.
  • Fig.16B is a micrograph of a Western blot gel loaded with Calhm2 knock out (KO) and Vector control (Ctrl), showing the presence of gel bands corresponding to Calhm2 and Vinculin, with relative intensity (Rel. intensity 0.29, 1.0) indicated above.
  • Fig.16C is a bar graph of CALHM2 protein level showing fold change in each of CALHM2-KO and AAVS1-KO.
  • Figures 16D-16E are bar graphs showing % of lysis over time (hr) for each of the AAVS1 Knock Out (KO) and CALHM2-KO in each of MDA-MB-231 cells (Fig.16D) and NAML6-GL- CD22S cells (Fig.16E), respectively.
  • Figures 17A-17B are schematic representations of the genetic constructs for ⁇ - BCMA ( Figure 17A) and ⁇ -HER2 CAR ( Figure 17B), respectively.
  • Figures 18A-18F are flow cytometry plots of the percentages of AAVS1-KO and CALHM2-KO NK92 cells with (Figs.18B-18C) and ⁇ -HER2 CAR (Figs.18E-18F) expression, respectively compared to wildtype (Figs.18A; 18D)
  • Figures 19A-19E are bar graphs showing % of lysis over time (hr) for each of the NK92 WT cells, AAVS1 Knock Out (KO) cells, and CALHM2-KO cells in each of MM.1R-PL-BCMA-OE (0.5:1 E:T; 1 Fig.19A; and 0.1:1 E:T; Fig.19B), and MDA- MB-23cells (Fig.19C); MCF-7-PL cells (Fig.19D); and MCF-7-
  • Figures 20A-20I are flow cytometry plots of parental NK cells (Figs.20A-20C), AAVS1-KO ⁇ -HER2-CAR cells (Figs.20D-20F) and CALHM2-KO HER2-CAR-NK92 cells (Figs.20G-20I), showing ⁇ -HER2-CAR degranulation when stimulated, gated for ⁇ -CD107 and ⁇ -CD56 at each of 2 hrs, 4 hrs and 6 hrs, respectively.
  • Figure 21 is a bar graph of in vivo tumor cytotoxicity analysis, showing % CD56+ ⁇ -CD107+ cells over Time post stimulation (2-6 hr) for each of NK92-WT, AAVS1-KO ⁇ -HER2-CAR and CALHM2-KO ⁇ -HER2-CAR cells.
  • Figures 22A-22B show Calhm2 KO enhanced tumor infiltration and persistence capability of ⁇ -HER2 CAR-NK cells.
  • Fig.22A is a schematic representation of the generation of ⁇ -HER2 CAR-NK92-hIL2 cells with CALHM2 and AAVS1 editing and functional validation with in vitro co-culture and in vivo tumor models.
  • Figure 23 is a bar graph of enhanced cytotoxicity of CALHM2-KO ⁇ -HER2- CAR-NK92-hIL2 cells to HT29-GL cells, showing % of Lysis over E:T ratio for each of NK92-WT, AAVS1-KO ⁇ -HER2-CAR-NK92-hl2 and CALHM2-KO ⁇ -HER2-CAR- NK92-hl2 line of cells, respectively.
  • Figures 24A-24F depict the in vivo tumor infiltration assay of CALHM2 KO- ⁇ - HER2 CAR-NK92 cells.
  • Fig.24A is a schematic representation of the in vivo tumor infiltration validation assay in mice treated with CAR-NK92 cells at 19 days-post-HT29 inoculation (FACS on 21 and 28 days, respectively).
  • Figs.24B-24E are bar plots for percent (Figs.22B, 22D) and total (Figs.22C, 22E) tumor CD56+ NK cell in each of AAVS1-KO ⁇ -HER2-CAR-NK92-hIL2, and CALHM2-KO ⁇ -HER2-CAR-NK92-hIL2 groups at days 21 (Figs.22B, 22C) and 28 (Figs.22D, 22E), respectively.
  • n 3-5 tumors per group.
  • Fig.24F is a line graph of the tumor growth curve of mice treated with CALHM2-KO ( ⁇ ) or AAVS1-KO ⁇ -HER2-CAR- NK92-hIL2 ( ⁇ ) cells, as well as a Control (No treatment ( ⁇ ). Showing Tumor volume (mm 3 ) over days post implant (dpi) for each group, respectively.
  • Figure 25 is a Volcano plot of the differential expression (DE) analysis of CALHM2-deficiency in human donor ⁇ -HER2 CAR-NK cells, showing Adj. p value (- log10) over Log-FC for each of the indicated genes.
  • DE differential expression
  • RNA-seq expression data was assessed by quasi-likelihood F tests.
  • Upregulated and downregulated transcripts are shown by respective red and blue dots (q ⁇ 0.05, absolute log-FC > 0.8), and the top significant DE gene names are labeled.
  • Figure 26 is a circular bar plot of meta-pathway analysis results for DE genes of the CALMH2-KO analysis in CAR-NK cell expression data. Meta-pathways are shown for relevant immune-related categories, and pathway significance is represented by bar height.
  • FIGS. 27A-27C are heatmaps of select meta-pathways from the enrichment analysis of CALMH2-KO in CAR-NK cells.
  • the selected meta-pathways were grouped by functional pathways (Pos. regulation of cytokine production, T cell activation, Regulation of cell adhesion, and Chemotaxis for stimulated or unstimulated cells; Fig. 27A) and signaling pathways (Regulation of MAPK cascade; Dephsophorylation and Pos. regulation of protein phosphorylation for stimulated or unstimulated cells; Fig.
  • Natural killer (NK) cells are an innate immune cell type that serves at the first level of defense against pathogens and cancer. NK cells have clinical potential, however, their effector function, persistence, and tumor infiltration naturally hinder the successful implementation of NK cell therapy against cancer.
  • compositions and methods for perturbomics mapping of tumor infiltrating NK cells by joint in vivo AAV-CRISPR screens and single cell sequencing have been established. The methods implement AAV-SleepingBeauty(SB)-CRISPR screening leveraging a custom high-density sgRNA library targeting cell surface genes.
  • CALHM2 a calcium homeostasis modulator emerged from both screen and single cell analyses, as having enhancement in in vitro cytotoxicity, in vivo tumor infiltration, and in vivo anti-tumor efficacy enhancement when perturbed in chimeric antigen receptor (CAR)-NK cells.
  • CAR chimeric antigen receptor
  • transposon or “transposable element” means a nucleic acid sequence, such as a chromosomal segment, that can undergo “transposition”, i.e., to change its position within a genome, especially a segment of DNA encoding one or more genes that can be translocated within a host cell, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size.
  • exemplary transpositions include introduction of one or more components of plasmid DNA into chromosomal DNA in the absence of a complementary sequence in the host DNA.
  • transposase means an enzyme that binds to the end of a transposon and catalyses its movement, e.g., into a genome at a specific point part, by a cut and paste mechanism or a replicative transposition mechanism.
  • “Introduce” in the context of genome modification refers to bringing in to contact.
  • to introduce a gene editing composition to a cell is to provide contact between the cell and the composition.
  • the term encompasses penetration of the contacted composition to the interior of the cell by any suitable means, e.g., via transfection, electroporation, transduction, gene gun, nanoparticle delivery, etc.
  • operably linked refers to functional linkage between a regulatory sequence (e.g., promoter, enhancer, silencer, polyadenylation signal, 5’ or 3’ untranslated region (UTR), splice acceptor, IRES, triple helix, 2A self- cleaving peptides such as F2A, E2A, P2A and T2A) and a heterologous nucleic acid sequence permitting them to function in their intended manner (e.g., resulting in expression of the latter).
  • a regulatory sequence e.g., promoter, enhancer, silencer, polyadenylation signal, 5’ or 3’ untranslated region (UTR), splice acceptor, IRES, triple helix, 2A self- cleaving peptides such as F2A, E2A, P2A and T2A
  • a heterologous nucleic acid sequence permitting them to function in their intended manner (e.g., resulting in expression of the latter).
  • the term encompasses positioning of a regulatory region (sequence), a sequence to be transcribed, and/or a sequence to be translated in a nucleic acid so as to influence transcription or translation of such a sequence.
  • the regulatory sequence can be positioned at any suitable distance from the sequence being regulated (e.g., 1 nucleotide – 10,000 nucleotides).
  • the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site.
  • a promoter typically includes at least a core (basal) promoter.
  • the term “complementary” refers to the degree of anti-parallel alignment between two nucleic acid strands. Complete complementarity requires that each nucleotide be across from its opposite. No complementarity requires that each nucleotide is not across from its opposite. The degree of complementarity determines the stability of the sequences to be together or anneal/hybridize. Furthermore various DNA repair functions as well as regulatory functions are based on base pair complementarity.
  • a DNA or RNA nucleotide sequence as recited refers to a polynucleotide molecule comprising the indicated bases in a 5' to 3' direction, from left to right.
  • CRISPR/Cas or “clustered regularly interspaced short palindromic Repeats” or “CRISPR” refers to DNA loci containing short repetitions of base sequences followed by short segments of spacer DNA from previous exposures to a virus or plasmid.
  • Bacteria and archaea have evolved adaptive immune defenses termed CRISPR/CRISPR associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids.
  • CRISPR/Cas In bacteria, the CRJSPR system provides acquired immunity against invading foreign DNA via RNA-guided DNA cleavage.
  • the “CRISPR/Cas” system or “CRISPR/Cas-mediated gene editing” refers to a CRISPR/Cas system that has been modified for genome editing/engineering. For a type II CRISPR/Cas system, it is typically comprised of a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9).
  • gRNA guide RNA
  • sgRNA short guide RNA
  • sgRNA single guide RNi
  • the sgRNA is a short synthetic RNA composed of a “scaffold” sequence necessary for Cas9-hinding and a user-defined , ⁇ 20 nucleotide “spacer” or “targeting” sequence which defines the genomic target to be modified.
  • the genomic target of Cas9 can be modified by changing the targeting sequence present in the sgRNA.
  • cleavage refers to the breakage of covalent bonds, such as in the backbone of a nucleic acid molecule or the hydrolysis of peptide bonds. Cleavage can be initiated by a variety of methods, including, but not limited to, enzymatic or chemical hydrolysis of a phosphodiester bond.
  • Double-stranded cleavage can occur as a result of two distinct single-stranded cleavage events. DNA cleavage can result in the production of either blunt ends or staggered ends.
  • fusion polypeptides can be used for targeting cleaved double stranded DNA.
  • knockdown refers to a decrease in gene expression of one or more genes.
  • knockout or “KO” refers to the ablation of gene expression of one or more genes.
  • Endogenous refers to any material from or produced inside an organism, cell, tissue or system.
  • Exogenous refers to any material introduced from or produced outside an organism, cell, tissue or system.
  • the term “antigen” as used herein is defined as a molecule capable of being bound by an antibody or T-cell receptor.
  • An antigen can additionally be capable of provoking an immune response. This immune response can involve either antibody production, or the activation of specific immunologically competent cells, or both.
  • any macromolecule including virtually all proteins or peptides, can serve as an antigen.
  • antigens can be derived from recombinant or genomic DNA.
  • any DNA which includes a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the disclosed compositions and methods includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all.
  • an antigen can be generated synthesized or can be derived from a biological sample.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.
  • antigen refers to an antigenic substance that is produced in a tumor cell, which can therefore trigger an immune response in the host.
  • cancer antigens can be useful as markers for identifying a tumor cell, which could be a potential candidate/target during treatment or therapy.
  • TSA tumor specific antigens
  • TAA tumor associated antigens
  • the chimeric antigen receptors are specific for tumor specific antigens. In some forms, the chimeric antigen receptors are specific for tumor associated antigens. In some forms, the chimeric antigen receptors are specific both for one or more tumor specific antigens and one or more tumor associated antigens. “Bi-specific chimeric antigen receptor” refers to a CAR that includes two domains, wherein the first domain is specific for a first ligand/antigen/target, and wherein the second domain is specific for a second ligand/antigen/target. In some forms, the ligand is a B-cell specific protein, a tumor-specific ligand/antigen/target, a tumor associated ligand/antigen/target, or combinations thereof.
  • a bispecific CAR is specific to two different antigens.
  • a multi-specific or multivalent CAR is specific to more than one different antigen, e.g., 2, 3, 4, 5, or more.
  • a multi-specific or multivalent CAR targets and/or binds three or more different antigens.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • both the coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • the non-coding strand used as the template for transcription of a gene or cDNA
  • locus is the specific physical location of a DNA sequence (e.g., of a gene) on a chromosome.
  • locus of interest can not only qualify a nucleic acid sequence that exists in the main body of genetic material (i.e., in a chromosome) of a cell but also a portion of genetic material that can exist independently to said main body of genetic material such as plasmids, episomes, virus, transposons or in organelles such as mitochondria as non-limiting examples. “Isolated” means altered or removed from the natural state.
  • nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • isolated nucleic acid refers to a nucleic acid segment or fragment which has been separated from sequences which flank it in a naturally occurring state, e.g., a DNA fragment which has been removed from the sequences which are normally adjacent to the fragment, i.e., the sequences adjacent to the fragment in a genome in which it naturally occurs.
  • nucleic acids which have been substantially purified from other components which naturally accompany the nucleic acid, e.g., RNA or DNA or proteins, which naturally accompany it in the cell.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (i.e., as a cDNA or a genomic or cDNA fragment produced by PCR or restriction enzyme digestion) independent of other sequences.
  • a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence, complementary DNA (cDNA), linear or circular oligomers or polymers of natural and/or modified monomers or linkages, including deoxyribonucleosides, ribonucleosides, substituted and alpha- anomeric forms thereof, peptide nucleic acids (PNA), locked nucleic acids (LNA), phosphorothioate, methylphosphonate, and the like.
  • cDNA complementary DNA
  • PNA peptide nucleic acids
  • LNA locked nucleic acids
  • phosphorothioate phosphorothioate
  • methylphosphonate and the like.
  • isolated cell is meant to include cells that are within samples that are substantially enriched for the cell of interest and/or in which the cell of interest is partially or substantially purified.
  • transformed,” “transduced,” and “transfected” encompass the introduction of a nucleic acid or other material into a cell by one of a number of techniques known in the art.
  • a “vector” is a composition of matter which includes an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell.
  • vectors include but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses.
  • vector encompasses an autonomously replicating plasmid or a virus.
  • the term is also construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like.
  • viral vectors include, but are not limited to, adenoviral vectors, adeno- associated virus (AAV) vectors, retroviral vectors, and the like.
  • AAV adeno- associated virus
  • Tumor burden refers to the number of cancer cells, the size or mass of a tumor, or the total amount of tumor/cancer in a particular region of a subject. Methods of determining tumor burden for different contexts are known in the art, and the appropriate method can be selected by the skilled person. For example, in some forms tumor burden can be assessed using guidelines provided in the Response Evaluation Criteria in Solid Tumors (RECIST). As used herein, “subject” includes, but is not limited to, animals, plants, parasites and any other organism or entity.
  • the subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, pig, rabbit, dog, sheep, goat, non-human primate, cow, cat, guinea pig or rodent), a fish, a bird or a reptile or an amphibian.
  • the subject can be an invertebrate, more specifically an arthropod (e.g., insects and crustaceans).
  • arthropod e.g., insects and crustaceans.
  • a patient refers to a subject afflicted with a disease or disorder.
  • patient includes human and veterinary subjects.
  • the subject can be any organism in which the disclosed method can be used to genetically modify the organism or cells of the organism.
  • the term “inhibit” or other forms of the word such as “inhibiting” or “inhibition” means to decrease, hinder or restrain a particular characteristic such as an activity, response, condition, disease, or other biological parameter. It is understood that this is typically in relation to some standard or expected value, i.e., it is relative, but that it is not always necessary for the standard or relative value to be referred to. “Inhibits” can also mean to hinder or restrain the synthesis, expression or function of a protein relative to a standard or control. Inhibition can include, but is not limited to, the complete ablation of the activity, response, condition, or disease.
  • “Inhibits” can also include, for example, a 10% reduction in the activity, response, condition, disease, or other biological parameter as compared to the native or control level.
  • the reduction can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 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%, or any amount of reduction
  • “inhibits expression” means hindering, interfering with or restraining the expression and/or activity of the gene/gene product pathway relative to a standard or a control.
  • “Treatment” or “treating” means to administer a composition to a subject or a system with an undesired condition (e.g., cancer).
  • the condition can include one or more symptoms of a disease, pathological state, or disorder.
  • Treatment includes medical management of a subject with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder.
  • this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological state, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological state, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological state, or disorder.
  • treatment while intended to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder, need not actually result in the cure, amelioration, stabilization or prevention.
  • the effects of treatment can be measured or assessed as described herein and as known in the art as is suitable for the disease, pathological condition, or disorder involved. Such measurements and assessments can be made in qualitative and/or quantitative terms.
  • characteristics or features of a disease, pathological condition, or disorder and/or symptoms of a disease, pathological condition, or disorder can be reduced to any effect or to any amount.
  • “Prevention” or “preventing” means to administer a composition to a subject or a system at risk for an undesired condition (e.g., cancer).
  • the condition can include one or more symptoms of a disease, pathological state, or disorder.
  • the condition can also be a predisposition to the disease, pathological state, or disorder.
  • the effect of the administration of the composition to the subject can be the cessation of a particular symptom of a condition, a reduction or prevention of the symptoms of a condition, a reduction in the severity of the condition, the complete ablation of the condition, a stabilization or delay of the development or progression of a particular event or characteristic, or reduction of the chances that a particular event or characteristic will occur.
  • the terms “effective amount” or “therapeutically effective amount” means a quantity sufficient to alleviate or ameliorate one or more symptoms of a disorder, disease, or condition being treated, or to otherwise provide a desired pharmacologic and/or physiological effect. Such amelioration only requires a reduction or alteration, not necessarily elimination. The precise quantity will vary according to a variety of factors such as subject-dependent variables (e.g., age, immune system health, weight, etc.), the disease or disorder being treated, as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.
  • subject-dependent variables e.g., age, immune system health, weight, etc.
  • the disease or disorder being treated as well as the route of administration, and the pharmacokinetics and pharmacodynamics of the agent being administered.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject along with the selected compound without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • variant or “active variant” refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties (e.g., functional or biological activity).
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Modifications and changes can be made in the structure of the polypeptides of the disclosure and still obtain a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution).
  • amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity. Because it is the interactive capacity and nature of a polypeptide that defines that polypeptide’s biological or functional activity, certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties (e.g., functional or biological activity).
  • Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. Use of the term “about” is intended to describe values either above or below the stated value in a range of approx.
  • compositions Compositions for the analysis and development of genetically modified NK cells that have enhanced anti-tumor activity are described. Libraries of sgRNAs targeting a range of genes associated with the membrane of Natural Killer (NK) cells have been developed.
  • the libraries include multiple sgRNAs targeting genes within NK cells. Genetically modified NK cells that have enhanced anti-tumor activity are also described.
  • A. Single Guide RNA (sgRNA) Libraries Single guide RNA (sgRNA, gRNA) libraries including a plurality of nucleic acids are provided.
  • a library has a size of between about 30,000 and about 100,000 different sgRNAs represented.
  • a library has a size of between about 40,000 and about 90,000 different sgRNAs represented.
  • a library has a size of between about 40,000 and about 80,000 different sgRNAs represented.
  • a library has a size of between about 50,000 and about 70,000 different sgRNAs represented.
  • a library has a size of between about 55,000 and about 70,000 different sgRNAs represented. In some forms, a library has a size of about 65,536 distinct sgRNAs, or about 69,747 distinct sgRNAs. As demonstrated in the Examples, a library having 65,536 distinct sgRNAs was identified as the optimal size for screening, having both high recovery rate and enabling coverage of a meaningful gene set, given that guide RNA redundancy needs to be factored in a CRISPR library. Although a library including between about 55,000 and about 70,000 different sgRNAs has a certain inevitable library representation loss, a library of this size can still consistently be used to recover a substantial fraction of the library without selection pressure.
  • a library having between about 55,000 and about 70,000 different sgRNAs can identify meaningful hits with strong selection and genetic perturbation phenotypes, despite partial library loss, even though the screen is not saturated, based on library representation. Therefore, in some forms, a high-density CRISPR library having between about 55,000 and about 70,000 different sgRNAs includes extensive sgRNA redundancy (i.e., >10 sgRNA / gene) to target collections of genes belonging to certain classes or annotated pathways, in a relatively unbiased manner. Exemplary molecular pathways that are targeted include all surface proteins, all kinases / phosphatases, all transcription factors, all KEGG enzymes, and combinations of two or more of these.
  • the library includes one or more sgRNAs including a nucleotide sequence selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (also referred to herein as “mSurfeome2” “mSurfeome2” and referred to herein as such).
  • the libraries include a multiplicity of different sgRNAs within a single pool or group of pools.
  • the libraries include at least one copy of each sgRNA represented within the library.
  • the libraries include multiple copies of each sgRNA.
  • the numbers of copies of each sgRNA within in library are equal or are similar. In other forms, the numbers of copies of each sgRNA are not equal.
  • sgRNA libraries are enriched for one or more of the multiplicity of sgRNAs within the library.
  • the sgRNA library includes between about 1 and about 100 or more sgRNA sequences. In some forms, the library includes about 1,000, or more than 1,000 sgRNA sequences, up to 10,000 sgRNA sequences. In some forms, the library includes about 10,000 or more sgRNA sequences. In further forms, the library includes about 20,000 or more sgRNA sequences.
  • the library includes about 30,000 or more than 30,000, up to 50,000 sgRNA sequences. In further forms, the library includes about 40,000 sequences or more than 40,000, up to 50,000 sgRNA sequences. In further forms, the library includes about 50,000 sequences or more than 50,000, up to 60,000 sgRNA sequences. In yet further forms, the library includes about 40,000 sequences or more than 60,000, up to 70,000 sgRNA sequences. Typically, the library includes a sufficient number of sgRNA sequences to enable coverage of a target gene set, whilst enabling complete (100%) or a high-degree (i.e., greater than 50%) representation in a screen.
  • a screen according to the described methods includes at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, up to 100% representation of all the distinct species of sgRNAs present in a library.
  • An exemplary library of sgRNAs is described in International Patent Application Publication No. WO 2020/028533 by YALE university, the content of which is hereby incorporated by reference in its entirety, including its sequence listing and all the sequences provided therein.
  • WO 2020/028533 describes an sgRNA library (“mSurfeome2”), including 69,747 sgRNAs.
  • the sgRNA sequence library includes one or more of the 69,747 sgRNAs described in WO 2020/028533, or any specific sequence of set of subsequences provided therein. In some forms, the library includes all of the 69,747 sgRNAs described in WO 2020/028533.
  • crRNA/sgRNA sequences The structure and function of sgRNAs is known in the art. Each sgRNA is made up of two parts: (i) a crispr RNA (crRNA), a 17-20 nucleotide sequence complementary to the target DNA; and (ii) a Trans-activating CRISPR RNA (tracr RNA), which serves as a binding scaffold for the Cas nuclease.
  • the tracrRNA pairs with complementary repeat sequences within the pre-crRNA primary transcript and forms an RNA duplex, pre-crRNA:tracrRNA, which is recognized and cleaved by RNase III in the presence of Cas9 protein.
  • the crRNA includes 17-20 contiguous nucleic acids that specifically bind to one gene with thin the NK cell genome.
  • the different species of sgRNAs in a library are combined to have a level of redundancy with respect to coverage of one or more target genes, such as a group of genes associated with one or more molecular pathway or cellular function.
  • the different species of sgRNAs in a library include at least 1% redundancy, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or more than 10%, such as 15% or 20% redundancy in the coverage of one or more target genes, such as a group of genes associated with one or more molecular pathway or cellular function
  • a high-density sgRNA library targeting the mouse homologs of the human surface proteome (Surf-v2) has been developed.
  • Surf-v2 targets 2,863 genes with up to 20 sgRNAs per gene (56,911 gene-targeting sgRNAs).
  • Surf-v2 includes 5,000 non- targeting control (NTC) sgRNAs, reverse-ranked by potential off-targets from 500,000 random 20 nt sequences, that were spiked into the final library containing a total of 61,911 sgRNAs.
  • NTC non- targeting control
  • This library is provided in WO 2020/028533 as SEQ ID NOs:1-61,911. Therefore, in some forms, the library includes one or more sgRNAs including a nucleotide sequence selected from SEQ ID NOs:1-61,911 of the sequence listing of WO 2020/028533 (mSurfeome2), which is specifically incorporated by reference herein in its entirety.
  • the sgRNA library includes a plurality of nucleic acid sequences selected from SEQ ID NOs:1-61,911 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the sgRNA library includes one or more sgRNAs including a nucleic acid sequence selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2), or an specific sequence or specific subset of sequences therein.
  • the sgRNA library includes a plurality of nucleic acid sequences selected from SEQ ID NOs: 61,912-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • sgRNAs target/are specific for one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43, KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3, VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2.
  • the sgRNA targets/is specific for Calhm2.
  • the sgRNA library includes one or more sgRNAs including a nucleic acid sequence that targets/is specific for one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43, KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3, VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2. 2.
  • the library includes one or more vectors.
  • the sgRNA library is packaged into a vector. Any vector known to one of ordinary skill in the art can be used.
  • the vector is a viral vector, including but not limited to lentiviral vectors, adenoviral vectors, and adeno-associated viral (AAV) vectors.
  • Adeno-associated virus vector A preferred vector for packaging the sgRNA library is an adeno-associated viral (AAV) vector.
  • AAV is a non-pathogenic, single-stranded DNA virus that has been actively employed for delivering therapeutic genes in both in vitro and in vivo systems (Choi, et al., Curr. Gene Ther., 5:299-310, (2005)).
  • AAV is a replication-deficient virus that belongs to the parvovirus family, and is dependent on co-infection with other viruses, mainly adenoviruses, to replicate.
  • ITR inverted terminal repeat
  • the single-stranded AAV genome contains three genes, Rep (Replication), Cap (Capsid), and aap (Assembly). These three genes give rise to at least nine gene products through the use of three promoters, alternative translation start sites, and differential splicing.
  • the Rep gene encodes four proteins (Rep78, Rep68, Rep52, and Rep40), while Cap expression gives rise to the viral capsid proteins (VP; VP1/VP2/VP3), which form the outer capsid shell that protects the viral genome, as well as being actively involved in cell binding and internalization. It is estimated that the viral coat includes 60 proteins, arranged into an icosahedral structure with the capsid proteins in a molar ratio of 1:1:10 (VP1:VP2:VP3).
  • Recombinant AAV which lacks viral DNA, is essentially a protein- based nanoparticle engineered to traverse the cell membrane, where it can ultimately traffic and deliver its DNA cargo into the nucleus of a cell.
  • ITR-flanked transgenes encoded within rAAV can form circular concatemers that persist as episomes in the nucleus of transduced cells. Because recombinant episomal DNA does not integrate into host genomes, it will eventually be diluted over time as the cell undergoes repeated rounds of replication. This will eventually result in the loss of the transgene and transgene expression, with the rate of transgene loss dependent on the turnover rate of the transduced cell.
  • AAV can be advantageous over other viral vectors due to low toxicity (this can be due to the purification method not requiring ultra centrifugation of cell particles that can activate the immune response) and low probability of causing insertional mutagenesis because AAV does not integrate into the host genome (primarily remaining episomal).
  • the AAV vector used in the disclosed compositions and methods is a naturally occurring serotype of AAV including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, artificial variants such as AAV.rhlO, AAV.rh32/33, AAV.rh43, AAV.rh64Rl, rAAV2- retro, AAV-DJ, AAV-PHP.B, AAV-PHP.S, AAV-PHP.eB, or other engineered versions of AAV.
  • the AAV used in the disclosed compositions and methods is AAV6 or AAV9.
  • AAV serotypes of AAV have thus far been identified, with the best characterized and most commonly used being AAV2. These serotypes differ in their tropism, or the types of cells they infect, making AAV a very useful system for preferentially transducing specific cell types.
  • AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof can be used for targeting brain or neuronal cells; AAV4 can be selected for targeting cardiac cells.
  • AAV8 is useful for delivery to the liver cells.
  • researchers have further refined the tropism of AAV through pseudotyping, or the mixing of a capsid and genome from different viral serotypes.
  • AAV2/5 indicates a virus containing the genome of serotype 2 packaged in the capsid from serotype 5.
  • Use of these pseudotyped viruses can improve transduction efficiency, as well as alter tropism.
  • AAV2/5 targets neurons that are not efficiently transduced by AAV2/2, and is distributed more widely in the brain, indicating improved transduction efficiency.
  • One of skill in the art would be able to determine the optimal AAV serotype to be used for the respective application.
  • the AAV can be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, artificial variants such as AAV.rhlO, AAV.rh32/33, AAV.rh43, AAV.rh64Rl, rAAV2-retro, AAV-DJ, AAV-PHP.B, AAV-PHP.S, and AAV-PHP.eB, or combinations thereof.
  • the AAV vector is AAV6 or AAV9.An exemplary nucleic acid sequence for a vector for use with the AAV system is: b.
  • the sgRNA library is packaged within or otherwise associated with another viral vector.
  • suitable viral vectors include, without limitation, vectors derived from bacteriophages, baculoviruses, retroviruses (such as lentiviruses), adenoviruses, poxviruses, and Epstein-Barr viruses.
  • the viral vector is derived from a DNA virus (e.g., dsDNA or ssDNA virus) or an RNA virus (e.g., an ssRNA virus).
  • the viral vector is a lentivirus vector.
  • Lentivirus is produced by HEK293T cells, the supernatant was collected and precipitated using Lenti-X Concentrator (Takara).
  • Lentiviral pellets are resuspended with NK92 complete culture media, then aliquoted and stored at -80°C.
  • the viral vector when the viral vector is a lentiviral vector, cells are transduced with lentivirus at 1-2e6 cells / ml in a 12-well plate, which is pre-coated with Retronectin (Takara) in PBS, overnight at 4°C. In some forms, spin-infection is performed (e.g., at 32°C at 900 x g for 90 min).
  • the vector is a viral vector such as a vesicular stomatitis (VSV) vector, a Bocavirus vector, such as a human bocavirus 1 (HBoV1) vector, a Herpes simplex virus (HSV) vector, or an adenovirus vector (AdV).
  • VSV vesicular stomatitis
  • Bocavirus vector such as a human bocavirus 1 (HBoV1) vector, a Herpes simplex virus (HSV) vector, or an adenovirus vector (AdV).
  • the viral vector is a Herpes simplex virus (HSV) vector.
  • Herpes simplex viruses HSV are large, enveloped dsDNA viruses characteristic of their lytic and latent nature of infection, which result in life-long latent infection of neurons and allows for long-term transgene expression. Deletion of HSV genes has generated expression vectors with low toxicity and an excellent packaging capacity of >30 kb foreign DNA.
  • the viral vector is a Vesicular stomatitis virus (VSV) vector.
  • VSV Vesicular stomatitis virus
  • Vesicular stomatitis virus is a non-segmented, negative-stranded RNA virus that belongs to the family Rhabdoviridae, genus Vesiculovirus.
  • VSV infects a broad range of animals, including cattle, horses, and swine.
  • the genome of the virus codes for five major proteins, glycoprotein (G), matrix protein (M), nucleoprotein (N), large protein (L), and phosphoprotein (P).
  • G protein mediates both viral binding and host cell fusion with the endosomal membrane following endocytosis.
  • the L and P proteins are subunits of the viral RNA-dependent RNA polymerase.
  • the simple structure and rapid high-titer growth of VSV in mammalian and many other cells has made recombinant VSV a useful tool in the fields of cellular and molecular biology and virology.
  • the viral vector is a human Bocavirus vector (HBoV).
  • Exemplary human bocavirus vectors include human bocaviruses 1-4 (HBoV1-4), As well as Gorilla BoV.
  • the viral vector is an adenovirus vector.
  • the vector is a chimeric vector, such as a vector that is based on a chimeric virus formed from a combination of one or more components from two or more different viral vectors.
  • An exemplary chimeric viral vector is a chimeric bocavirus/adeno-associated virus vector. Therefore, in some forms, the vector is a chimeric HBoV1/AAV2 vector (e.g., rAAV2/HBoV1 chimeras). 3.
  • the sgRNA library is a vector-sgRNA library than includes a multiplicity of vectors each encapsulating or otherwise associated with one or more sgRNA.
  • the library includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the vector is AAV. Therefore, in some forms the library is an AAV-sgRNA library.
  • the library includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs:1-61,911 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the AAV library includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the AAV library (AAV- sgRNA) includes a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs:1- 61,911 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • B. CRISPR Vectors for Gene Editing of NK Cells Vectors including the described sgRNA libraries are described for gene editing and high-throughput screen in NK cells. Therefore, gene editing compositions for use in methods of modifying the genome of a cell are disclosed.
  • an sgRNA library is encapsulated or associated with a vector configured for CRISPR-based gene editing in target NK cells.
  • the CRISPR vector is configured for the delivery of sgRNA in the context of the Sleeping Beauty (SB) transposon system (SB-CRISPR vector).
  • a preferred vector is an AAV vector, such as AAV6. Therefore, in some forms, the vector is a AAV-CRISPR vector, such as an AAV-SB-CRISPR vector.
  • the AAV-SB-CRISPR vectors are configured for in vivo CRISPR-mediated knockout screening directly in primary NK cells using the single guide RNA (sgRNA, gRNA) library.
  • sgRNA, gRNA single guide RNA
  • each CRISPR vector within the library includes one or more of an antibiotic resistance sequence, two ITRs, two sleeping beauty (SB) IR/DR repeats, a RNA pol-III promoter (e.g., U6), an sgRNA from the library (spacer and tracrRNA backbone), a promoter (EFS), a Thyl.l selection marker, an SB 100x transposase, and a short poly A region.
  • RNA pol-III promoter e.g., U6
  • an sgRNA from the library spacer and tracrRNA backbone
  • EFS promoter
  • Thyl.l selection marker a Thyl.l selection marker
  • SB 100x transposase an SB 100x transposase
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the prokaryotic CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) for use in eukaryotes (see, for example, Cong, Science, 15:339(6121):819–823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • Methods of preparing compositions for use in genome editing using the CRISPR/Cas systems are described in detail in WO 2013/176772 and WO 2014/018423, which are specifically incorporated by reference herein in their entireties.
  • Cas generally refers to an effector protein of a CRISPR Cas system or complex.
  • the term “Cas” may be used interchangeably with the terms “CRISPR” protein, “CRISPR Cas protein,” “CRISPR effector,” CRISPR Cas effector,” “CRISPR enzyme,” “CRISPR Cas enzyme” and the like, unless otherwise apparent.
  • CRISPR system 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 other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a “spacer” in the context of an endogenous CRISPR system
  • One or more tracr mate sequences operably linked to a guide sequence can also be referred to as pre- crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease.
  • pre-CRISPR RNA pre-CRISPR RNA
  • the described vectors include an sgRNA from the described libraries, together with a Crispr-Cas effector protein.
  • the described sgRNA libraries include a tracrRNA and crRNA that are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA:tracrRNA duplex as described in Cong, Science, 15:339(6121):819–823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)).
  • a single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)).
  • the crRNA portion can be identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’.
  • Crispr-Cas effector protein The Crispr-Cas effector protein may be without limitation a type II, type V, or type VI Cas effector protein.
  • Non-limiting examples of Crispr-Cas effector proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • the unmodified CRISPR enzyme has DNA cleavage activity.
  • the Crispr-Cas effector protein is mutated with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • the Type II CRISPR enzyme is a Cas9 enzyme such as disclosed in International Patent Application Publication No. WO/2014/093595.
  • the Cas9 enzyme is S. pneumoniae, S. pyogenes or S. thermophilus Cas9, and may include mutated Cas9 derived from these organisms.
  • the enzyme may be a Cas9 homolog or ortholog. Additional orthologs include, for example, Cas9 enzymes from Corynebacter diptheriae, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaeroachaeta globus, Azospirillum B510, Gluconacetobacter diazotrophicus, Neisseria cinereal, Roseburia intestinalis, Parvibaculum lavamentivorans, Staphylococcus aureus, Nitratifractor salsuginis DSM 16511, Camplyobacter lari CF8912, and Streptococcus thermophilus LMD 9.
  • the Cas9 effector protein and orthologs thereof may be modified for enhanced function.
  • improved target specificity of a CRISPR Cas9 system may be accomplished by approaches that include, but are not limited to, designing and preparing guide RNAs having optimal activity, selecting Cas9 enzymes of a specific length, truncating the Cas9 enzyme making it smaller in length than the corresponding wild-type Cas9 enzyme by truncating the nucleic acid molecules coding therefor and generating chimeric Cas9 enzymes wherein different parts of the enzyme are swapped or exchanged between different orthologs to arrive at chimeric enzymes having tailored specificity.
