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WO2024145478A1 - Facteurs de transcription régulant la différenciation et la perturbation des lymphocytes t dans le cadre de la lutte contre les tumeurs et les virus - Google Patents

Facteurs de transcription régulant la différenciation et la perturbation des lymphocytes t dans le cadre de la lutte contre les tumeurs et les virus Download PDF

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WO2024145478A1
WO2024145478A1 PCT/US2023/086236 US2023086236W WO2024145478A1 WO 2024145478 A1 WO2024145478 A1 WO 2024145478A1 US 2023086236 W US2023086236 W US 2023086236W WO 2024145478 A1 WO2024145478 A1 WO 2024145478A1
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cell
zscan20
jdp2
nfil3
expression
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Susan M. KAECH
Hokyung CHUNG
Wei Wang
Cong Liu
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Salk Institute for Biological Studies
University of California Berkeley
University of California San Diego UCSD
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Salk Institute for Biological Studies
University of California Berkeley
University of California San Diego UCSD
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Definitions

  • T cells serve as an example: these cells exist in different activation states, which arise in response to different stimuli, yet they maintain their T-cell identity.
  • Cell state is directly associated with the function of the given cell type. Cells in the same state will share the pathways that are active, the genes that are expressed, and the functions that are being performed. Transcription factors (TFs) are fundamental regulators of both cell type and cell state differentiation.
  • Naive CD8 + T cells differentiate into heterogeneous states to offer immune defense against intracellular pathogens and cancer.
  • T cells assume diverse effector and memory states.
  • MP highly plastic memory precursor
  • TCM cells at secondary lymphoid, T EM in blood, T RM at peripheral tissues.
  • T cells in chronic virus infections or tumors progressively become dysfunctional T cells and lose memory potential, a phenomenon known as T cell exhaustion.
  • the CD8 + T cells in the exhaustion trajectory include multiple cell states that serve different roles.
  • FIGs. 1A-1H depict a transcription and epigenomic atlas of CD8+ T cell differentiation states and cell-state specific TFs identification pipeline.
  • A Pipeline of integrative analysis. The matched RNA-seq and ATAC-seq data were used as input for Taiji algorithm to construct a regulatory network and output PageRank scores matrix representing the activity of transcription factors (TFs). The downstream analysis included the identification of cell-type-specific TFs, differentiationspecific TFs, and the construction of temporal transcriptional waves.
  • B PageRank scores of 151 bona fide cell-state-specific TFs. TFs in rows (z-normalized), samples in columns, and the color of the cell in the matrix indicates the normalized PageRank scores.
  • FIGs. 5A-5B depict a catalog of TFs in 9 different CD8+ T cell states.
  • A Summary table of identified TFs in 9 cell states.
  • B UpSetR plot shows the intersection size between multi-taskers along with single-taskers size.
  • FIG. 11 depicts bubble plots for the TexProg cell state. Circle size represents the logarithm of gene expression, and the color represents the normalized PageRank score.
  • FIGs. 15A-15C depict transcription factor wave analysis
  • A Analysis pipeline.
  • B, C Selecting algorithms and parameters for clustering analysis for transcription factor wave.
  • B Plotting the cumulative proportion of variance explained against the number of principal components (PCs). The first 20 PCs were kept for the following K-means clustering, which explained ⁇ 70% variance.
  • FIG. 18 depicts a heat map of biological pathways enriched in each transcription wave. Color represents p value.
  • A Pipeline of TF interaction network analysis.
  • B communities of TFs in TexTerm. Communities consist of TexTerm TFs and their interaction partners. Dots represent each TF and TexTerm TFs with high interaction scores are labeled. Lines represent the interactions between TexTerm-specific TFs.
  • C Intersection size of the regulatees of communities. ToplOOO regulatees for each community were selected by the average edge weight. Community (Cm.) Prdml shares the most regulatees with other communities (orange highlight).
  • FIGs. 27A-27L demonstrate how disruption of transcription factor Zscan20 provides enhanced virus and tumor control.
  • A-E LCMV chronic strain-infected mice
  • A-E Experiment timeline. Cas9+ P14 T cells are transduced with gRNA Zscan20 or scramble gRNA.