  • C. Modified Natural Killer Cells Genetically-modified NK cells are provided.
  • the genetically modified NK cells are for use in cell-based immunotherapy applications.
  • the NK cells are modified by knock-down or knock-out of one or more of the genes or gene expression products of the NK cell.
  • Natural killer (NK) cells are lymphocytes with critical effector functions in innate immunity (Vivier, et al., Nat Immunol 9, 503-510 (2008).) that do not require sensitization or specific antigens to initiate an effective immune response (Ben-Shmuel, et al., Frontiers in immunology 11, 275-275 (2020)).
  • NK Cells are lymphocytes in the same family as T and B cells, coming from a common progenitor.
  • NK cells are classified as group I Innate Lymphocytes (ILCs) and respond quickly to a wide variety of pathological challenges.
  • ILCs Innate Lymphocytes
  • NK cells are best known for killing virally infected cells, and detecting and controlling early signs of cancer.
  • Effector populations of NK cells are able to lyse adjacent cells based on the expression of oncogenic transformation-associated surface markers (Shimasaki, et al., Nat Rev Drug Discov 19, 200-218 (2020)).
  • NK populations can influence the functions of DCs (Peterson, et al., Frontiers in Immunology 11 (2021)., Fernandez, et al., Nature Medicine 5, 405-411 (1999).), monocytes, T cells, and B cells via cytokine production or through direct cell-cell contact in a receptor-ligand interaction-dependent manner (Abel, et al., Front Immunol 9, 1869 (2016), Zwirner, et al., Front Immunol 8, 25 (2017)).
  • specialized NK cells are also found in the placenta and may play an important role in pregnancy.
  • CD56, CD161, CD16, CD94 or CD57 represent prototypic markers of NK cells.
  • NK cells were first noticed for their ability to kill tumor cells without any priming or prior activation (in contrast to cytotoxic T cells, which need priming by antigen presenting cells). They are named for this ‘natural’ killing. Additionally, NK cells secrete cytokines such as IFN ⁇ and TNF ⁇ , which act on other immune cells like Macrophage and Dendritic cells to enhance the immune response. While on patrol NK cells constantly contact other cells. Whether or not the NK cell kills these cells depends on a balance of signals from activating receptors and inhibitory receptors on the NK cell surface. Activating receptors recognize molecules that are expressed on the surface of cancer cells and infected cells, and ‘switch on’ the NK cell.
  • cytokines such as IFN ⁇ and TNF ⁇
  • Inhibitory receptors act as a check on NK cell killing. Most normal healthy cells express MHC I receptors which mark these cells as ‘self’. Inhibitory receptors on the surface of the NK cell recognize cognate MHC I, and this ‘switches off’ the NK cell, preventing it from killing. Cancer cells and infected cells often lose their MHC I, leaving them vulnerable to NK cell killing. Once the decision is made to kill, the NK cell releases cytotoxic granules containing perforin and granzymes, which leads to lysis of the target cell. NK-based cell therapy is a promising emerging branch of cancer immunotherapies.
  • NK cell therapy leverages the advantages of rapid cytotoxic anti- tumor immune responses, TCR-independence, enhanced safety, simplicity in generating off-the-shelf allogeneic products, reduced off-target immune responses (Zhang, Y. et al., Immunology 121, 258-265 (2007)), and reduced production of molecules associated with cytokine release syndrome (CRS) relative to other cell types (Chou, et al., Bone Marrow Transplant 54, 780-784 (2019), Hunter, et al., J Natl Cancer Inst 111, 646-654 (2019), Xie, et al., EBioMedicine 59, 102975 (2020).).
  • the NK cell to be modified is a human cell.
  • the cell is from an established cell line, or a primary cell.
  • the term “primary cell,” refers to cells and cell cultures derived from a subject and allowed to grow in vitro for a limited number of passages, i.e. splitting, of the culture.
  • the genetically modified cell is modified by knock-down and/or knock out of at least one or more of the genes or gene expression products of the NK cell according to the described methods.
  • An exemplary genetically modified Natural Killer (NK) cell includes a mutation in, or loss of at least one gene selected from the group consisting of TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43, KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2.
  • NK Natural Killer
  • NK cells Natural Killer
  • the disclosed gene editing compositions and methods can be configured to achieve genomic modification of cells such as Natural Killer (NK) cells, however alternative approaches can also be used.
  • the gene that is modified by knock-down and/or knock out has a function selected from a marker of exhaustion, positive regulation of apoptotic processes, cell adhesion, and leukocyte proliferation.
  • knock-down and/or knock out refers to reducing or eliminating gene expression, or modifying gene expression to reduce or eliminate gene product expression or bioactivity.
  • nucleic acid sequences including deletions, substitutions, insertions, and combinations thereof, to promoters and/or coding regions of the gene or genes that reduce or prevent expression of functional protein.
  • non- functional protein or protein with reduced bioactivity for example truncated or mutated protein, is expressed. Additionally or alternatively wildtype protein is reduced or absent.
  • NK cells are genetically modified to knock-down and/or knock out expression of one or more genes selected from ZEB2, S1PR5, SPN, LY6C2, CX3CR1, PRDM1, ITGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2, or the full-length gene product thereof.
  • genes selected from ZEB2, S1PR5, SPN, LY6C2, CX3CR1, PRDM1, ITGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2, or the full-length gene product thereof.
  • the genetic modification(s) to the NK cells reduce expression and/or bioactivity of the full-length protein(s) encoded by ZEB2, S1PR5, SPN, LY6C2, CX3CR1, PRDM1, ITGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and/or CALHM2.
  • the gene that is modified by knock-down and/or knock out is selected from VNN3, CD59B, CCR2, SLC2A8, PRNP, CEACAM14, AND CALHM2.
  • the gene that is modified by knock-down and/or knock out is Calhm2. Therefore, genetically modified NK cells lacking expression and/or function of one or more of the genes TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD- 1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2 are provided.
  • the expression and/or bioactivity of the protein(s) encoded by one or more of TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and/or CALHM2 is reduced.
  • the genetically modified NK cells lack expression and/or have diminished function of a Calcium homeostasis modulator (CALHM) gene or gene product. As described in the Examples, it has been established that lack or reduction in the expression of CALHM2 particularly enhances the anti-tumor efficacy of NK cells.
  • CALHM Calcium homeostasis modulator
  • genetically modified NK cells lack of have diminished expression of one or more CALHM genes or gene expression products relative to a control NK cell.
  • the NK cells lack of have diminished expression of the Calhm1 gene, and/or the Calhm2 gene, and/or the CALHM3 gene relative to a control NK cell.
  • genetically modified NK cells lack expression and/or have diminished function of the Calhm2 gene or its gene product, the CALHM2 transmembrane protein.
  • the Calhm2 gene is also known as FAM26B (NCBI accession number: 51063) located in humans on Chromosome 10, (see NCBI Reference No: NC_000010.11, positions 103446786..103452370, complement).
  • CALHMs Calcium homeostasis modulators
  • Ca2+-inhibited nonselective ion channels that act as major ATP release channels and have important roles in gustatory signaling and neuronal toxicity.
  • Dysfunction of CALHMs has previously been linked to neurological disorders.
  • Extracellular Ca2+ (Ca2+ o) plays important roles in physiology. Changes of Ca2+o concentration ([Ca2+]o) have been observed to modulate neuronal excitability in various physiological and pathophysiological settings, but the mechanisms by which neurons detect [Ca2+]o are not fully understood.
  • CALHM Calcium homeostasis modulator protein
  • the human CALHM2 gene (NCBI accession number: 51063) located in humans on Chromosome 10 (see NCBI Reference No: NC_000010.11, positions 103446786..103452370, complement) that is knocked down or knocked out has the nucleic acid sequence:
  • the gene expression product of the human CALHM2 gene that is knocked down or knocked out is the 323 amino acid CALHM2 transmembrane protein having UNIPROT accession ID No. Q9HA72 and having an amino acid sequence of: Therefore, in some forms, the genetically modified human NK cells lack expression of a gene having the nucleic acid sequence of SEQ ID NO:2. In some forms, the genetically modified human NK cells have reduced or lack expression or bioactivity of a protein having the amino acid sequence of SEQ ID NO:3. 2. Sources of NK cells In preferred forms, the NK cells to be genetically modified are obtained from a human subject.
  • the cells are autologous cells, i.e., cells obtained from a subject prior to genetic modification and re-introduction to the same subject following modification.
  • the cells are heterologous cells, i.e., cells obtained from a different subject than the intended recipient.
  • the cells are frozen prior to or after genetic modification. Methods and compositions for freezing and thawing viable eukaryotic cells are known in the art.
  • the cells are autologous immune cells, such as T cells or progenitor cells/stem cells. a. Autologous human NK cells
  • the NK cells are obtained from a human subject, prior to modification and reintroduction to the same human subject for use as cell therapy.
  • NK cells are obtained from a healthy subject.
  • cells are obtained from a subject identified as having or at risk of having a disease or disorder, such as cancer and/or an auto-immune disease.
  • prior to expansion and/or genetic modification NK cells are obtained from a diseased or healthy subject.
  • NK cells can be obtained from a number of samples, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • NK cells are obtained from a unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as FICOLLTM separation.
  • NK cells from the circulating blood of an individual are obtained by apheresis.
  • the apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets.
  • the cells collected by apheresis can be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps.
  • the cells are washed with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the wash solution lacks calcium and can lack magnesium or can lack many if not all divalent cations.
  • the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2+-free, Mg2+-free PBS, PLASMALYTE A, or other saline solution with or without buffer.
  • buffers such as, for example, Ca2+-free, Mg2+-free PBS, PLASMALYTE A, or other saline solution with or without buffer.
  • the undesirable components of the apheresis sample are removed and the cells directly resuspended in culture media.
  • the NK cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLLTM gradient or by counterflow centrifugal elutriation.
  • a specific subpopulation of NK cells is further isolated by positive or negative selection techniques.
  • 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, for a time period sufficient for positive selection of the desired T cells.
  • anti-CD3/anti-CD28 i.e., 3 ⁇ 28
  • conjugated beads such as DYNABEADS® M-450 CD3/CD28 T
  • the genetically modified cell is an NK cell that expresses or includes a chimeric antigen receptor (CAR), i.e., a CAR-NK Cell.
  • NK cells genetically engineered to express a chimeric antigen receptor is rapidly emerging as a promising new treatment for hematological and non-hematological malignancies.
  • CAR-NK cells The development of CAR-NK cells has increased the therapeutic potential of CAR-reprogramming by adding a reduced risk for alloreactivity and Graft-vs-Host Disease, potentially allowing for CAR-NK to be mass produced in a more cost-effective manner than CAR-T cells.
  • NK cell-based immunotherapies require effective anti-tumor function, exhaustion, durable immune responses (persistence), and tumor infiltration. This requires rational engineering of substantially enhanced NK cells, particularly by modification of endogenous genes.
  • CAR Chimeric antigen receptor
  • a CAR refers to an engineered receptor that is expressed on a NK cell or any other effector cell type capable of cell-mediated cytotoxicity.
  • a CAR includes an extracellular domain having an antigen binding domain that is specific for a ligand or receptor.
  • a CAR also includes a transmembrane domain, and a costimulatory signaling domain.
  • a CAR includes a hinge.
  • the antigen binding domain is specific for EGFRvlll.
  • the costimulatory signaling domain is a 4-lBB signaling domain.
  • a CAR further includes a CD3 zeta signaling domain.
  • a CAR- NK cell is a NK cell engineered to express a CAR.
  • CARs are engineered receptors that possess both antigen-binding and cell- activating functions. Based on the location of the CAR in the membrane of the cell, the CAR can be divided into three main distinct domains, including an extracellular antigen- binding domain, followed by a space region, a transmembrane domain, and the intracellular signaling domain.
  • the antigen-binding domain typically contains VH and VL chains that are joined up by a linker to form the so-called “scFv.”
  • the segment interposing between the antigen-binding domain (e.g., scFv) and the transmembrane domain is a “spacer domain.”
  • the spacer domain can include the constant IgG1 hinge-CH2–CH3 Fc domain. In some cases, the spacer domain and the transmembrane domain are derived from CD8.
  • the intracellular signaling domains mediating T cell activation can include a CD3 ⁇ co-receptor signaling domain derived from C-region of the TCR ⁇ and ⁇ chains and one or more costimulatory domains.
  • the antigen-binding domain of a CAR is derived from an antibody.
  • antibody herein refers to natural or synthetic polypeptides that bind a target antigen.
  • the term includes polyclonal and monoclonal antibodies, including intact antibodies and functional (e.g., antigen-binding) antibody fragments, including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • functional antibody fragments including Fab fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • the term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antigen-binding domain of a CAR can contain complementary determining regions (CDR) of an antibody, variable regions of an antibody, and/or antigen binding fragments thereof.
  • CDR complementary determining regions
  • the antigen-binding domain for a CD19 CAR can be derived from a human monoclonal antibody to CD19, such as those described in U.S. Patent 7,109,304, for use in accordance with the disclosed compositions and methods.
  • the antigen-binding domain can include an F(ab')2, Fab', Fab, Fv or scFv.
  • the CAR includes one or more spacer domain(s) (also referred to as hinge domain) that is located between the extracellular antigen-binding domain and the transmembrane domain.
  • a spacer domain is an amino acid segment that is generally found between two domains of a protein and may allow for flexibility of the protein and movement of one or both of the domains relative to one another. Any amino acid sequence that provides such flexibility and movement of the extracellular antigen- binding domain relative to the transmembrane domain can be used.
  • the spacer domain can be a spacer or hinge domain of a naturally occurring protein.
  • the hinge domain is derived from CD8a, such as, a portion of the hinge domain of CD8a, e.g., a fragment containing at least 5 (e.g., 5, 10, 15, 20, 25, 30, 35, or 40) consecutive amino acids of the hinge domain of CD8a.
  • Hinge domains of antibodies such as an IgG, IgA, IgM, IgE, or IgD antibodies can also be used.
  • the hinge domain is the hinge domain that joins the constant CH1 and CH2 domains of an antibody.
  • Non-naturally occurring peptides may also be used as spacer domains.
  • the spacer domain can be a peptide linker, such as a (GxS)n linker, wherein x and n, independently can be an integer of 3 or more, including 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more.
  • the CAR includes a transmembrane domain that can be directly or indirectly fused to the antigen-binding domain.
  • the transmembrane domain may be derived either from a natural or a synthetic source.
  • a “transmembrane domain” refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane.
  • the transmembrane domain of the CAR includes a transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD8, CD4, CD28, CD137, CD80, CD86, CD152 or PD1, or a portion thereof.
  • Transmembrane domains can also contain at least a portion of a synthetic, non-naturally occurring protein segment.
  • the transmembrane domain is a synthetic, non-naturally occurring alpha helix or beta sheet.
  • the protein segment is at least about 15 amino acids, e.g., at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more amino acids. Examples of synthetic transmembrane domains are known in the art, for example in U.S. Patent No.7,052,906 and PCT Publication No. WO 2000/032776.
  • the intracellular signaling domain is responsible for activation of at least one of the normal effector functions of the immune effector cell expressing the CAR.
  • effector function refers to a specialized function of a cell.
  • Effector function of a T cell may be cytolytic activity or helper activity including the secretion of cytokines.
  • an intracellular signaling domain includes the zeta chain of the T cell receptor or any of its homologs (e.g., eta, delta, gamma or epsilon), MBl chain, B29, Fc RIII, Fc RI and combinations of signaling molecules such as CD3 ⁇ and CD28, 4-1BB, OX40 and combination thereof, as well as other similar molecules and fragments.
  • Intracellular signaling portions of other members of the families of activating proteins can be used, such as Fc ⁇ RIII and Fc ⁇ RI.
  • the CAR includes at least one co-stimulatory signaling domain.
  • co-stimulatory signaling domain refers to at least a portion of a protein that mediates signal transduction within a cell to induce an immune response such as an effector function.
  • the co-stimulatory signaling domain can be a cytoplasmic signaling domain from a co-stimulatory protein, which transduces a signal and modulates responses mediated by immune cells, such as T cells, NK cells, macrophages, neutrophils, or eosinophils.
  • the co-stimulatory signaling domain is derived from a co-stimulatory molecule selected from CD27, CD28, CD137, 0X40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands of CD83 and combinations thereof.
  • CARs can be used in order to generate immuno-responsive cells, such as NK cells, specific for selected targets, such as malignant cells, with a wide variety of receptor chimera constructs having been described (see U.S. Patent No.11,207,350 and PCT Publications WO 2016123333 A1 and WO 2016201300 A1).
  • Alternative CAR constructs can be characterized as belonging to successive generations.
  • First-generation CARs typically include a single-chain variable fragment of an antibody specific for an antigen, for example including a VL linked to a VH of a specific antibody, linked by a flexible linker, for example by a CD8 ⁇ hinge domain and a CD8 ⁇ transmembrane domain, to the transmembrane and intracellular signaling domains of either CD3 ⁇ or FcR ⁇ (scFv-CD3 ⁇ or scFv- FcR ⁇ ; 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-CD3 ⁇ ; 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 CD3 ⁇ -chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3 ⁇ or scFv-CD28-OX40-CD3 ⁇ ; 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).
  • costimulatory endodomains such as CD3 ⁇ -chain, CD97, GDI la-CD18, CD2, ICOS, CD27, CD154, CDS, OX40, 4-1BB, or CD28 signaling domains (for example scFv-CD28-4-1BB-CD3 ⁇ or sc
  • co-stimulation can be orchestrated by expressing CARs in antigen-specific T cells, chosen so as to be activated and expanded following engagement of their native ⁇ TCR, for example by antigen on professional antigen-presenting cells, with attendant co-stimulation.
  • Any of the first, second, or third generation CARs described above can be used in accordance with the disclosed compositions and methods.
  • the gene of interest within a transposon encodes a CAR targeting one or more antigens specific for cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, an autoimmune disease, or combinations thereof.
  • the genetically-modified CAR-NK cells include a CAR component that targets a cancer antigen.
  • Exemplary antigens specific for cancer that could be targeted by the CAR include, but are not limited to, 4-1BB, 5T4, adenocarcinoma antigen, alpha-fetoprotein, BAFF, B-lymphoma cell, C242 antigen, CA-125, carbonic anhydrase 9 (CA-IX), C-MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA-4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain-B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF-1 receptor, IGF-I, IgGl, Ll-CAM
  • the CAR targets CD19, CD22, or both CD19 and CD22.
  • Exemplary antigens specific for an inflammatory disease that could be targeted by the CAR include, but are not limited to, AOC3 (VAP-1), CAM-3001, CCL11 (eotaxin-1), CD 125, CD 147 (basigin), CD 154 (CD40L), CD2, CD20, CD23 (IgE receptor), CD25 (a chain of IL-2 receptor), CD3, CD4, CD5, IFN-a, IFN- ⁇ , IgE, IgE Fc region, IL-1, IL-12, IL-23, IL-13, IL-17, IL-17A, IL-22, IL-4, IL-5, IL-5, IL-6, IL-6 receptor, integrin a4, integrin ⁇ 4 ⁇ 7, Lama glama, LFA-1 (CD 11a), MEDI-528, myostatin, OX-40, rhuMAb ⁇ 7, scleroscin
  • Exemplary antigens specific for a neuronal disorder that could be targeted by the CAR include, but are not limited to, beta amyloid, MABT5102A, and combinations thereof.
  • Exemplary antigens specific for diabetes that could be targeted by the CAR include, but are not limited to, L- ⁇ ⁇ , CD3, and combinations thereof.
  • Exemplary antigens specific for a cardiovascular disease that could be targeted by the CAR include, but are not limited to, C5, cardiac myosin, CD41 (integrin alpha-lib), fibrin II, beta chain, ITGB2 (CD 18), sphingosine-1 -phosphate, and combinations thereof.
  • Exemplary antigens specific for an infectious disease that could be targeted by the CAR include, but are not limited to, anthrax toxin, CCR5, CD4, clumping factor A, cytomegalovirus, cytomegalovirus glycoprotein B, endotoxin, Escherichia coli, hepatitis B surface antigen, hepatitis B virus, HIV-1, Hsp90, Influenza A hemagglutinin, lipoteichoic acid, Pseudomonas aeruginosa, rabies virus glycoprotein, respiratory syncytial virus, TNF-a, and combinations thereof.
  • the CAR targets one or more antigens selected from an antigen listed in Table 1. Table 1.
  • Compositions of Genetically Modified NK Cells are formulated into pharmaceutical compositions for administration in vivo.
  • pharmaceutical compositions include a plurality of genetically modified NK cells combined with excipients and/or other reagents suitable for administration to a subject in the form of a “living drug” or therapeutic agent.
  • a plurality of NK cells that may or may not express a chimeric antigen receptor
  • genetically modified according to the methods are combined with excipients and/or other reagents suitable for administration to a subject to provide a NK cell therapy for a subject in need thereof.
  • compositions containing NK and/or CAR NK cells include between about 10 4 and about 10 9 cells per kg body weight of the intended recipient (i.e., between 7x 10 5 and 7x10 10 cells for an average adult), preferably 10 5 to 10 7 cells/kg body weight, including all integer values within those ranges.
  • Pharmaceutical compositions containing a genetically modified NK cell, or a population of genetically modified NK cells are provided.
  • the pharmaceutical compositions include one or more of a pharmaceutically acceptable buffer, carrier, diluent, or excipients.
  • the pharmaceutical compositions include a specific number or population of cells, for example, expanded by culturing and expanding an isolated genetically modified NK cell (e.g., Calhm2 KO/CAR NK cell), e.g., a homogenous population. Therefore, in some forms, pharmaceutical compositions include a homogenous population of modified NK cells. In other forms, the pharmaceutical compositions include populations of cells that contain variable or different genetically modified NK cells, e.g., a heterogeneous population. In some forms, the pharmaceutical compositions include CAR-NK cells that are bispecific or multi-specific. In some forms, the NK cells have been isolated from a diseased or healthy subject prior to genetic modification.
  • an isolated genetically modified NK cell e.g., Calhm2 KO/CAR NK cell
  • pharmaceutical compositions include a homogenous population of modified NK cells.
  • the pharmaceutical compositions include populations of cells that contain variable or different genetically modified NK cells, e.g., a heterogeneous population.
  • compositions e.g., more AAV-sgRNA vectors
  • the term “Pharmaceutically acceptable carrier” describes a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body.
  • the carrier is a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof.
  • Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation.
  • compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, phosphate buffered saline and the like
  • carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol
  • proteins polypeptides or amino acids such as glycine
  • antioxidants chelating agents
  • adjuvants e.g., aluminum hydroxide
  • preservatives e.g., aluminum hydroxide
  • the term “Route of administration” can refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and/or injections.
  • the pharmaceutical compositions are preferably formulated for intravenous administration.
  • the disclosed pharmaceutical compositions are administered in a manner appropriate to a disease to be treated (or prevented).
  • the quantity and frequency of administration is typically determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.
  • the disclosed pharmaceutical compositions can be delivered in a therapeutically effective amount.
  • the precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration.
  • One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly.
  • Methods of Screening/Producing Genetically Modified NK Cells Methods for screening genes associated with the membrane and surface of NK cells to identify candidate targets that enhance the anti-cancer efficacy of NK cells have been developed. Methods for providing genetically modified NK cells based on data obtained from the screening methods are also provided. Methods of formulating pharmaceutical formulations including genetically modified NK cells for treating diseases and disorders are also provided. Methods of treating diseases and disorders in a subject in need thereof by administering formulations of genetically modified NK cells to the subject are also described. A.
  • NK cells A high-throughput gene perturbation screen for highly efficient identification and engineering of therapeutic NK cells has been established.
  • the screen identifies genes whose perturbation enhances the anti-tumor properties of NK cells.
  • the methods employ a library of sgRNAs designed to target membrane and cell-surface components of NK cells to knock-out the genes through CRISPR-based gene editing.
  • the methods combine the AAV vector-encapsulated sgRNAs and CRISPR components into one composite system.
  • the methods introduce user-defined genetic modifications into NK cells in a controllable and highly efficient manner to target one or more genes.
  • the gene or genes that are targeted can be transcriptionally repressed and/or translationally repressed and/or undergo targeted degradation and/or targeted by other targeting methods.
  • Other targeting methods include, but are not limited to, dCas9 coupled with transcriptional repressors, antibodies, small molecule inhibitors, and the like.
  • the methods include stimulating a NK cell by contacting the NK cell with a therapeutically effective amount of an inhibitor of at least one gene selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2.
  • the inhibitor is selected from the group consisting of an antibody, an siRNA, and a CRISPR system.
  • the CRISPR system includes a Cas9, and at least one sgRNA complementary to TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2.
  • the methods perform genome editing and screening of a NK cell for a mutation in vitro.
  • An exemplary method includes contacting the NK cell with Cas9 and an AAV library.
  • the AAV library (AAV-SurfV2) includes a plurality of vectors, whereby each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs: 1-61,911 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the NK cell undergoes genome editing and is then screened for a mutation in vitro.
  • the methods edit the genome and screen NK cells for a mutation in vivo.
  • the methods contact an NK cell with Cas9 and an AAV library including a plurality of vectors, each of which includes an expression cassette for an sgRNA including a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1-61,911 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the methods contact an NK cell with Cas9 and an AAV library (AAV-sgRNA) including a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs: 61,912-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the methods contact an NK cell with Cas9 and an AAV library (AAV-sgRNA) including a plurality of vectors, wherein each vector includes an expression cassette for an sgRNA including a nucleotide sequence selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • AAV-sgRNA AAV library
  • the methods modify the NK cell in vitro and administer the NK cell to a subject and the NK cell is screened for a mutation in vivo.
  • the sgRNA is complementary to/targets at least one gene selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2.
  • the methods include one or more steps for identifying and isolating or selecting genetically modified NK cells having one or more desirable characteristics.
  • the desirable characteristics can be phenotypic, such as reduced exhaustion, enhanced tumor penetration, reduced apoptosis, enhanced tumor killing, etc.
  • All the characteristics of genetically modified NK cells can be compared to a control NK cell or population of control NK cells.
  • Methods for assessing phenotypic characteristics of cells are known to those skilled in the art.
  • the methods identify genetically modified NK cells having enhanced tumor killing efficacy.
  • the methods include one or more steps for genetically characterizing the genetically modified NK cells that are identified as having one or more desirable characteristics. Identifying the genetically modified NK cells can include any method commonly known to one of ordinary skill in the art including but not limited to methods of nucleotide sequencing, sgRNA PCR, and/or flow cytometry.
  • NGS Next-generation sequencing
  • B. Exemplary Methods for Identifying Anti-Cancer NK Cells An exemplary screening method is set forth in Figure 1. Typically, the methods include one or more steps as follows: i. Provide the library of sgRNAs configured to target and perturb membrane bound genes in NK cells (e.g., Surf-V2); ii.
  • the methods screen the modified cells by injection into a tumor-bearing animal model and identify animals with enhanced survival/reduced tumor burden. For example, in some forms, the methods isolate target NK cells from the tumor, and characterize the most abundant mutants within the isolated NK cells, e.g., using single cell transcriptome analysis. The methods optionally sequence the pool of selected KO mutants and repeat the screen steps using only best sgRNAs for one or more additional rounds.
  • AAV-Surf-v2 viral library including one or more of SEQ ID NOs:1-61,911 of the sequence listing of WO 2020/028533 was successful to identify mutant NK cells that enhance the anti-cancer activity of NK cells.
  • AAV-CRISPR screening is performed using na ⁇ ve NK cells isolated from a subject.
  • Cas9+ NK cells are transduced with an AAV-Surf-v2 viral library including one or more of SEQ ID NOs:1-61,911 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the methods screen for anti-cancer activity using cancer cells in vitro, ex vivo or in vivo.
  • the methods screen the NK cells for anti-tumor activity using in vivo using an animal model.
  • An exemplary animal model is a syngeneic mouse model of melanoma, GBM, and colon cancer. Methods for establishing mouse models of diseases such as cancer are known in the art.
  • the methods include subcutaneous injection of native B16F10, GL261, and Pan02 into different experimental animals to establish four different cancer models.
  • syngeneic mouse models of breast cancer are established by fat-pad injection of E0771 cells into C57BL/6J mice.
  • the methods introduce AAV-Surf-v2 infected NK cells via adoptive cell transfer into the models (e.g., tumor burden mice) via i.v. (tail vein) injection.
  • the models e.g., tumor burden mice
  • i.v. (tail vein) injection e.g., four screen models are performed, using B16F10 melanoma and E0771 breast cancer models, and GL261 GBM and Pan02 colon cancer models.
  • the methods typically include one or more controls, e.g., by injecting wild-type/non-modified NK cells.
  • the methods investigate the resulting effects upon the tumors within the model animals, for example, by investigation of tumor tissues removed up to 24 days post tumor inoculation.
  • the NK cell screen is different from T cell or other screen due to the distinct biology, culture condition, gene editing and the nature of the NK cell type.
  • Methods of Treatment including administering the genetically modified NK cells as therapeutic agents are described.
  • the methods include Adoptive Cell Therapy (ACT) employing the genetically modified NK cells prepared according to the described methods for screening and genetic manipulation of NK cells.
  • An exemplary method involves treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including genetically modified NK cells prepared according to the described methods and compositions.
  • the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition including NK cells modified according to the disclosed methods.
  • the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen by administering to the subject an effective amount of a pharmaceutical composition including genetically modified NK cells modified to exhibit one or more characteristics that enhance the therapeutic activities of the NK cells in the context of the disease or disorder that is to be treated.
  • Methods of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells engineered to enhance therapeutic efficacy are provided.
  • the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells engineered to knock down or knock out expression of one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2.
  • the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells engineered to knock down or knock out expression of CALHM2.
  • the methods treat a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen.
  • the methods include administering to the subject an effective amount of a genetically modified NK cell modified to express a CAR that targets the antigen.
  • the methods screen and/or genetically modify a NK cell that is a CAR-NK cell.
  • the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR-NK cells engineered to knock down or knock out expression of one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2.
  • a pharmaceutical composition including live, viable CAR-NK cells engineered to knock down or knock out expression of one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2.
  • the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable CAR NK cells engineered to knock down or knock out expression of CALHM2, and/or having diminished or no function of the CALHM2 gene product.
  • the NK cell can have been isolated from the subject having the disease, disorder, or condition, or from a healthy donor, prior to genetic modification.
  • A. Diseases to be treated Methods of treating a subject having a disease or disorder are provided. Typically, the methods administer the genetically modified NK cells and/or CAR NK cells to the subject in an amount effective to treat and/or prevent the disease, or disorder.
  • the subject to be treated can have a disease, disorder, or condition such as but not limited to, cancer, an immune system disorder such autoimmune disease, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or combinations thereof, or can be identified as being at increased risk of developing the disease or disorder.
  • the disease, disorder, or condition can be associated with an elevated expression or specific expression of an antigen.
  • the methods treat or prevent cancer and/or autoimmune disease in a subject in need thereof. 1. Cancer In some forms, the methods treat or prevent a cancer in a subject.
  • Cancer is a disease of genetic instability, allowing a cancer cell to acquire the hallmarks proposed by Hanahan and Weinberg, including (i) self-sufficiency in growth signals; (ii) insensitivity to anti-growth signals; (iii) evading apoptosis; (iv) sustained angiogenesis; (v) tissue invasion and metastasis; (vi) limitless replicative potential; (vii) reprogramming of energy metabolism; and (viii) evading immune destruction (Cell.,144:646–674, (2011)).
  • Tumors which can be treated in accordance with the disclosed methods, are classified according to the embryonic origin of the tissue from which the tumor is derived.
  • Carcinomas are tumors arising from endodermal or ectodermal tissues such as skin or the epithelial lining of internal organs and glands. Sarcomas, which arise less frequently, are derived from mesodermal connective tissues such as bone, fat, and cartilage.
  • the leukemias and lymphomas are malignant tumors of hematopoietic cells of the bone marrow. Leukemias proliferate as single cells, whereas lymphomas tend to grow as tumor masses. Malignant tumors may show up at numerous organs or tissues of the body to establish a cancer.
  • Methods of treating a subject having cancer including administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells and/or CAR NK cells engineered to knock down or knock out expression of one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2 are provided.
  • a pharmaceutical composition including live, viable NK cells and/or CAR NK cells engineered to knock down or knock out expression of one or more genes selected from TGA1/CD49A, ITGA2/CD49B, ITGA3/CD49C, SPN/CD43KLRK1/NKG2D, CD27, TIGIT, PDCD1/PD-1, LAG3 VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, or CALHM2
  • the methods treat a subject having cancer by administering to the subject an effective amount of a pharmaceutical composition including live, viable NK cells and/or CAR NK cells engineered to knock down or knock out expression of CALHM2.
  • the NK cell can have been isolated from the subject having the cancer, or from a healthy donor, prior to genetic modification.
  • Table 2 provides a non-limiting list of cancers for which the CAR of the disclosed methods and compositions can target a specific or an associated antigen. Table 2.
  • the disclosed compositions and methods can be used in the treatment of one or more cancers provided in Table 2.
  • the disclosed compositions and methods of treatment thereof are generally suited for treatment of carcinomas, sarcomas, lymphomas and leukemias.
  • compositions and methods are useful for treating, or alleviating subjects having benign or malignant tumors by delaying or inhibiting the growth/proliferation or viability of tumor cells in a subject, reducing the number, growth or size of tumors, inhibiting or reducing metastasis of the tumor, and/or inhibiting or reducing symptoms associated with tumor development or growth.
  • the types of cancer that can be treated with the provided compositions and methods include, but are not limited to, cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine.
  • cancers such as vascular cancer such as multiple myeloma, adenocarcinomas and sarcomas, of bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine.
  • the compositions are used to treat multiple cancer types concurrently.
  • the compositions can also be used to treat metastases or tumors at multiple locations.
  • tumor cells include, but are not limited to, tumor cells of cancers, including leukemias including, but not limited to, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemias such as myeloblastic, promyelocytic, myelomonocytic, monocytic, erythroleukemia leukemias and myelodysplastic syndrome, chronic leukemias such as, but not limited to, chronic myelocytic (granulocytic) leukemia, chronic lymphocytic leukemia, hairy cell leukemia; polycythemia vera; lymphomas such as, but not limited to, Hodgkin’s disease, non-Hodgkin’s disease; multiple myelomas such as, but not limited to, smoldering multiple myeloma, nonsecretory myeloma, osteosclerotic myeloma, plasma cell leukemia, solitary plasmacytoma and extramedul
  • the methods administer genetically modified NK cells and/or CAR-NK cells to treat one or more non-cancer disease or disorder in a subject in need thereof.
  • the methods treat one or more genetic disease or disorders in a subject, such as a hereditary genetic disease or disorder, or a somatic genetic disease or disorder in a subject.
  • the methods administer genetically modified NK cells and/or CAR-NK cells to treat or prevent an autoimmune disease or disorder in a subject.
  • Any of the methods can include treating a subject having an underlying disease or disorder.
  • the methods treat a disease or disorder, such as a cancer or auto-immune disease in a patient having another disease or disorder, such as diabetes, a bacterial infection (e.g., Tuberculosis), viral infection (e.g., Hepatitis, HIV, HPV infection, etc.), or a drug-associated disease or disorder.
  • the methods treat an immunocompromised subject.
  • the methods treat a subject having a disease of the kidney, liver, heart, lung, brain, bladder, reproductive system, bowel/intestines, stomach, bones or skin.
  • the effective amount or therapeutically effective amount of a pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, such as a cancer or autoimmune disease, or to otherwise provide a desired pharmacologic and/or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying a disease or disorder, such as cancer or autoimmune disease.
  • the amount administered can be expressed as the amount effective to achieve a desired anti-cancer effect in the recipient.
  • the amount of the pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells is effective to inhibit the viability or proliferation of cancer cells in the recipient.
  • the amount of the pharmaceutical composition including modified cells, such as therapeutic NK cells and/or CAR NK cells is effective to reduce the tumor burden in the recipient, or reduce the total number of cancer cells, and combinations thereof.
  • the amount of the pharmaceutical compositions including modified cells is effective to reduce one or more symptoms or signs of cancer in a cancer patient, or signs of an autoimmune disease in a patient having an autoimmune disease or disorder.
  • Signs of cancer can include cancer markers, such as PSMA levels in the blood of a patient.
  • the effective amount of the pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells, that is required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for every pharmaceutical composition.
  • an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • effective dosages and schedules for administering the pharmaceutical compositions including therapeutic NK cells and/or CAR NK cells can be determined empirically, and making such determinations is within the skill in the art.
  • the dosage ranges for the administration of the compositions including therapeutic NK cells and/or CAR NK cells are those large enough to effect reduction in cancer cell proliferation or viability, or to reduce tumor burden for example. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, and sex of the patient, route of administration, whether other drugs are included in the regimen, and the type, stage, and location of the disease to be treated.