  • B gZscan20 RV group shows lower serum virus level.
  • C gZscan20 RV group shows higher effector- associated markers (CX3CR1, KLRG1).
  • D Quantification of (C).
  • E T cells with gRNA Zscan20 are more polyfunctional and release both IFNy and TNFa.
  • FIGs. 28A-28D demonstrate that Jdp2 disrupted CD8+ T cell offers better virus control in LCMV chronic strain-infected mice (A-D).
  • A Experiment timeline. Cas9+ P14 T cells are transduced with gRNA Jdp2 or scramble gRNA.
  • B gJdp2 RV group shows lower serum virus level.
  • C gjdp2 RV group shows higher effector-associated markers (CX3CR1, KLRG1).
  • T cells with gRNA Zscan20 are more polyfunctional and release both IFNy and TNFa. All data include more than three biological replicates, n > 5. Data are expressed as mean ⁇ SEM. Statistical analysis was performed using Student’s t test (two-tailed) comparing TF gRNA vs scramble gRNA.
  • FIGs. 29A-29C show tumor control by T cells with Jdp2 deficiency.
  • Jdp2 KO or control P14 cells were adoptively transferred to melanoma-bearing mice that are implanted with 5 X 105 Bl 6- gp33 cells) followed by anti-PDl or isotype IgG2a control treatment.
  • B Tumor growth
  • C survival curve and show improved tumor control only when Jdp2 KO is combined with anti-PDl treatment. Data are expressed as mean ⁇ SEM. Statistical analysis was performed using Student’s t test (two-tailed) comparing TF gRNA vs scramble gRNA within anti-PDl group.
  • Each gene was targeted with four gRNAs, expressed by two retrovirus gRNA vectors, each expressing dual gRNAs with a GFP expression marker.
  • Cas9-expressing LCMV gp33 peptide-specific TCR T cells (Cas9+ Pl 4) were transduced with the retroviral gRNA library at an MOI of 0.3 and adoptively transferred to mice infected with the chronic LCMV virus (Clone 13 strain) on post-day 1. On day 23 post-transfer, mice spleens were isolated, and GFP-positive P14 T cells were sorted. Additionally, library-transduced P14 cells were cultured in vitro for three days and sequenced to normalize gRNA distribution from in vivo.
  • SEQ ID NO: 1 is an exemplary amino acid sequence of human JDP2, from NCBI reference sequence NP_001128520.1. mmpgqipdpsvttgslpglgpltglpssaltveelkyadirnlgamiaplhf levklgkrpqpvks eldeeeerrkrrreknkvaaarcrnkkkertef Iqreserlelmnaelktqieelkqerqqlilml nrhrptcivrtdsvktpesegnplleqlekk
  • an “effective amount” of a therapeutic agent is an amount sufficient to reduce signs or symptoms of the viral infection in a subject, reduce the viral load in a subject, reduce infectivity of a virus, reduce cytopathic effect in the subject’s cells, or combinations thereof, for example by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, or at least 99% (as compared to a suitable control, such as no administration of the therapeutic agent).
  • Increase or Decrease A positive or negative change, respectively, in quantity from a control value (such as a value representing no therapeutic agent).
  • An increase is a positive change, such as an increase at least 25%, at least 50%, at least 100%, at least 200%, at least 300%, at least 400% or at least 500%, as compared to the control value.
  • a decrease is a negative change, such as a decrease of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or at least 100% decrease as compared to a control value.
  • the increase or decrease is statistically significant relative to a suitable control.
  • Isolated An “isolated” biological component (e.g., a cell, PBMC, nucleic acid, protein) has been substantially separated, produced apart from, or purified away from other biological components in the cell or tissue of an organism in which the component occurs, such as other cells (e.g., RBCs), chromosomal and extrachromosomal DNA and RNA, and proteins.
  • Nucleic acids and proteins that have been “isolated” include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids and proteins.
  • parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, 5% human serum albumin, glycerol, or the like as a vehicle.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • Supplementary active compounds can also be incorporated into the compositions.
  • promoters include, but are not limited to the SV40 promoter, the CMV enhancer-promoter, the CMV enhancer/p-actin promoter, EFla promoter, or PGK promoter.