  • the dosage can be adjusted by the individual physician in the event of any counter-indications.
  • the effective dosage of the composition including therapeutic NK cells and/or CAR NK cells used for treatment can increase or decrease over the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic assays. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.
  • Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject or patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual pharmaceutical compositions and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. It can generally be stated that a pharmaceutical composition containing NK cells and/or CAR NK cells described herein can be administered at a dosage of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 7 cells/kg body weight, including all integer values within those ranges.
  • patients can be treated by infusing a disclosed pharmaceutical composition containing CAR expressing cells (e.g., genetically modified CAR NK cells lacking expression of the CALHM2 gene) in the range of about 10 4 to 10 12 or more cells per square meter of body surface (cells/m).
  • CAR expressing cells e.g., genetically modified CAR NK cells lacking expression of the CALHM2 gene
  • the infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved.
  • Compositions of NK cells and/or CAR NK cells can also be administered once or multiple times at these dosages.
  • the cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.319:1676, 1988).
  • the unit dosage is in a unit dosage form for intravenous injection.
  • the unit dosage is in a unit dosage form for oral administration.
  • the unit dosage is in a unit dosage form for inhalation.
  • the unit dosage is in a unit dosage form for intra-tumoral injection.
  • Treatment can be continued for an amount of time sufficient to achieve one or more desired therapeutic goals, for example, a reduction of the amount of cancer cells relative to the start of treatment, or complete absence of cancer cells in the recipient.
  • Treatment can be continued for a desired period of time, and the progression of treatment can be monitored using any means known for monitoring the progression of anti-cancer treatment in a patient.
  • administration is carried out every day of treatment, or every week, or every fraction of a week.
  • treatment regimens are carried out over the course of up to two, three, four or five days, weeks, or months, or for up to 6 months, or for more than 6 months, for example, up to one year, two years, three years, or up to five years.
  • the efficacy of administration of a particular dose of the pharmaceutical compositions including modified cells, such as therapeutic T cells, according to the methods described herein can be determined by evaluating the aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of cancer or other diseases and/or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field.
  • a subject for example, if, based on a comparison with an appropriate control group and/or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a tumor has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious. In some forms, efficacy is assessed as a measure of the reduction in tumor volume and/or tumor mass at a specific time point (e.g., 1-5 days, weeks, or months) following treatment. C.
  • any of the disclosed genetically modified cells can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • the compositions described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington's Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapeutics described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans.
  • these include solutions such as sterile water, saline, and buffered solutions at physiological pH.
  • Other therapeutics can be administered according to standard procedures used by those skilled in the art.
  • the pharmaceutical compositions including modified cells, such as therapeutic NK cells and/or CAR NK cells, described herein can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the therapeutic(s) of choice.
  • Pharmaceutical compositions containing one or more modified cells, such as therapeutic T cells, and optionally one or more additional therapeutic agents can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated.
  • a pharmaceutical composition including modified cells can be administered as an intravenous infusion, or directly injected into a specific site, for example, into or surrounding a tumor.
  • a pharmaceutical composition can be administered to a subject as an ophthalmic solution and/or ointment to the surface of the eye, vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes.
  • compositions are administered directly into a tumor or tissue, e.g., stereotactically.
  • Parenteral administration if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No.3,610,795, which is incorporated by reference herein.
  • Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intra-tissue injection (e.g., intraocular injection, intra-retinal injection, or sub-retinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g., an implant including a porous, non-porous, or gelatinous material).
  • intravascular administration e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature
  • peri- and intra-tissue injection e.g., intraocular injection, intra-retinal injection, or sub-retinal injection
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Administration of the pharmaceutical compositions containing one or more genetically modified cells can be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic.
  • compositions are not limited to specific synthetic methods, specific analytical techniques, or to particular reagents unless otherwise specified, and, as such, can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
  • D. Combination therapy Any of the disclosed pharmaceutical compositions including modified cells, such as therapeutic NK cells (e.g., containing a population of genetically modified CAR NK cells lacking expression of the CALHM2 gene), can be used alone, or in combination with other therapeutic agents or treatment modalities, for example, chemotherapy or stem-cell transplantation. As used herein, “combination” or “combined” refer to either concomitant, simultaneous, or sequential administration of the therapeutics.
  • the pharmaceutical compositions and other therapeutic agents are administered separately through the same route of administration. In other forms, the pharmaceutical compositions and other therapeutic agents are administered separately through different routes of administration.
  • the combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject; one agent is given orally while the other agent is given by infusion or injection, etc.,), or sequentially (e.g., one agent is given first followed by the second).
  • preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder or condition.
  • the therapeutic agent is one or more other targeted therapies (e.g., a targeted cancer therapy) and/or immune-checkpoint blockage agents (e.g., anti-CTLA-4, anti-PD1, and/or anti-PDL1 agents such as antibodies).
  • targeted therapies e.g., a targeted cancer therapy
  • immune-checkpoint blockage agents e.g., anti-CTLA-4, anti-PD1, and/or anti-PDL1 agents such as antibodies.
  • the compositions and methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio-frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.
  • the disclosed pharmaceutical compositions and/or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of remission or less active disease.
  • the pharmaceutical compositions can be administered before the additional treatment, concurrently with the treatment, post- treatment, or during remission of the disease or disorder.
  • the disclosed pharmaceutical compositions and the additional therapeutic agents e.g., second or third agent
  • the disclosed pharmaceutical compositions and the additional therapeutic agents can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy.
  • the administered amount or dosage of the disclosed pharmaceutical composition, the additional therapeutic agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy (e.g., required to achieve the same therapeutic effect).
  • the methods administer genetically modified NK or CAR NK cells lacking expression of the CALHM2 gene in combination with one or more additional anti-cancer agents to a subject.
  • targeted therapies are therapeutic agents that block the growth and spread of cancer by interfering with specific molecules (“molecular targets") that are involved in the growth, progression, and spread of cancer.
  • chemotherapeutic or antineoplastic drug can be used in combination with the disclosed pharmaceutical compositions.
  • the additional therapeutic agent is a chemotherapeutic or antineoplastic drug.
  • the majority of chemotherapeutic drugs can be divided into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, monoclonal antibodies, and other anti-tumor agents.
  • the methods administer genetically modified NK or CAR NK cells lacking expression of the CALHM2 gene in combination with one or more conventional therapies for autoimmune diseases to the subject.
  • exemplary therapies for autoimmune diseases include immunosuppressive agents, such as steroids or cytostatic drugs, analgesics, non-steroidal anti-inflammatory drugs, glucocorticoids, immunosuppressive and immunomodulatory agents, such as methotrexate, leflunomide, hydroxychloroquine, and sulfasalazine.
  • the methods administer one or more disease-modifying antirheumatic drugs (DMARDs).
  • DMARDs disease-modifying antirheumatic drugs
  • the methods administer one or more biologic agents for localized treatment (i.e., agents that do not affect the entire immune system), such as TNF- ⁇ inhibitors, belimumab and rituximab depleting B cells, T-cell co-stimulation blocker, anti- interleukin 6 (IL-6), anti-IL-1, and protein kinase inhibitors.
  • the methods also administer one or more monoclonal antibodies (mAbs), such as anti-TNF ⁇ , anti-CD19, anti-CD20, anti-CD22, and anti-IL6R, or other mAbs that target multiple B cell subtypes, and other aberrant cells in autoimmune diseases.
  • mAbs monoclonal antibodies
  • kits The compositions, reagents, and other materials for cellular genomic engineering can be packaged together in any suitable combination as a kit useful for performing, or aiding in the performance of, the methods. It is useful if the components in a kit are designed and adapted for use together in the method.
  • kits with one or more compositions for administration to a subject may include a pre-measured dosage of the composition in a sterile needle, ampule, tube, container, or other suitable vessel.
  • the kits may include instructions for dosages and dosing regimens.
  • kits containing an sgRNA library for example, including a multiplicity of RNAs having a spacer and tracrRNA backbone, the tracrRNA including one or more sequences selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • kits include one or more of an AAV- CRISPR NK cell vector for efficient gene editing and high-throughput screening in NK cells (e.g., the vector including one or more of an antibiotic resistance sequence, two ITRs, two sleeping beauty (SB) IR/DR repeats, a RNA pol III promoter (e.g., U6), a promoter (EFS), a Thyl.l selection marker, an SB lO0x transposase, and a short poly A), a transposase enzyme (e.g., SB100X transposase) or a vector suitable for expressing the transposase enzyme, and instructional material for use thereof.
  • an AAV- CRISPR NK cell vector for efficient gene editing and high-throughput screening in NK cells
  • the vector including one or more of an antibiotic resistance sequence, two ITRs, two sleeping beauty (SB) IR/DR repeats, a RNA pol III promoter (e.g., U6), a promoter (EF
  • the kit includes a plurality of vectors, where each vector independently contains a single sgRNA having a spacer and tracrRNA backbone, the tracrRNA including one or more sequences selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2).
  • the kit contains a population of NK cells (e.g., naive NK cells or CAR NK cells) collectively containing the AAV and/or dCas9.
  • the instructional material can include a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the kit.
  • the instructional material may provide instructions for methods using the kit components, such as performing transfections, transductions, infections, and conducting screens.
  • the kit includes a transposon including a CAR that is specific for an antigen that is selected from a cancer antigen selected from 41BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB
  • Exemplary CARs include CD19BBz or CD22BBz.
  • the kits include a viral vector that is AAV6 or AAV9, and/or cells.
  • a viral vector that is AAV6 or AAV9, and/or cells.
  • the disclosed compositions and methods can be further understood through the following numbered paragraphs.
  • NK Natural Killer
  • the genetically modified NK cell of paragraph 1 or 2 wherein the mutation causes lack or reduction of the expression of one or more of the genes and/or the full- length protein(s) encoded by the gene(s) VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2, and wherein the mutation enhances the anti-cancer efficacy of the NK cell as compared to a non-genetically modified NK cell. 4.
  • CAR Chimeric Antigen Receptor
  • the cancer antigen is selected from the group including 41BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, Ll CAM, IL 13, IL 6, insulin-
  • the cancer antigen is selected from the group
  • a method of treating a subject having a disease, disorder, or condition including administering to the subject an effective amount of the pharmaceutical composition of paragraph 14. 16.
  • a method of treating a subject having a disease, disorder, or condition associated with an elevated expression or specific expression of an antigen the method including administering to the subject an effective amount of the population of genetically modified NK cells of paragraph 13, wherein the NK cells include a CAR that targets the antigen.
  • a method of treating cancer in a subject in need thereof including administering to the subject an effective amount of a population of genetically modified NK cells, wherein at least one gene selected from the group including VNN3, CCR2, SLC2A8, PRNP, CD59B, CEACAM14, and CALHM2 has been mutated in the NK cell. 18.
  • NK cell has been mutated to reduce or knock out the expression of CALHM2 and/or the full-length protein encoded by CALHM2. 19.
  • the NK cell expresses or encodes a Chimeric Antigen Receptor (CAR).
  • CAR Chimeric Antigen Receptor
  • any one of paragraphs 17-20 wherein the cancer is selected from the group including leukemia, vascular cancer such as multiple myeloma, adenocarcinomas and bone, bladder, brain, breast, cervical, colorectal, esophageal, kidney, liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach, and uterine cancer. 22. The method of any one of paragraphs 17-21, wherein the cancer is triple negative breast cancer or glioblastoma. 23. The method of any one of paragraphs 15-22, wherein the NK cells are derived from the subject prior to genetic modification. 24.
  • any one of paragraphs 17-23 wherein the administration includes injection of the composition of cells into or directly adjacent to a tumor, or into the blood stream, or into the brain or into a ventricle of the heart of the subject.
  • 25 The method of any one of paragraphs 17-24, further including administering to the subject one or more additional therapeutic agents and/or procedures.
  • 26 The method of paragraph 25, wherein the additional treatment is selected from the group including a chemotherapeutic agent, and antimicrobial agent, an immune checkpoint inhibitor, a PD-I inhibitor, a CTLA-4 inhibitor, radiation treatment and surgery.
  • a method of performing genome perturbation screening of a Natural Killer (NK) cell including: (i) contacting an NK cell with Cas9 and an sgRNA nucleic acid targeting membrane-bound molecules of Natural Killer (NK) cells, (ii) causing the NK cell to be genetically modified by CRISPR-mediated genome editing of a gene targeted by the sgRNA; and (iii) screening the NK cell for a mutation.
  • the sgRNA includes (i) a guide sequence; and (ii) a tracrRNA including a nucleic acid sequence selected from SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2), which is specifically incorporated by reference herein in its entirety.
  • 29. The method of paragraph 27 or 28, wherein the sgRNA is included within a vector.
  • 30. The method of paragraph 29, wherein the vector is an adeno-associated vector (AAV), and wherein the vector further includes an expression cassette for the sgRNA. 31.
  • AAV adeno-associated vector
  • steps (i)-(iii) are carried out using a plurality of NK cells, and wherein each of the plurality of NK cells is contacted by one or more sgRNAs including one or more of SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2), which is specifically incorporated by reference herein in its entirety.
  • sgRNAs including one or more of SEQ ID NOs:1-69,747 of the sequence listing of WO 2020/028533 (mSurfeome2), which is specifically incorporated by reference herein in its entirety.
  • a genetically modified Natural Killer (NK) cell including a mutation that causes lack or reduction of the expression of the CALHM2 gene and/or the full-length protein encoded by the CALHM2 gene in the cell as compared to a non-genetically modified NK cell, wherein the mutation enhances the anti-cancer efficacy of the genetically modified NK cell as compared to a non-genetically modified NK cell, and wherein the genetically modified NK cell expresses or encodes a Chimeric Antigen Receptor (CAR) that targets a cancer antigen.
  • CAR Chimeric Antigen Receptor
  • the genetically modified NK cell of paragraph 41 wherein the cancer antigen is selected from 41BB, 5T4, adenocarcinoma antigen, alpha fetoprotein, BAFF, B lymphoma cell, C242 antigen, CA 125, carbonic anhydrase 9 (CA IX), C MET, CCR4, CD 152, CD 19, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD28, CD30 (TNFRSF8), CD33, CD4, CD40, CD44 v6, CD51, CD52, CD56, CD74, CD80, CEA, CNT0888, CTLA 4, DR5, EGFR, EpCAM, CD3, FAP, fibronectin extra domain B, folate receptor 1, GD2, GD3 ganglioside, glycoprotein 75, GPNMB, HER2/neu, HGF, human scatter factor receptor kinase, IGF 1 receptor, IGF I, IgGl, Ll CAM, IL 13, IL 6, insulin-like growth
  • the oligo sequences were generated by the Yale Keck facility, and pooled oligos were cloned into double BbsI restriction digest sites of sgRNA vector by Gibson Assembly (NEB), after which, assembly products were transformed into high-efficiency competent cells (Endura) by electroporation.
  • the vector used in this study was a hybrid AAV-SB-CRISPR plasmid for targeting primary mouse NK cells (AAV-SB100x) which is the same one for generating the AAV-SB-Surf-v2 plasmid library was.
  • AAV-SB in vivo barcoding screen The AAV-barcoded libraries were packaged similarly to a previously described approach (Ye, L.
  • Na ⁇ ve NK cells were isolated from the spleens of C57BL/6Ncr mice and cultured with cRPMI medium supplemented with mIL-2 and mIL-15 cytokines for 6 days in vitro. NK cells were transduced with AAV-barcoded viral libraries and cultured for another 4 days. The NK cells were then adoptively transferred into C57BL/6Ncr mice via intravenous (i.v.) tail vein injections, using 4e6 cells / mouse and 8 mice per barcode library. After 48 hours, spleens were extracted, and genomic DNA were isolated using previously described methods (Slattery, K.
  • AAV-SB-CRISPR and barcoding screen readout and sequencing Library readout was performed by nested PCR reactions to decrease the effect of PCR-amplification bias on the screen.
  • the first-round PCR amplified the sgRNA sequence of the AAV-SB vector from genomic DNA ( ⁇ 2.5 ⁇ g per 50 ⁇ L reaction, 24 reactions per sample), and the second-round PCR added barcoded sequencing adapters (2 ⁇ L per 30 ⁇ L reaction, 6 reactions per sample).
  • Primers for PCR#1 including the following oligonucleotide sequences: Forward: 5’-aatggactatcatatgcttaccgtaacttgaaagtatttcg-3’ (SEQ ID NO:4); and Reverse: 5’-actcctttcaagacctagtcgacg-3’ (SEQ ID NO:5) were used.
  • Each round of PCR was performed using the following thermocycler conditions: 98 °C for 1 min, 25 cycles of (98 °C for 1s, 60 °C for 5 s, 72 °C for 12 s), and 72 °C for 2 min. All PCR reactions were performed using Phusion Flash High Fidelity Master Mix.
  • the final PCR products were pooled and normalized for each biological sample before combining individual biological samples.
  • the pooled product 150 ng per sample was then gel purified from a 2% E-gel EX (Life Technologies) using the QiaQuick Gel Extraction kit (Qiagen).
  • the purified, pooled library was then quantified with a gel-based method using the Low-Range Quantitative Ladder (Life Technologies), dsDNA High- Sensitivity Qubit (Life Technologies), BioAnalyzer (Agilent), and/or qPCR. Libraries were sequenced with 5-20% PhiX using an Illumina NovaSeq 4000 sequencer at the Yale Center for Genomic Analysis (YCGA).
  • AAV libraries (AAV-SB-BC-6, AAV-SB-BC-7, AAV-SB-BC-8, AAV-SB-BC- 9) were generated and used to infect, NK cells, and the cells were adoptively transferred into mice.
  • Genomic DNA gDNA
  • Fig.1A Genomic DNA
  • NGS readout data revealed the library representation of all libraries in vivo and their correlations between mice and results from barcoded library representation analyses showed that, while all libraries have substantial fractions of the barcode pools being captured in vivo, there is a clear trend of decreased representation as the library size increases (Fig.1B). All plasmid and cell samples in all four libraries have full coverage (100%).
  • AAV-SB-BC-9 has lost the majority of library in all mice, an in vivo screen with this size of library is challenging. While AAV-SB-BC-6 and AAV-SB-BC-7 have good coverage, their sizes are relatively small to target a meaningful gene set, given that guide RNA redundancy needs to be factored in a CRISPR library. Although AAV- SB-BC-8 has a certain inevitable library representation loss, a library of this size can still consistently be used to recover a substantial fraction of the library without selection pressure, as in this case, of all-theoretically-neutral barcodes in primary NK cells.
  • Example 2 Establishment of Multi-model, high-density in vivo perturbomics assay for tumor infiltrating NK cells
  • a perturbomics assay was established directly in primary NK cells, to systematically map thousands of genes for their quantitative effects in tumor infiltration, with a custom-designed high-density CRISPR library, targeting the surface proteome encoding genes embedded in an AAV-SB vector, in four different in vivo tumor models.
  • the transcriptomic landscapes of tumor-infiltrating NK cells were characterized through single-cell RNA-sequencing (scRNA-seq) of tumor-infiltrating NK cells.
  • scRNA-seq single-cell RNA-sequencing
  • mice constitutive Cas9-expression mice, known as Rsky/Cas9 ⁇ mice (Rosa26-Cas9- 2A-EGFP in C57BL/6, B6), as well as C57BL/6, B6 CD45.1, and NOD-scid IL2R- gamma-null (NSG) mice.
  • Rsky/Cas9 ⁇ mice Rosa26-Cas9- 2A-EGFP in C57BL/6, B6
  • C57BL/6, B6 CD45.1 NOD-scid IL2R- gamma-null mice.
  • NSG NOD-scid IL2R- gamma-null mice.
  • Each mouse strain was purchased from JAX and bred in-house for in vivo tumor model experiments.
  • Cell lines NK-92 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).
  • NK-92 and CAR-NK92 cells were cultured with MEM- ⁇ (no nucleosides), supplemented with 2 mM L-glutamine, 0.2 mM myo-inositol, 0.02 mM folic acid, 0.1 mM 2-mercaptoethanol, 200 IU/ml human recombinant IL-2 (Biolegend), 12.5% FBS, 12.5% horse serum, and 1% penicillin/streptomycin (Gibco, Life Technologies, America).293T human embryonic kidney (HEK) cells were bought from ATCC and cultured with DMEM, supplemented with 10% FBS and 1% penicillin/streptomycin (D10 media).
  • Human cancer cell lines (HT-29, MCF-7, MDA- MB-231, NALM6, and MM.1R) were infected with lentivirus with firefly luciferase and either puromycin or EGFP selection genes (pXD023 and pXD024 plasmids, respectively) to generate the GFP-Luciferase (GL) and Puromycin-Luciferase (PL) cell lines, used for the in vivo tumor assays.
  • Adherent cancer cell lines were cultured in D10 media, while the leukemia cell lines (NALM6-GL and MM.1R-PL) were cultured in RPMI medium, supplemented with 10% FBS and 1% penicillin/streptomycin.
  • Cancer- antigen overexpression models were used for indicated experiments, and these cell lines were generated by infection with CD22-Blasticidin, BCMA-Blasticidin or HER2- Blasticidin lentiviral vectors for NALM6-CD22-GL, MM.1R-BCMA-PL, and MCF7- HER2-PL, respectively.
  • Mouse NK cells isolation and culture Spleens were dissected from B6.Rosa26-Cas9 (also noted as Rsky/Cas9 ⁇ mice) or C57BL/6J mice and placed into ice-cold PBS supplemented with 2 % FBS.
  • Lymphocytes were released by grinding organs through a 100 mm filter, washed with 2 % FBS, treated with 1 mL ACK lysis buffer (Lonza) per 2 spleens at 1-2 min at room temperature, neutralized with 2 % FBS, filtered through 40 mm filters, and then NK cell were purified using EasySepTM Mouse NK Cell Isolation Kit (Stem Cell), according to the manufacturer’s protocol.
  • NK cells were cultured at 5e6 cells / mL density in plates or dishes with RPMI-1640 (Gibco) media with 10 % FBS, 2 mM L-Glutamine, 200 U / mL penicillin–streptomycin (Gibco), 49 mM 2-mercaptoethanol (Sigma) (cRPMI), supplemented with 50 ng / mL IL-2 (Biolegend), and 50 ng / mL IL-15 (Biolegend).
  • RPMI-1640 Gibco
  • FBS 10 % FBS
  • 2 mM L-Glutamine 200 U / mL penicillin–streptomycin
  • cRPMI 49 mM 2-mercaptoethanol
  • sgRNAs were then identified using FlashFry68, following default settings and using the scoring metrics “deonch2014ontarget”, “rank”, “minot”, “doench2016cfd”, and “dangerous”. With the resultant scoring matrix, sgRNAs were first filtered for those that did not have high GC content, no polyT tracts, and exactly one match in the mm10 genome.
  • the sgRNAs targeting a given gene were then ranked by using the “doench2014ontarget” and “doench2016cfd” scores, by first converting each score to nonparametric ranks where high “doench2014ontarget” scores correspond to high ranks, while low “doench2016cfd” scores correspond to high ranks. The two nonparametric ranks were then added together, weighting the “doench2014ontarget” rank twice as heavily as the “doench2016cfd” rank.
  • all of the sgRNAs that are contained in the Brie library69 were first selected, then the composite ranks described above were used to choose the top scoring sgRNAs, up to a total of 20 sgRNAs per gene.
  • the final set of on-target sgRNAs was composed of 56,911 sgRNAs targeting 2863 murine genes.
  • a set of non-targeting control sgRNAs was designed by generating 500,000 random 20 nt sequences, followed by sgRNA scoring in FlashFry. The top 5000 non-targeting control sgRNAs were selected by choosing sgRNAs with a “doench2016cfd” score ⁇ 0.2 and ⁇ 100 total potential off-targets (maximum 4 mismatches). These 5000 control sgRNAs were added to the library, for a total of 61,911 sgRNAs.
  • the custom sgRNA vector used in this study was a hybrid AAV-SB-CRISPR plasmid for targeting primary mouse NK cells (AAV-SB100x) that was constructed by gBlock fragments (IDT) followed by Gibson Assembly (NEB).
  • the Surf-v2 library (sgRNAs of SEQ ID NOs:1-69,747 the sequence listing of WO 2020/028533 (mSurfeome2), which is specifically incorporated by reference herein in its entirety) was cloned into the AAV-SB- CRISPR vector by pooled cloning to generate the AAV-SB-Surf-v2 plasmid library.
  • AAV production The AAV-SB-Surf-v2 and barcode libraries were packaged similarly to another described approach (Ye, et al. Cell Metab 34, 595-614.e514 (2022)) . Low-passage HEK293FT cells were used for AAV production.
  • D10 medium DMEM (Gibco) medium supplemented with 10% FBS (Sigma) and 200 U/mL penicillin-streptomycin (Gibco)
  • FBS-free pre-warmed DMEM
  • HEK293FT cells were transiently transfected with 5.4 ⁇ g transfer, 8.7 ⁇ g serotype (AAV6), and 10.4 ⁇ g packaging (pDF6) plasmids, using 130 ⁇ L PEI. After 6-12 hrs of transfection, DMEM was replaced with 20 mL pre-warmed D10 medium.
  • the mixture was incubated on the ice for 1 hr followed by centrifugation at 20,000 x g at 4 °C for 15 min. The supernatant was discarded, and the pellet was resuspended with 5-15 mL PBS, 1 mM MgCl 2, and 250 U / ml Benzonase (Sigma), incubated at 37 °C for at least 30 min.
  • One volume of chloroform was added, shaken vigorously, and spun down at 15,000 x g at 4 °C for 15 min. The aqueous layer was collected carefully and concentrated using AmiconUltra 100 kD ultracentrifugation units (Millipore). Virus was aliquoted and stored at -80 °C.
  • RT-qPCR was performed using Taqman assays (ThermoFisher), targeted to the human EFS promoter engineered in the AAV vector.
  • AAV-SB-Surf-v2 NK cell in vivo screen in syngeneic tumor models The AAV-CRISPR screen was performed with >400x coverage, in which > 5e7 Cas9+ NK cells were transduced with an approximate ⁇ 50% infectivity rate, using the AAV-Surf-v2 viral library (0.5 infectivity * 5e7 cells / 61,911 sgRNAs > 400-fold coverage).
  • Na ⁇ ve NK cells were isolated from the spleens of Rsky/Cas9 ⁇ mice.
  • Syngeneic mouse models of melanoma, GBM, and pancreatic cancers were setup with subcutaneous injections of 2e6 B16F10, 5e6 GL261, or 4e6 Pan02 cells, respectively.
  • Syngeneic mouse models of breast cancer were established by fat-pad injections of 2e6 E0771 cells into C57BL/6J mice.
  • AAV-Surf-v2-infected NK cells were adoptively transferred into tumor burden mice via i.v. tail vein injections.
  • B16F10 melanoma and E0771 breast cancer models were euthanized by 20 days post tumor implantation (dpi), while GL261 GBM and Pan02 pancreatic cancer models were euthanized at 27 dpi and 24 dpi, respectively.
  • dpi days post tumor implantation
  • Pan02 pancreatic cancer models were euthanized at 27 dpi and 24 dpi, respectively.
  • 4e6 AAV-Surf-v2 infected NK cells were injected into 9 and 10 tumor-burden mice, respectively; 2e6 AAV-Surf-v2 infected NK cells were injected into 7 Pan02 pancreatic cancer and 11 GL261 GBM mouse models.
  • Lysis Buffer 50 mM Tris, 50 mM EDTA, 1% SDS, pH adjusted to 8.0
  • 30 ⁇ L of 20 mg/mL Proteinase K (Qiagen) was added, then incubated at 55 °C overnight. After tissue digestion, 30 ⁇ L of 10 mg/mL RNase
  • Genomic DNA was air dried for 30-60 min, and then resuspended in 0.5-1 mL nuclease-free water overnight at room temperature.
  • 100-200 ⁇ L QuickExtract solution (Epicentre) was directly added to cells and incubated at 65 °C for 30 min.
  • QIAmp Fast DNA Tissue Kit (Qiagen) was used for gDNA extraction following the manufacturer’s protocol.
  • Single-cell RNA-sequencing of tumor-infiltrating NK cells NK cells were isolated from the spleen C57BL/6J mice and expanded through in vitro culture.
  • NK cells were adoptively transferred into tumor burden mice via i.v. (tail vein) injection. At 7 and 15 days after NK cells adoptively transferred, one mouse was sacrificed and the spleen and dissected tumor were collected.
  • NKp46 and NK1.1 double positive NK cells were isolated by fluorescence-activated cell sorting (FACS), using FACS-Aria (BD). Sorted NK cells were counted and processed for single- cell RNA-sequencing library preparation by YCGA following manufacturer’s protocols.
  • Lentivirus production Lentivirus was produced using low-passage HEK239FT cells.
  • HEK293FT or HEK293T cells were seeded in 15 cm-dish at 50-60 % confluency.
  • D10 media was replaced with 13 mL pre- warmed Opti-MEM medium (Invitrogen).
  • Opti-MEM was mixed with 20 ⁇ g CAR containing plasmid, 15 ⁇ g psPAX2 (Addgene), 10 ⁇ g pMD2.G (Addgene) and 100 ⁇ L lipofectamine 2000 (Thermo Fisher).
  • crRNA and tracrRNA were mixed in 1:1 ratio (final concentration 50 ⁇ M), heated at 95 °C for 5 min in a thermal cycler, then cooled to room temperature.3 ⁇ L HiFi Cas9 protein (61 ⁇ M; Invitrogen) was mixed with 2 ⁇ L Buffer R for each reaction (Neon Transfection System Kit, Invitrogen), then mixed with 5 ⁇ L annealed crRNA:tracrRNA duplex, incubated the mixture at room temperature for 15 min.3e6 of NK92 cells per reaction were resuspended in 100 ⁇ L Buffer R which included 10 ⁇ L RNP complex.100 ⁇ L of cell:RNP mixture was loaded into the Neon Pipette without bubbles.
  • the electroporation parameter was set at 1600 V, 10 ms for 3 pulses.
  • Cells were immediately transferred to a 24-well plate with pre-warmed media after electroporation.
  • KO efficiency for each target was examined after 5 days with T7E1 assay.
  • Lentivirus was produced by HEK293T cells, and the supernatant was collected and precipitated using Lenti-X Concentrator (Takara). Lentiviral pellets were resuspended with NK92 complete culture media, then aliquoted and stored at -80°C.
  • AAVS1-KO and CALHM2-KO NK92 cells were transduced with lentivirus at 1-2e6 cells / ml in a 12-well plate, which was pre-coated with Retronectin (Takara) in PBS, overnight at 4°C. The spin-infection was performed at 32°C at 900 x g for 90 min.
  • the CAR-positive AAVS1-KO and CALHM2-KO NK92 cells were selected with 3 ⁇ g /mL puromycin for 3 days and measured at day 7, after transduction. Then CAR-NK92 cells were used for different assays.
  • CAR-NK92 cytotoxicity assay To detect the cytotoxic capability of CALHM2-KO CAR-NK92 cells, cancer cell lines of NALM6-GL, MCF7-PL, MCF7-PL-HER2-OE, MDA-MB-231-PL, and MM.1R-PL were established as described above. The cancer cells were seeded in a 96- well plate first, then different Effector (NK92 cells): Target (cancer cells) ratio (E: T ratio) co-cultures were set up. Cytolysis was measured by adding 150 ⁇ g / mL D- Luciferin (PerkinElmer) using a multi-channel pipette. Luciferase intensity was measured by luminometer (PerkinElmer).
  • mRNA-seq Bulk mRNA sequencing (mRNA-seq) library preparation
  • the mRNA library preparations were performed using a NEBNext® UltraTM RNA Library Prep Kit, and samples were multiplexed using barcoded primers provided by NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 2).
  • CALHM2-KO anti-HER2 CAR-NK92-hIL2 cells and AAVS1-KO anti-HER2-CAR-NK92-hIL2 cells were stimulated with HT29-PL cancer cells with 1:1 Effector (NK cells): Target (cancer cells) ratio.4 hrs later, CAR-NK92 cells were sorted out and subsequently went for RNA extraction and mRNA-seq library preparations.
  • Membranes were blocked with 5 % non-fat milk in TBS-T for 1 hr at room temperature, followed by the primary antibody incubation at 4 °C overnight, including ⁇ -CALHM2 polyclonal antibody (1:400) (Invitrogen, PA5-53219) and ⁇ -Vinculin Recombinant Rabbit Monoclonal antibody (1:400) (Invitrogen, 700062).
  • the membrane was washed with PBS-T 3 times, each for 10 min with agitation.
  • the membrane was incubated with a goat anti-Rabbit IgG (H+L) secondary antibody with HRP (1:2500 ⁇ 1:5000) (Invitrogen, 65-6120) for 1 hr at room temperature with agitation.
  • the membrane was washed with PBS-T 3 times, each for 10 min with agitation.
  • the membrane was treated with ECL substrate (Bio-Rad), and the relative levels of Calhm2/CALHM2 protein were quantified by greyscale analysis.
  • NOD-scid IL2R-gamma-null (NSG) mice were purchased from JAX and bred in- house. Eight-to-twelve-week-old male mice were inoculated with 2e6 HT29-GL cells through subcutaneous injection. After 12 days, 5e6 AAVS1-KO or CALHM2-KO anti- HER2-CAR-NK92-hIL2 were injected intravenously into tumor burden mice.
  • In vivo tumor infiltration assay of Calhm2 KO mouse NK cells Syngeneic mouse models of breast cancer were established by injecting 1e6 E0771 cells into the fat-pads of CD45.1 mice. Tumor-bearing mice were randomly assembled into different treatment groups.
  • Na ⁇ ve NK cells were isolated from the spleens of Cas9-expressing mice, transduced with AAV-Calhm2 and AAV-pLY017b vectors, separately, after 6 days of in vitro culture. Cells wre cultured for an additional 4 days in vitro before being adoptively transferred into tumor burden mice via tail vein injection (7e6 NK cells per mouse).2 days later, all mice were euthanized. Spleen and tumors were dissected and used for following assays.
  • NOD-scid IL2R-gamma-null (NSG) mice were purchased from JAX and bred in- house.
  • mice Eight-to-twelve-week-old female mice were inoculated with 4e6 HT29-GL cells through subcutaneous injection. After 19 days, tumor-bearing mice were randomized into two groups that were treated with either 1e7 AAVS1-KO or CALHM2-KO anti- HER2-CAR-NK92-hIL2 intravenously. Mice were euthanized at 21 and 28 dpi, as indicated, and tumors and spleens were collected for flow cytometry analyses. All mice were sacrificed at 28 dpi. Isolation of splenocytes and TILs Mice were euthanized at indicated time point. Tumors and spleens were collected and kept in ice-cold 2% FBS.
  • spleens For spleens, they were placed in ice-cold 2% FBS and mashed through a 100- ⁇ m filter. Splenocytes were washed once with 2% FBS. Tumors were minced into 1- to 3-mm size pieces using a scalper and then digested using Collagenase IV for 30–60 min at 37 °C. Tumor suspensions were filtered through a 100- ⁇ m cell strainer to remove large bulk masses.
  • Red blood cells were lysed with 1 mL ACK Lysis Buffer (Lonza) per spleen, 2 mL ACK Lysis Buffer (Lonza) per tumor sample by incubating 2–5 min at room temperature, which was followed by dilution with 10 ml 2% FBS and a pass through a 40- ⁇ m filter. Splenocytes were resuspended in 2% FBS buffer, counted for flow cytometry staining. Single-cell suspensions of tumors were used for flow cytometry staining. FACS analysis of tumor infiltrating NK cells Single tumor cell suspensions were prepared using the Collagenase IV digestion with the method described above.
  • Tumor cells were blocked using anti-Fc receptor anti- CD16/CD32. Live cells were distinguished from dead cells in flow cytometry by staining with a LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit, following the manufacturer’s instructions.
  • mouse NK cells cells at a density of 107 ml –1 were stained with dimethyl sulfoxide-dissolved live/ dead staining dye and PE/Cy7 conjugated anti-CD3, FITC conjugated anti-NKp46, BV421 conjugated anti-CD45.2 antibody in MACS buffer (PBS + 0.5% BSA+2 mM EDTA) and incubated on ice for 30 min. Stained cells were washed three times before being analyzed on a BD FACSAria.
  • CAR NK cells For CAR NK cells, cells at a density of 107 ml –1 were stained with dimethyl sulfoxide-dissolved live/ dead staining dye and APC conjugated anti-human CD56 antibody in MACS buffer and incubated on ice for 30 min. Stained cells were washed three times, and resuspended in 200 ⁇ L MACS with 50 ⁇ L Precision Count Beads TM (Biolegend) before being analyzed on a BD FACSAria. Cell proliferation assay Cells were collected, counted, and adjusted to 1 e7 cells / ml with CellTraceTM Far Red staining (1:1000 dilution) staining solution. Cells were incubated at 37 °C for 20 mins.