  • expression of a gRNA is driven by a polymerase III promoter, such as U6 or Hl, such as human or mouse U6 or Hl promoter. Both constitutive and inducible promoters are included (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987).
  • promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue-specific, or inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Promoters produced by recombinant DNA or synthetic techniques can also be used to provide for transcription of the nucleic acid sequences.
  • Preventing a condition refers to reducing, delaying, or inhibiting the full development of a condition, for example preventing, reducing, or slowing the progression of a T cell to an exhausted T cell.
  • an agent that reduces Zscan20 expression, or a non- naturally occurring genetic modification that reduces an amount of functional ZSCAN20, when present in a PBMC, such a T cell, such as a CAR or TCR prevents or reduces the likelihood that the cell will become exhausted (e.g., prevents or reduces the likelihood a T cell will overexpress programmed cell death 1 (PDl hl ), become positive for T cell immunoglobulin and mucin domaincontaining protein 3 (TIM3 + ), express CTLA-4, express lymphocyte-activation gene 3 (LAG-3), express TIGIT, and/or express CD160) or may slow the progression of the cell to an exhausted state.
  • a T cell such as a CAR or TCR
  • the disclosed modified PBMCs do not become exhausted.
  • the disclosed modified PBMCs, such as modified T cells show a reduction in exhaustion, such as a reduction of least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 99%, or at least 99.9%, relative to an unmodified PBMC or T cell.
  • the disclosed modified PBMCs, such as modified T cells show a slower progression to exhaustion, such as an increase in the number of days to exhaustion of least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 99%, slower relative to an unmodified PBMC or T cell.
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, MD) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.
  • Short hairpin RNA A sequence of RNA that makes a tight hairpin turn and can be used to silence gene expression via the RNAi pathway.
  • the shRNA hairpin structure is cleaved by cellular machinery into siRNA.
  • a shRNA that is “specific” for a target sequence (such as Zscan20) has sufficient complementarity to the target sequence that it binds the target and does not significantly hybridize with other unrelated sequences.
  • siRNA Small interfering RNA
  • siRNA molecules are generally 15 to 40 nucleotides in length, such as 20-30 or 20-25 nucleotides in length, with 0 to 5 (such as 2)-nucleotide overhangs on each 3' end.
  • siRNAs can also be blunt ended.
  • one strand of a siRNA molecule is at least partially complementary to a target nucleic acid, such as a target mRNA.
  • siRNAs are also referred to as “small inhibitory RNAs.”
  • a siRNA that is “specific” for a target sequence (such as Zscan20) has sufficient complementarity to the target sequence that it binds the target and does not significantly hybridize with other unrelated sequences.
  • a vertebrate such as a mammal, for example a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • the subject is a non-human mammalian subject, such as a monkey or other non-human primate, mouse, rat, rabbit, pig, goat, sheep, dog, cat, horse, or cow.
  • the subject has cancer (or a tumor), that can be treated using the modified PBMCs disclosed herein.
  • the subject is a laboratory animal/organism, such as a mouse, rabbit, or rat.
  • T cells A white blood cell (lymphocyte) that is an important mediator of the immune response.
  • T cells include, but are not limited to, CD3+ T cells, CD4+ T cells and CD8+ T cells.
  • a CD4+ T cell is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses.
  • CD8+ T cells carry the “cluster of differentiation 8” (CD8) marker.
  • a CD8+ T cell is a cytotoxic T lymphocyte (CTL).
  • CD3+ T cells carry the “cluster of differentiation 3” (CD3) marker, a multimeric protein complex historically known as the T3 complex.
  • Activated T cells can be detected by an increase in cell proliferation and/or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFN- ⁇ , or TNF ⁇ ). Activation of CD8+ T cells can also be detected by an increase in cytolytic activity in response to an antigen. “Exhausted T cells” are dysfunctional T cells (hyporesponsive) commonly found in cancer environments.
  • T cell exhaustion is characterized by a progressive loss of effector function (for example, loss of IL-2, TNF- ⁇ , and IFN- ⁇ production) and sustained expression of inhibitory receptors such as PD-1, T cell immunoglobulin domain and mucin domain-containing protein 3 (Tim-3), CTLA-4, lymphocyte-activation gene 3 (LAG-3), and CD160.
  • the exhausted T cell is a CD3+ T cell or CD8+ T cell.
  • the exhausted T cell is a terminally exhausted T cell (a terminally differentiated T cell that is exhausted).
  • the exhausted cell is an exhausted progenitor (Tex Prog ) or an exhausted effector-like (Tex Eff-like ) cell.
  • T cells may express PD1, and may lack expression of SLAMF6 and/or CX3CR1 relative to other T cells (PD1+, SLAMF6-, CX3CR1-).
  • T cells that are PD1+, SLAMF6- and/or CX3CR1- can be determined by FACs analysis, for example, by FACs analysis of a population of T cells.
  • terminally exhausted T cells express ZSCAN20.
  • a possible cause of T cell exhaustion is chronic activation or prolonged antigen stimulation.
  • the modified PBMC is an exhausted T cell (including a terminally exhausted T cell).
  • terminally exhausted cells are defined as PD1+, SLAMF6-, CX3CR1- cells.
  • terminally exhausted cells are defined as PD1+, CD101+ cells.
  • terminally exhausted cells are CD39+, CD38+.
  • terminally exhausted T cells are defined as PD1+ LAG3+ CTLA4+ and CD45RA low .
  • a “Therapeutic T Cell” is a T cell that is used for therapy, such as immunotherapy (e.g., cancer immunotherapy).
  • Therapeutic T cells are administered to a subject for treatment of a particular disease, for example, cancer or an immune disease.
  • the therapeutic T cell recognizes and kill target cells, for example, cancerous cells, thereby treating a disease, such as cancer.
  • Therapeutic T cells may be autologous or allogeneic to the subject.
  • the therapeutic T cell is a T cell to be used for Adoptive Cell Transfer (ACT) immunotherapy.
  • the therapeutic T cell expresses a Chimeric Antigen Receptor (CAR) or Engineered T Cell Receptor (TCR), and/or is a Tumor-Infiltrating Lymphocyte (TIL).
  • TIL Tumor-Infiltrating Lymphocyte
  • the T cell is an exhausted T cell or a tissue resident memory (TRM) T cell.
  • T cell receptor A receptor found on the surface of T lymphocytes (or T cells) responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is composed of two different protein chains. In humans, in 95% of T cells the TCR consists of an alpha (a) and beta (
  • a TCR When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction, that is, a series of biochemical events mediated by associated enzymes, co-receptors, specialized adaptor molecules, and activated or released transcription factors.
  • a TCR is a recombinant TCR, such as one used in TCR-engineered T cells for ACT therapy.
  • TRM Tissue Resident Memory
  • CD8+ TRM cells Immune memory subset cells that reside in situ, typically in nonlymphoid tissues, rather than recirculating.
  • the TRM are CD8+ TRM cells.
  • the TRM are CD4+ TRM cells.
  • Exemplary TRM genetic markers include one or more of Itgae, It gal, Runx3, Cxcr3, Prdml, Notch.2, 117 r, Id3, or Cd69 and/or reduced expression of one or more of Slprl, Klf2, Klf3, Tox, Entpdl, Eomes, Tbx21, Tigit, Cd38, Lag3, Cx3crl, CdlOl, and/or Havcr2.
  • CD8+ TRM in some examples include CD69 and/or CD 103 on the cell surface.
  • a transformed cell is a cell (such as a PBMC, such as a T cell) into which a nucleic acid molecule has been introduced by molecular biology techniques.
  • the term transformed and the like encompass all techniques by which a nucleic acid molecule might be introduced into such a cell, including viral vectors, plasmid vectors, nucleic acid-protein complexes (e.g., ribonucleoprotein), or naked nucleic acids (e.g., oligonucleotides).
  • Exemplary methods of transformation include chemical methods (e.g., calcium-phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), lipofection, nucleofection, receptor-mediated endocytosis (e.g., DNA- protein complexes, viral envelope/capsid-DNA complexes), particle gun accelerator (gene gun), and by biological infection by viruses such as recombinant viruses (Wolff, J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)).