  • Statistical Genomics Methods and Protocols 1418, 391-416 (2016) to calculate sgRNA enrichment, after which, sgRNA statistics are aggregated to acquire FDR-corrected p values and z-scores for each gene.
  • the CRISPR-SAMBA analysis was performed with tumor-infiltrating, splenic, and pre-injection control NK cell screen readout samples (default settings). Briefly, sample-variability of thesgRNA measurements was estimated as common and trended dispersion, representing dataset-wide variability and sgRNA mean- dispersion relationship, respectively.
  • the estimated dispersion was used to fit to a quasi- likelihood (QL) generalized log-linear model ( ⁇ InVivo + Tumor, where InVivo is any tumor or spleen sample), and the unbiased raw dispersion was squeezed towards the estimated mean-dispersion abundance trendline to adjust for uncertainty, related to low sample numbers.
  • QL quasi- likelihood
  • the fitted QL models showed strong separation of InVivo and Tumor coefficients, based on heatmaps of the top 1000 highly-variable gRNAs (top trended dispersion).
  • the sgRNA-level results were then determined with a QL-F test, using “Tumor” as the coefficient.
  • Gene-level results were attained using a meta-analysis of the sgRNA-level results, including sgRNAs with log-FC values > the top 10% of non- targeting control sgRNAs, representing a 10% FDR, and a minimum of 2 sgRNAs were used per gene.
  • the meta-analysis p values for each gene were calculated using the Fisher method (sum of logs), and the gene-level results were adjusted using the Benjamini-Hochberg FDR method.
  • enriched genes were those with an FDR-adjusted p value ⁇ 0.05 and an absolute z-score > 0.8.
  • the enriched genes were narrowed down to include those with detectable expression in NK cells, based on 18 NK samples from the ImmGen project (GEO: GSE122597) (Aguilar, et al., Nature Immunology 21, 700-703 (2020); Benoist, et al., Nature Immunology 17, 741-741 (2016);Gal-Oz, et al. Nature Communications 10 (2019); Zemmour, et al., Nature Immunology 23, 643-644 (2022)). ImmGen NK count data were processed by calculating the log2-transformed (0.5 pseudocount) gene-averaged counts-per-million, and log-expression > 1 was considered detectable expression.
  • NK cells Single cell profiling Splenocytes were collected from mice, and NK cells were purified by flow sorting, selecting NKp46+NK1.1+ cells with a FACSAria sorter. NK cells were then normalized to 1000 cells/ ⁇ L. Standard volumes of cell suspension were loaded to achieve targeted cell recovery to 10000 cells. The samples were subjected to 14 cycles of cDNA amplification. Following this, gene expression (GEX), TCR-enriched and BCR- enriched libraries were prepared according to the manufacturer’s protocol (10x Genomics). All libraries were sequenced using a NovaSeq 6000 (Illumina) with 2x150 read length.
  • GEX gene expression
  • TCR-enriched and BCR- enriched libraries were prepared according to the manufacturer’s protocol (10x Genomics). All libraries were sequenced using a NovaSeq 6000 (Illumina) with 2x150 read length.
  • Single cell transcriptomics data processing Single-cell RNA-seq data were pre-processed with Cell Ranger v6.0.1 (10x Genomics) pipeline, using a standard pipeline that aligned reads to the mm10 mouse reference transcriptome and aggregated multiple datasets with the “agg” function. The aggregated datasets were subsequently processed using the Seurat v4.0.5 package for the R statistical programming language (Satija, et al., Nat Biotechnol 33, 495-502 (2015)).
  • each dataset was filtered to include cells with (1) 200-2500 RNA features, (2) ⁇ 5% mitochondrial RNA, (3) ⁇ 0.1% expression of Kcnq1ot1 (representing low-quality cells) (Jordao, et al., Science 363, 365-+ (2019)), and (4) ⁇ 5% combined expression of Gm26917 and Gm42418 (representing rRNA contamination)(Liu, et al., Iscience 23 (2020)).
  • Integrated data were rescaled, and dimensional reduction was performed by uniform manifold approximation and projection (UMAP)(Becht, et al., Nat Biotechnol (2016)) using the first 27 dimensions from PCA, which were chosen by the inflection point of an elbow plot.
  • UMAP uniform manifold approximation and projection
  • SNN shared nearest neighbor
  • the labeled NK cell populations were assessed for within-cluster homogeneity by (a) performing Wilcoxon rank sum analyses of scaled expression data in each cluster compared to all other cells, (b) selecting the top 100 DE genes for each cluster (FDR-adjusted p value ⁇ 0.01, absolute log-FC > 1, cluster detection rate > 20%), and (c) determining the presence of discreet cluster-specific transcriptional patterns by hierarchical clustering and heatmap visualization.
  • Single-cell differential expression analyses Differential expression (DE) analyses of single-cell transcriptomics data were performed using a custom R pipeline, as previously described.
  • GLMs Gamma-Poisson generalized log-linear models
  • QLF quasi-likelihood F
  • DE genes were those with an FDR-adjusted p value ⁇ 0.01 and an absolute log2 fold-change (log-FC) > 2.
  • Bulk mRNA sequencing Bulk mRNA sequencing was performed in HT29-stimulated and unstimulated ⁇ HER2-CAR-NK cells, in which there were paired AAVS1-KO and CALHM2-KO samples.
  • Raw sequencing data were filtered and had adapters removed by Trimmomatic v0.39 in paired-end mode, clipping Illumina TruSeq adapters with the following settings: LEADING:3 TRAILING:3 SLIDINGWINDOW:4:20 MINLEN:3085.
  • Trimmed, filtered reads were then aligned to the human transcriptome (GRCh38 Gencode version 96) using Kallisto v0.45.0 with the default settings (Bray, Nat Biotechnol 34, 525-527 (2016)). Aligned reads were imported in R using the EdgeR package (Lun, et al., Statistical Genomics: Methods and Protocols 1418, 391-416 (2016)), and the data were processed by removing low-expression transcripts with the filterByExpr command (default settings), normalized by trimmed mean of M-values method87. GLM dispersions were then calculated, and the data were fit to a quasi-likelihood (QL) negative binomial GLM for paired samples ( ⁇ CALHM2-KO*Stimulation + Sample).
  • QL quasi-likelihood
  • DE analyses were performed using empirical-Bayes QL F tests (Lun, et al., Statistical Genomics: Methods and Protocols 1418, 391-416 (2016)), using either CALHM2-KO, Stimulation, or CALHM2-KO:Stimulation interaction as the coefficient.
  • DE genes were those with an FDR-adjusted p value ⁇ 0.05 and an absolute log2 fold-change (log-FC) > 0.80.
  • Meta-pathway analyses Meta-pathway analyses were performed using a modified pipeline of previously described strategy (Covid var paper).
  • upregulated or downregulated DE genes were sorted by p-value and used as input for gene set enrichment analyses by the gProfiler2 R package with gene ontology (GO) terms for biological processes and known genes as the analysis domain (Kolberg, et al., Elife 9 (2020), Raudvere, et al., Nucleic Acids Res 47, W191-W198 (2019)).
  • the threshold of DE was lowered to include more genes (screens: absolute z-score > 0.8, q ⁇ 0.05; bulk RNA-seq: absolute log-FC > 0.8, q ⁇ 0.01).
  • Leiden clustering modulearity optimization method, 500 iterations
  • circular bar plots were used to display functionally relevant meta-pathways, which (a) had at least one of the selected GO ancestor terms (leukocyte proliferation, cell activation, leukocyte differentiation, cell adhesion, chemotaxis, immune effector process, leukocyte migration, transport, cell communication, response to cytokine, defense response, metabolic process, regulation of apoptotic process, cell motility, cell death, and cell population proliferation) and were (b) filtered to include only the most significant of any meta-pathways with 100% DE gene overlap.
  • NGS experiments were performed with biological replications as indicated in the manuscript. Randomization and blinding statements Regular in vitro experiments were not randomized or blinded. Mouse experiments were randomized by using littermates, and blinded using generic cage barcodes and eartags where applicable. High-throughput experiments and analyses were blinded by barcoded metadata. Standard statistical analysis Standard statistical analyses were performed using regular statistical methods. GraphPad Prism, Excel and R were used for all analyses. Different levels of statistical significance were accessed based on specific p values and type I error cutoffs (0.05, 0.01, 0.001, 0.0001). Further details of statistical tests were provided in figure legends and/or supplemental information.
  • Flow cytometry data was collected by BD FACSAria. All deep sequencing data were collected using Illumina Sequencers at Yale Center for Genome Analysis (YCGA). Co-culture killing assay data were collected with PE Envision Plate Reader. Flow cytometry data were analyzed by FlowJo v.10.7. Results High-density in vivo perturbomics of tumor infiltrating NK cells To systematically map the quantitative contribution of factors that influence NK cell tumor infiltration, in vivo CRISPR-mediated knockout screens were performed directly in primary NK cells using a custom high-density single guide RNA (sgRNA, gRNA) library, in four different tumor models (Fig.2A).
  • sgRNA, gRNA single guide RNA
  • Surf-v2 a high-density sgRNA library targeting the mouse homologs of the human surface proteome were designed.
  • Surf-v2 targets 2,863 genes with up to 20 sgRNAs per gene (56,911 gene- targeting sgRNAs) which were used to increase gene representation and mitigate the considerable drop-out that is expected from in vivo screening.
  • a total of 5,000 non- targeting control (NTC) sgRNAs reverse-ranked by potential off-targets from 500,000 random 20 nt sequences were spiked into the final library, for a total of 61,911 sgRNAs.
  • Surf-v2 was cloned into the chimeric AAV-SB-CRISPR vector, which has high gene editing efficiency in primary immune cells.
  • the AAV-SB-Surf-v2 viral library was then produced by packaging with AAV6 serotype system and transduced primary Cas9- expressing splenic NK cells from constitutive transgenic Cas9 mice with a C57BL/6 (B6) background (Fig.2A). These donor NK cells were intravenously (i.v.) injected into syngeneic host B6 mice pre-implanted with tumors. These experiments were performed with four tumor models in parallel, B16F10 melanoma, E0771 triple negative breast cancer (TNBC), GL261 glioblastoma and Pan02 pancreatic cancer (Fig.2A; Methods).
  • TNBC triple negative breast cancer
  • Fig.2A Pan02 pancreatic cancer
  • genomic DNA (gDNA) samples were extracted from pre-injection NK cells as well as tumors and spleens of tumor-bearing animals for screen readout (Fig.2A).
  • NGS next-generation sequencing
  • sgRNA library representations across all samples produced a dataset of multi-model, high-density in vivo perturbomics of tumor infiltrating NK cells.
  • NGS next-generation sequencing
  • GLM negative binomial generalized linear models
  • SAMBA then calculated a gene-level scores as a weighted sum from the coefficient-of-interest (i.e., Tumor) in a way that considers only top-performing guides, removing the effect of poorly detected sgRNAs that can adversely affect gene scores in other algorithms, such as MAGeCK-RRA (Li, et al. Genome Biol 15, 554 (2014)).
  • the sgRNA-level statistics demonstrated strong overall enrichment in tumor samples from the E0771 and B16F10 models, with moderate enrichment observed in GL261 and Pan02 models. Greater similarity between the overall log-fold changes of the B16F10 and E0771, as well as between GL261 and Pan02 models was observed
  • the sgRNA statistics were then aggregated into gene-level results, and subsequently filtered include those with detectable expression in primary NK cells, based on ImmGen project data 32, 33, 34, 35, to identify enrichment in 327, 336, 54, and 10 genes in the B16F10, E0771, GL261, and Pan02 models, respectively (z-score > 1, q value ⁇ 0.01) (Figs.2B-2E).
  • NK tumor-infiltration screens include (1) “cell adhesion”, as well as (2) “leukocyte proliferation” and (3) “positive regulation of apoptotic processes”, as a respective increase in NK cell expansion or survival could also result in increased numbers of NK cells within tumors (Fig.5).
  • Several representative genes include markers of exhaustion, such as Tigit, Pdcd1/PD-1, and Lag3 (Judge, et al. Frontiers in Cellular and Infection Microbiology 10 (2020)).
  • Figs.6A-6C Other notable hits include the Klrk1/NKG2D, an NK cell activation receptor and marker of different NK cell developmental phases; CD27 of immature NK cells (iNK); as well as Itga1/Cd49a, Itga2/Cd49b, Itga3/Cd49c, and Spn/Cd43 of mature NK (mNK) (Figs.6A-6C). Noted that since the library started with a defined set of surface / membrane genes, there is natural limitation of these pathway analyses.
  • this in vivo primary NK cell AAV-CRISPR screen dataset provided an overall quantitative perturbomics of surfaceome encoding genes in tumor infiltrating NK cells in four syngeneic tumor models, revealing a diverse collection of enriched hits.
  • screen hits were then assessed by generating individual gene knockouts in primary Cas9+ NK cells, using the same AAV-SB-CRISPR vector expressing individual gene-targeting sgRNAs. These individual gene knockout NK cells were tested for their cancer lysis ability with in vitro co-culture assays using different cancer cell lines.
  • Example 3 Single-cell transcriptomic investigation of tumor-infiltrating NK cells Results To further understand the tumor infiltration and behaviors of NK cell subsets, and to gain independent global functional maps of the NK cells in the tumor micro- environment, single-cell RNA-seq analyses were performed. Due to the strength of overall selection in the in vivo CRISPR screens above, two tumor models, B16F10 and E0771 were chosen for single cell analysis of primary tumor infiltrating NK cells. Again, these orthotopic syngeneic tumor models were established by subcutaneous transplantation of B16F10 cells, and mammary fat pad transplantation of E0771 cells, into B6 mice.
  • donor splenic NK cells were then isolated, also from B6 mice, without perturbations, and adoptively transferred them into tumor-bearing mice. After 7 days, NK cells were isolated from tumors and spleens at 7 and 15 days-post-injection by fluorescence assisted cell sorting (FACS) and subjected them to single-cell transcriptomics profiling using the 10X Genomics platform (Fig.7). Pre-transfer donor NK cells were also sequenced in parallel to serve as a control/baseline while exploring the effects of (1) time, (2) tumor type, and (3) tissue localization on NK cell phenotype . A total of nine different scRNA-seq datasets were generated, represented by the various factors of time, tumor type, and tissue/localization (Fig.7).
  • the cell populations were clustered by shared nearest neighbors (SNN) modularity optimization (Luvain algorithm with multilevel refinement) with the resolution optimized to ensure a unique transcriptional pattern between clusters, using highly variable genes (Stuart, et al., Cell 177, 1888-1902 e1821 (2019), Satija, et al., Nat Biotechnol 33, 495-502 (2015)).
  • Cell populations were classified by the expression of known cell type-specific markers (Figs.8D-8E), leading to the identification of various immune cell subtypes that were excluded from further analysis.
  • the remaining Ncr1+ cells were re-processed, visualized, and unbiasedly clustered.
  • NK sub-populations were broadly classified using relatively conservative definitions for murine NK cells, based on the expression of Cd27, Itgam, and Cd3e. This resulted in the detection of 5 groups of immature NK cells (iNK; Ncr1+Cd27+Itgam-), 1 group of NK-T cells (Ncr1+Cd3e+), 1 group of transitional NK (tNK; Ncr1+Cd27+Itgam+), 2 groups of mature NK cells (mNK; Ncr1+Cd27-Itgam+), ILC1 (Ncr1+CD160+ Eomes+) and NCR+ ILC3 (Ncr1+Il7r+Kit+Rora+Gpr183+) (Figs 8A-C, Fig 10).
  • tNK and mNK populations were considered to be Itgam+, if more than 10% of the population had detectable expression.
  • all of the iNK cells express some level of granzyme b gene (GzmB), while Ifng expression is only high in the tNK and iNK4 cells (Figs.8A-8C), which is the only iNK subset with significantly increased Cxcr4 expression, as well as no detectable expression of the Spn (Cd43) adhesion gene (Figs. 8F-8I).
  • iNK2 and iNK4 have effector phenotypes marked by upregulation of cytotoxic genes (Gzmc in iNK2; Gzmb and Ifng in iNK4) and effector function pathways.
  • the iNK1 cells display reduced effector phenotype among the iNK subpopulations, and iNK5 displays a proliferative phenotype with upregulated Top2a gene expression and related pathways, including mitotic cell process and cell division.
  • ILC cell populations each expressing Lag3 and the Tox exhaustion TF, but Tox expression higher in ILC1 cells (Figs.8A-8C).
  • the ILC1 cells also express Ctla4 and higher levels of Pdcd1. Both exhausted populations express at similar levels of activating and inhibitory receptors, yet there are distinguishable differences in chemokine receptor expression, whereby ILC1 favors Cxcr3 and ILC3 favors Cxcr6.
  • Single-cell analysis also revealed an mNK cell population that, uniquely among all other NK populations, expressed high levels of Sirpa, a gene recently identified as an NK immune checkpoint molecule (Deuse, et al., J Exp Med 218 (2021)).
  • the Sirpa-mNK cells are also marked by reduced detection of nearly all activating, inhibitory, chemokine, and adhesion receptors (Figs.8F-8I).
  • Example 4 NK tumor population changes in progressing tumor models
  • Methods NK cell population levels were quantified by single-cell transcriptomics across tumor progression at day 0, 7, and 15, in both melanoma and breast cancer models (B16F10 and E0771), and from different tissues. Results The results indicated that the vast majority of NK cell subsets were highly stable across all conditions, allowing straight-forward detection of specific population shifts. In particular, data showed that iNK4 cells (Figs.9A-9C) are also the only iNK subset specifically localized within the tumor (Figs.9A-9C).
  • the iNK2 cell population also exhibited tumor-specific trends with its presence significantly associated with the pre- injection in vitro cell culture conditions, given the decreased presence in (1) each tumor model, (2) over time, and (3) in the spleen/tumor extracts (Figs.9A-9C). Each of these associations were observed along with an increasing abundance of mNK cells, except in tissue localization, where the significant loss of iNK2 is matched with an increase in only the splenic mNK (Figs.9A-9C).
  • Example 5 Expression patterns of tumor infiltration among NK populations
  • the transcriptional programs of tumor infiltration were explored in all NK cell populations by differential expression (DE) analyses using a Gamma-Poisson GLM to account for celltype, in vivo status, tumor infiltration, and the scaled RNA molecule detection rate of single cells.
  • Results The DE analysis showed top upregulation in the early activation gene Ly6a/Sca- 136, and upregulation of the senescence-related Litaf gene (Fig.11).