  • retroviruses the infecting retrovirus particles are absorbed by the target cells, resulting in reverse transcription of the retroviral RNA genome and integration of the resulting provirus into the cellular DNA.
  • Treating, Treatment, and Therapy Any success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject’s physical or mental well-being, or prolonging the length of survival.
  • the treatment may be assessed by objective or subjective parameters; including the results of a physical examination, blood and other clinical tests, and the like.
  • treatment with the disclosed methods results in a decrease in the number, volume, and/or weight of a tumor and/or metastases.
  • treatment with the disclosed methods results in a decrease in signs or symptoms of the viral infection in a subject, and/or reduces viral load in a subject, and/or reduces infectivity of a virus, and/or reduce cytopathic effect in the subject’s cells.
  • TIL Tumor-Infiltrating Lymphocyte
  • ACT therapy generally involves isolating TILs from a patient tumor, activating and expanding the TILs in culture, and then reinfusing into the patient.
  • the modified PBMC disclosed herein is a TIL.
  • TILs express PD1, CD25, 0X40, CD69, CD44 and/or CTLA4.
  • Tumor, neoplasia, or malignancy A neoplasm is an abnormal growth of tissue or cells which results from excessive cell division. Neoplastic growth can produce a tumor. The amount of a tumor in an individual is the “tumor burden” which can be measured as the number, volume, or weight of the tumor.
  • a “non-cancerous tissue” is a tissue from the same organ wherein the malignant neoplasm formed, but does not have the characteristic pathology of the neoplasm. Generally, noncancerous tissue appears histologically normal.
  • a “normal tissue” is tissue from an organ, wherein the organ is not affected by cancer or another disease or disorder of that organ.
  • a “cancer-free” subject has not been diagnosed with a cancer of that organ and does not have detectable cancer.
  • Exemplary tumors such as cancers, that can be treated using the disclosed modified PBMCs include solid tumors, such as breast carcinomas (e.g. lobular and duct carcinomas, such as a triple negative breast cancer), sarcomas, carcinomas of the lung (e.g., non small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, for instance, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell aden
  • the disclosed modified PBMCs can also be used to treat liquid tumors, such as a lymphatic, white blood cell, or other type of leukemia.
  • the tumor treated is a tumor of the blood, such as a leukemia (for example acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell prolymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), a lymphoma (such as Hodgkin’s lymphoma or non-Hodgkin’s lymphoma), or a myeloma.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • Tumor-Specific Antigen antigens unique to cancer cells or much more abundant on them, as compared to other cells, such as normal cells.
  • Example tumor-specific antigens include, but are not limited to, CD19, CD20, BCMA, MUC1, PSA, CEA, HER1, HER2, TRP-2, EpCAM, GPC3, mesothelin l (MSLN), and EGFR.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements.
  • Viral vectors (such as AAV and lentiviral vectors) are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.
  • a replication deficient viral vector is a vector that requires complementation of one or more regions of the viral genome required for replication due to a deficiency in at least one replication-essential gene function.
  • Exemplary negative-strand RNA viruses include, but are not limited to: Orthomyxyoviruses (such as the influenza virus), Rhabdo viruses (such as Rabies virus), vesiculoviruses (such as VSV) and Paramyxoviruses (examples of which include measles virus, respiratory syncytial virus, and parainfluenza viruses).
  • Orthomyxyoviruses such as the influenza virus
  • Rhabdo viruses such as Rabies virus
  • vesiculoviruses such as VSV
  • Paramyxoviruses examples of which include measles virus, respiratory syncytial virus, and parainfluenza viruses.
  • DNA viruses include, but are not limited to: Hepadnaviridae (Hepatitis B virus) Herpesviruses (such as Varicella- zoster virus (VZV), for example the Oka strain; cytomegalovirus (CMV); epstein-barr virus (EBV), and Herpes simplex virus (HSV) types 1 and 2), Adenoviruses (such as Adenovirus type 1 and Adenovirus type 41), Poxviruses (such as Vaccinia virus), papillomavaridae (such as human papillomavirus (HPV)), (and Parvoviruses (such as Parvovirus B19).
  • Hepadnaviridae Hepatitis B virus
  • Herpesviruses such as Varicella- zoster virus (VZV), for example the Oka strain
  • CMV cytomegalovirus
  • EBV epstein-barr virus
  • HSV Herpes simplex virus
  • viruses which can establish a chronic infection include adenovirus (Ad), a herpes simplex virus (HSV), a hepatitis B virus (HBV), a hepatitis C virus (HCV), a vesicular stomatitis virus (VSV), a human immunodeficiency virus (HIV), an influenza virus, a varicella zoster virus (VZV), a human papillomavirus (HPV), an Epstein-Barr virus (EBV), a cytomegalovirus (CMV), an enterovirus, a togavirus, a SARS-CoV virus, a SARS- CoV-2 virus, or a flavivirus.
  • Ad adenovirus
  • HSV herpes simplex virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • VSV vesicular stomatitis virus
  • HAV vesicular stomatitis virus
  • Antiviral agents can work by a variety of mechanisms, including inhibiting any or all of: attachment, entry, uncoating, protease activity, polymerase activity, nucleoside and/or nucleotide reverse transcriptase activity, nonnucleoside reverse transcriptase activity, and integrase activity. Antiviral agents might also physically disrupt a virion.
  • antiviral agents that can be used with the methods provided herein include Lopinavir (for HIV), remdesivir (for SARS-CoV-2), acyclovir (for Herpes viruses), ribavirin (for viral hemomoragic fevers), emtricitabine/tenofovir (for HIV), and bamlanivimab/etesevimab (for SARS-CoV-2), ZMapp (for ebolavirus).
  • Antiviral agents can be given in combination, for example to prevent the target virus from developing resistance to the therapy.
  • compositions incorporating ZNF324 (ZNF324 )) or Zfp324 (Zfp324 ) may be adapted to the appropriate species by incorporating a species appropriate gene ortholog, for example, a composition incorporating a gRNA specific for Zfp324 might be adapted for use in humans by incorporating a gRNA specific for ZNF324. Also known as ZF5128, ZNF324A.
  • Zinc Finger and SCAN Domain- Containing Protein 20 (e.g., OMIM 611315) first reported by Thiesen, Multiple genes encoding zinc finger domains are expressed in human T cells, New Biol. (1990) 2:363-74, who speculated that 30 newly identified zinc finger domaincontaining proteins might bind to DNA or RNA based on sequence similarity to other zinc finger containing proteins. Zscan20 , s role as a TF was not previously studied.
  • Zscan20 sequences are publicly available, and exemplary sequences include Amino Acid NCBI Reference Sequences: NP_001364305.1 (human), NP_808426.2 (Mus musculus), XP_003127824.1 (Sus scrofa); Nucleotide NCBI Reference Sequence: NC_000001. l l (human), NM_001377376.1 (human), NM_177758.4 (Mus musculus), XM_003127776.4 (Sus scrofa), each of which is herein incorporated by reference in their entirety. NCBI Gene ID: 7579.
  • Zscan20 or ZSCAN20 includes the corresponding gene or protein in any species: human, mouse, or otherwise, such as any mammalian Zscan20. Also known as KOX29, ZNF31 , ZFP-31 , and ZNF360. II. Overview
  • the same types of cells can assume diverse states with varying functionalities.
  • Single-cell genomics and proteomics enable not only precise characterization of cell state, but also provide a stunningly high-resolution view of transitions between states.
  • Cell state differentiation can be regulated by TFs that relay environmental signals through control of gene expression (5, 6). Therefore investigation of TFs of each cell state enables efficient and precise regulation of cell programming.
  • an epigenomic and transcriptomic atlas was generated to systematically identify TFs that define different CD8 + T cell states. Novel TFs were discovered, which can improve T cell therapy.