  • the top downregulated genes include mNK cell markers and genes involved in terminal NK differentiation, such as Zeb2, Spn, S1pr5, Itgam, Prdm138 (Fig.11).
  • mNK tumor-infiltrating NK population was comprised of ⁇ 20% mNK (Figs.9A-9C), and the GLM should accounts for cell type. Therefore, tumor-infiltration was explored in only mNK cells populations via DE analysis and found a similar downregulation of terminally differentiated NK cell genes: Zeb2, S1pr5, Spn, Ly6c2, Cx3cr1, Prdm1 genes (Figs.14A-14C). The same trend was found in tumor-infiltrating iNK cells.
  • the Itgam marker of the mNK subset was decreased in both iNK and mNK tumor-infiltrating cells, yet it was only significantly downregulated in the iNK.
  • a consistent upregulation of the senescence-related Litaf gene (Pfefferle, et al., Cell Rep 29, 2284- 2294 e2284 (2019))
  • a consistent downregulation of Calhm2 a calcium-modulating enzyme gene (Choi, et al., Nature 576, 163-+ (2019)) that was also found as a hit in the in vivo AAV-CRISPR screens.
  • Tumor-infiltration by NK cells were then further investigated with a meta- pathway analysis.
  • tNK cells had distinctly higher expression Nr4a1/Nur77, strongly upregulated upon stimulation of NK activating receptors (Marcais, et al., Elife 6 (2017)).
  • the iNK4 tumor cells also exhibited a distinct upregulation of Cxcl10, encoding a ligand of CXCR3 that has been shown to be upregulated in NK and NKT in a model of mycoplasma-enhanced colitis (Singh, et al., BMC Immunol 9, 25 (2008)).
  • Cxcl10 encoding a ligand of CXCR3 that has been shown to be upregulated in NK and NKT in a model of mycoplasma-enhanced colitis
  • Example 6 Single cell and gene expression signatures of tNK and iNK4 subsets with unique tumor functions
  • Methods DE analyses of tumor infiltrating tNK and iNK4 were performed, as each of these NK subsets seemed to have distinct expression signatures with exploring tumor NK cells as a whole.
  • Results Although either DE analysis shared many of the top DE genes with tumor iNK/mNK, such as downregulated mNK markers (S1pr5, Zeb2, and Spn), and increased common activation genes (Crem and Litaf), there were unexpected changes in top upregulated genes, such as Hspa1a/Hspa1b heat shock protein genes in tNK (Figs.15C- 15D).
  • the tumor iNK4 cells express high levels of the Ctla4 checkpoint receptor, and Spp1, critical for long-lasting NK immune responses (Leavenworth, et al., Proceedings of the National Academy of Sciences of the United States of America 112, 494-499 (2015)), yet high expression is correlated with immunosuppression(Zheng, et al., Frontiers in Oncology 11 (2021)) (Figs.15C-15D).
  • tNK cells had negative enrichment for activation and positive enrichment for the regulation of defense response, leukocyte differentiation, and the regulation of cytokine production (Figs.15E-15F), for which tumor tNK had the highest expression of Ifng and Ikbiz, required for NK IFN- ⁇ production in response to Il-12/Il-18 stimulation (Miyake, et al., Proc Natl Acad Sci U S A 107, 17680-17685 (2010)).
  • the splenic tNK still expressed Ifng, as well as high expression of Egr3, important for T and B cell activation, yet less is known for its role in NK cells (Li, et al., Immunity 37, 685- 696 (2012)).
  • tumor iNK4 cells In the tumor iNK4 cells, there is a positive enrichment for the inflammatory response differentiation, and regulation of adhesion and cell activation (Figs.15E-15F). Within the meta-pathways, tumor iNK4 cells show a prominent upregulation of Ifi204 and Isg15 interferon response genes and of Irf7, the primary regulator of the type-I interferon response (Honda, et al., Nature 434, 772-777 (2005)).
  • Example 7 Calhm2-knockout enhanced anti-tumor infiltration of primary mouse NK cells The intersection between the CRISPR screen hits, single cell profiling differentially expressed genes, and a collection of critical pathways were analyzed.
  • CALHM2/Calhm2 and Spn/Cd43 are the only two genes that emerged as the common hits (Fig.13B). Given the known function of Spn/Cd43 in NK92 and CAR-NK92 cellsNK cells, it was decided to focus on Calhm2/CALHM2, whose role in NK cells is unclear. Results Although the effects of in vitro assay are modest for Calhm2-KO, it is one of few hits that scored firstly in the screen, survived in vitro validation, and scored again in tumor-infiltration single cell DE analysis. The role of Calhm2/CALHM2 in NK cells is unclear, although it was found to regulate proinflammatory activity of microglial cells and is a potential therapeutic target for diseases related to microglia-mediated neuroinflammation.
  • Calhm2 mutant mouse NK cells were first generated, using the AAV-SB- CRISPR vector same as in the screen, and verified gene editing by reduced protein levels via Immunoblot (Fig.13C). Proliferation was quantified by cell-trace dye assays and found that Calhm2-KO did not influence primary mouse NK proliferation during multiple timepoints, ranging from 24-168 hours (Fig.13D). Whether Calhm2 influences tumor infiltration was then investigated by tracking the numbers of Calhm2-KO CD45.2 donor NK cells in CD45.1 host mice with orthotopic E0771 tumors (Figs.13E-13N).
  • Example 8 CALHM2 perturbation enhanced anti-tumor function in NK92 and CAR-NK92 cells in vitro and in vivo
  • Results CALHM2 mutant NK92 cells were first generated via Cas9/gRNA RNP electroporation and verified gene editing in the CALHM2 locus by T7EI assay (Fig. 16A) and protein level knock down of CALHM2 by western blot (Figs.16B-16C) .
  • NK92 cells with CALHM2-KO showed significantly increased killing of both MDA- MB-231 (breast cancer cell line) and NALM6-GL-CD22S cells (leukemia cancer cell line) in co-culture assays (Figs.16D-16E).
  • CALHM2 can serve as an endogenous gene target to enhance CAR-NK function
  • two different CALHM2-KO CAR-NK92 systems were established: ⁇ -BCMA-CAR and ⁇ -HER2-CAR separately (Figs.17A-17B) by lentiviral delivery.
  • CALHM2-KO ⁇ -BCMA-CAR-NK92 cells significantly kill more MM.1R-PL cells with BCMA overexpression (OE) at different effector : target cell (E:T) ratios (Figs.19A-19B), while CALHM2-KO ⁇ -HER2-CAR NK92 cells displayed a higher killing capability toward different breast cancer cell lines (MDA- MB231-PL, MCF-7-PL, and MCF-7-PL-HER2-OE) (Figs.19C-19E).
  • CALHM2-KO ⁇ -HER2-CAR NK92 cells expressed more CD107a after stimulation by cognate cancer cells (Figs.20A-20I).
  • NK92 cells are human interleukin 2 (hIL2)-dependent
  • ⁇ -HER2-CAR-NK92- hIL2 cells were established via lentiviral delivery to assess in vivo efficacy (Figs.21-22A).
  • CALHM2-KO does not influence proliferation via CellTrace assay (Fig.22B).
  • Co-culture assay showed that CALHM2-KO significantly enhances ⁇ -HER2- CAR-NK92 cell’s cytotoxicity against HER2+ HT29-GL cancer cells at four different E : T ratios (Fig. 23).
  • CALHM2-KO ⁇ -HER2-CAR-NK92-hIL2 cells effect was then tested in vivo using a solid tumor model, induced by subcutaneous injection of an established human colon cancer cell line (HT29-GL) (Fig. 24A). Tumor infiltration and persistence were evaluated in the model and showed that CALHM2-KO increases CAR-NK infiltration at 21 dpi, and the data demonstrated that the tumor CAR-NK levels persisted at 28 dpi (Figs. 24B-24E)..
  • CALHM2-KO significantly enhanced in vivo anti-tumor efficacy of ⁇ -HER2-CAR-NK92-hIL2 cells, compared to AAVS1-KO controls, which showed limited to no efficacy compared with no treatment (Fig. 24F).
  • CALHM2 perturbation significantly enhanced the (a) anti-cancer cell cytotoxicity across different E : T ratios, (b) tumor infiltration at two time points, and (c) in vivo efficacy of human CAR- NK92 cells.
  • Example 9 CALHM2-knockout downregulates immune response pathways in unstimulated CAR-NK cells.
  • CALHM2-gRNA CALHM2-gRNA
  • AAVS1-gRNA control human ⁇ -HER2 CAR-NK92-hIL2 cells
  • the DE genes using the edgeR pipeline with a GLM for paired sample analysis were then identified along with CALHM2-KO, stimulation, and an interaction term between the two (Fig.25), allowing for the effect of CALHM2-KO to be explored independent of stimulation.
  • the CALHM2-KO effect in CAR-NK cells revealed 49 upregulated genes and 211 downregulated genes (FDR adjusted p ⁇ 0.05).
  • FCRL4 an IgA-specific Fc receptor-like protein
  • NKp44 an NK activating receptor that senses platelet-derived growth factor (PDGF-DD isoform) from tumor cells
  • NK cell therapy leverages the advantages of rapid cytotoxic anti-tumor immune responses, TCR-independence, enhanced safety, simplicity in generating off- the-shelf allogeneic products, reduced off-target immune responses (Zhang, et al., Immunology 121, 258-265 (2007)), and reduced production of molecules associated with cytokine release syndrome (CRS) relative to other cell types (Chou & Turtle, Bone Marrow Transplant 54, 780-784 (2019), Hunter & Jacobson, J Natl Cancer Inst 111, 646-654 (2019), Xie, et al., EBioMedicine 59, 102975 (2020)).
  • CAR-NK cells have increased the therapeutic potential of CAR- reprogramming by adding a reduced risk for alloreactivity and Graft-vs-Host Disease, potentially allowing for CAR-NK to be mass produced in a more cost-effective manner than CAR-T cells.
  • NK cell-based immunotherapies still have many obstacles to overcome, including effective anti-tumor function, exhaustion, durable immune responses (persistence), and tumor infiltration. This requires rational engineering of substantially enhanced NK cells, particularly by modification of endogenous genes. A small number of genes have been shown where the knockout or perturbations have strong effects in NK cell’s anti-tumor efficacy, such as CISH26.
  • Such endogenous inhibitors can have fundamental implications on NK cell-based cancer immunotherapy.
  • endogenous inhibitors or cellular checkpoints
  • NK cell-based cancer immunotherapy In order to systematically identify genes that can serve as endogenous targets to enhance NK function and thereby CAR-NK cell therapy, in this study, tumor infiltrating NK cells were functionally mapped with two independent, massive-scale investigations.
  • high-throughput, in vivo pooled AAV-SB-CRISPR knockout screens with a customized high-density sgRNA library, four separate in vivo tumor models, and functional genomics screen readout in tumor and spleen samples was leveraged to quantitatively identify genes involved in NK tumor infiltration.
  • NTC control the screen has a large pool of NTCs serving as internal controls, so any phenotypic shifts would be similarly present in NK cells transfected with NTC or gene-targeting sgRNAs; Therefore, a phenotypic effect would be cancelled out in the data analysis.
  • Statistical control the screen analysis strategy includes “in vivo” as a cofactor in both the full and reduced statistical model, so the initial NK phenotype and graft-host immunogenicity would not impact the results of the regression analysis.
  • Validation In the in vivo validation experiments of tumor infiltration, both human and mouse NK cells demonstrated that the Calhm2 / CALHM2-KO phenotype is robust.
  • tumor NK abundance as sampled at different time points might be a compound effect of infiltration, survival and/or proliferation. Therefore, validation experiments were needed to confirm that CALHM2/Calhm2 deficiency increases tumor infiltration and cytotoxicity, without affecting proliferation.
  • the in vivo screens unveiled a perturbation map of thousands of surface protein encoding genes, and identified significant hits, including the immune checkpoints and NK exhaustion markers (Tigit, Lag3 and Pdcd1), as analysis references (although it is challenging to have positive control benchmarks because the number of genes where the KOs are known to have strong effects, or NK cell checkpoints, are scarce), along with a large collection of previously unknown / under-studied genes in NK cells.
  • the screen results also showed tumor type-specific differences, based on the lack of strong correlation between log-FC values of sgRNA-level results from different tumors and differences between top hits of each cancer model.
  • this screen investigated only membrane-bound cell-surface genes to decrease library- complexity, future studies could benefit from exploring other categories of molecules in the context of tumor infiltration, such as members of signaling cascades, kinases, phoshotases, epigenetic regulators, and/or transcription factors, using respective customed CRISPR libraries.
  • the screen analysis revealed functionally relevant genes at the top of the list of candidate knockout genes.
  • the enrichment of mNK cell markers in the screen is interesting, given that the terminally differentiated NK cells have much slower proliferation, which would impact the readout of the screen. Knocking these genes out might potentially impede the differentiation of the NK cells.
  • Cd27 as an enriched hit, as it is both a costimulatory receptor and a marker for the more proliferative iNK cell population.
  • Certain annotated “positive regulators” could occasionally show up as hits in a CRISPR “knockout” screen, because of two plausible reasons: (1) sgRNAs / Cas9 don’t always simply “knockout” a gene, instead, it creates double-stranded breaks (DSBs) and the NHEJ repair mechanism kicks in that resulted in insertions and deletions (indels).
  • DSBs double-stranded breaks
  • indels insertions and deletions
  • Integrins can have cell type specific roles, for example, integrin ⁇ 2 promotes melanoma metastasis, but is considered a metastasis suppressor in breast cancer. A context specific role might also explain why integrin gene KOs could improve infiltration (Pickar-Oliver, Nat Rev Mol Cell Biol 20, 490-507 (2019); Shi, J. et al.
  • NK cells such as the iNK4 cells with distinct expression profiles of Cxcr4 chemokine receptor and Cxcr10, which could have a supportive role in the control of tumors and infection through the recruitment of Cxcr3+ T cells and dendritic cells (Singh, et al., BMC Immunol 9, 25 (2008)).
  • Tumor-infiltrating NK cells have significantly increased regulation of cytokine production with genes related to TGF- ⁇ -signaling (Tgfb1 and Smad7), which has a major immunosuppressive effect in NK cells (Ghiringhelli, et al., J Exp Med 202, 1075-1085 (2005), Smyth, et al., J Immunol 176, 1582-1587 (2006));
  • TGF- ⁇ -signaling genes related to TGF- ⁇ -signaling
  • CALHM2/Calhm2 emerged as both an enriched hit in the in vivo AAV-CRISPR infiltration screens, and as a consistently downregulated gene among tumor infiltrating NK cells from single cell sequencing, and CALHM2/Calhm2 disruption enhanced in NK cell’s anti-cancer function in vitro and in vivo.
  • CALHM2 has previously been studied in the context of Alzheimer’s Disease (AD)(Choi, et al., Nature 576, 163-+ (2019), Shibata, et al., J Alzheimers Dis 20, 417-421 (2010), Dreses-Werringloer, et al., Cell 133, 1149- 1161 (2008), Jun, et al., Mol Psychiatry 23, 1091 (2016)), and the known function is context-specific, as the protein is a Ca2+-inhibited nonselective ion channel involved in calcium homeostasis and ATP release in depolarized cells (Choi, et al.,
  • CAR-NK cells also have tonic signaling, the continuous antigen-independent CAR signaling that promotes lymphocyte exhaustion (Weber, et al., Science 372 (2021)), which involves intracellular Ca2+ flux (Shao, et al., Adv Sci (Weinh) 9, e2103508 (2022)).
  • the functional and RNA-seq data revealed that (1) these transcription signals are downregulated in unstimulated CAR-NK upon CALHM2-deficiency, (2) CALHM2-deficiency has little effect on stimulated CAR-NK, and (3) CALHM2-KO CAR-NK cells had significantly decreased exhaustion.
  • CALHM2- targeting may therefore decrease the unwanted activation in unstimulated CAR-NK cells without impacting effector function in stimulated cells, which is in line with the combination of data in in vitro cytotoxicity, degranulation, infiltration at different time points, changes in the transcriptome, and in vivo efficacy.
  • tumor infiltrating NK cells were functionally mapped with two independent, massive-scale investigations. Thes ingle cell transcriptomics analyses identified previously unexplored sub-populations of NK cells, as well as a dynamic shift from iNK to mNK cells within the TME, despite decreased expression of key mNK marker genes in mNK cells.
  • the in vivo pooled AAV-SB-CRISPR knockout screens mapped multiple potential regulatory genes involved in NK tumor infiltration, including Calhm2, which were validated in mouse and human NK models to improve tumor infiltration and cytotoxicity, independent of proliferation. Moreover, CALHM2-KO was demonstrated to achieve drastically improved tumor remission in clinically relevant ⁇ - HER2-CAR-NK92 immunotherapy in vivo. Peng, et al., “Perturbomics of tumor-infiltrating NK cells.” bioRxiv.2023 Mar 15:2023.03.14.532653. doi: 10.1101/2023.03.14.532653.
  • each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • any subset or combination of these is also specifically contemplated and disclosed.
  • the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D.
  • nucleic acid sequence includes a plurality of such nucleic acids
  • nucleic acids is a reference to one or more nucleic acid and equivalents thereof known to those skilled in the art, and so forth.
  • “Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present. Unless the context clearly indicates otherwise, use of the word “can” indicates an option or capability of the object or condition referred to.
  • use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to.
  • use of the word “may” indicate an option or capability of the object or condition referred to.
  • use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to.
  • use of “may” herein does not refer to an unknown or doubtful feature of an object or condition. Ranges can be expressed herein as from “about” one particular value, and/or to "about” another particular value.
  • ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint.
  • any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers.
  • a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.

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Abstract

Cellules NK génétiquement modifiées et compositions pharmaceutiques et procédés d'utilisation de celles-ci pour traiter des sujets présentant des maladies ou des troubles. Dans certaines formes, les cellules NK génétiquement modifiées présentent une expression réduite ou abrogée du gène Calhm2. Des cellules NK et des cellules CAR-NK dépourvues d'expression de Calhm2 sont fournies pour le traitement du cancer. L'invention fournit également des compositions et des procédés de criblage de perturbation génomique de cellules tueuses naturelles (NK). Les compositions et les procédés sont applicables au développement de cellules NK génétiquement modifiées pour une thérapie cellulaire adoptive (ACT) améliorée. Un procédé donné à titre d'exemple fournit une bibliothèque d'ARNsg spécifiques pour des gènes à la surface de cellules NK pour mettre en œuvre une édition génomique basée sur CRISPR d'une population de cellules NK.
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