  • TF network in Texierm cell state uncovers cooperation between group of TFs and revealed biological circuits that have not been appreciated such as cellular catabolic process, GTPase activity, hypoxia and oxidative stress, which provide interesting pathways for future study. Furthermore, this systemic analysis indicated that the same sets of TFs are used in the transition of both Naive — > MP — > TRM and Naive — > Texpr Og — > TexTerm, parallel differentiation trajectories from acute and chronic infection. Lastly, four different TFs (Zscan20, Jdp2, Nfil3, and Znf324) were discovered, in vivo validated, and are now reported as having roles in Texierm. The precise global analysis pipeline overcome the fact that TF activity is not proportional to the expression level of TF and identified TFs that have not yet been reported.
  • Singletasker TFs can be useful in designing therapeutic cell state.
  • Effective T cell therapy can be achieved by programming T cells to avoid dysfunctional Texierm, to favor afunctional effector state, and without compromising immunological memory potential.
  • this disclosure identifies TFs to perturb that are specifically active in Texierm and possibly regulate genes suppress effector state transition. Even though many studies have modulated the expression of TFs to improve anti-tumor immunity, they did not propose blocking Texierm or consider compromising TRM formation by doing so. This disclosure prevents the dysfunctional and terminally differentiated TexTerm state.
  • the modified PBMC includes (a) the agent that reduces Zscan20 expression or the non-naturally occurring genetic modification that reduces an amount of functional ZSCAN20 and either one or the other of (b) an agent that reduces Jdp2 expression or a non- naturally occurring genetic modification that reduces an amount of functional JDP2; (c) an agent that reduces Nfil3 expression or a non-naturally occurring genetic modification that reduces an amount of functional NFIL3.
  • RNAi specific for Zscan20 is a short hairpin RNA (shRNA) molecule, short interfering RNA (siRNA) molecule, or antisense RNA molecule.
  • RNAi specific for Jdp2 is a shRNA molecule, siRNA molecule, or antisense RNA molecule.
  • RNAi specific for Nfil3 is a shRNA molecule, siRNA molecule, or antisense RNA molecule.
  • RNAi specific for Znf324 is a shRNA molecule, siRNA molecule, or antisense RNA molecule.
  • the agent that reduces Zscan20, Jdp2, Nfil3, and/or Znf324 expression includes (a) a gRNA specific for Zscan20, including SEQ ID NOs: 7, 8, 9, 10, 19, 20, or 21 ; (b) a gRNA specific for Jdp2, including SEQ ID NOs: 11, 12, 13, 14, 22, 23, or 24; (c) the gRNA specific for Nfil3, including SEQ ID NOs: 15, 16, 17, 18, 25, 26, or 27 , and/or (d) the gRNA specific for Znf324, including SEQ ID NOs: 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46.
  • the modified PBMC includes an expression vector encoding a heterologous nucleic acid.
  • the modified PBMC includes a Cas nuclease.
  • the Cas nuclease is a Cas3, dCas3, Cas9, dCas9, Casl2, dCas!2, Casl 3a, dCas!3a, Casl3b, dCas l3b, Cas 13d, or dCasl3d nuclease.
  • the modified PBMC has a genetic modification that reduces ZSCAN20.
  • the mutation may be a point mutation, a partial deletion, full deletion, or insertion of Zscan20 that reduces expression of Zscan20 and/or reduces activity of ZSCAN20.
  • the modified PBMC has a genetic modification that reduces JDP2.
  • the mutation may be a point mutation, a partial deletion, full deletion, or insertion of Jdp2 that reduces expression of Jdp2 and/or reduces activity of JDP2.
  • the modified PBMC has a genetic modification that reduces NFIL3.
  • the mutation may be a point mutation, a partial deletion, full deletion, or insertion of Nfi 13 that reduces expression of Nfil3 and/or reduces activity of NFIL3.
  • the modified PBMC has a genetic modification that reduces ZNF324.
  • the mutation may be a point mutation, a partial deletion, full deletion, or insertion of Znf324 that reduces expression of Znf324 and/or reduces activity of ZNF324.
  • the modified PBMC is a T cell.
  • the T cell can be a CD3+ T cell, a CD4+ T cell, and/or a CD8+ T cell.
  • the PBMC is a therapeutic T cell, an exhausted T cell, tissue resident memory T cell (TRM), a chimeric antigen receptor (CAR) T cell, an engineered T cell receptor (TCR) T cell, a tumor-infiltrating lymphocyte (TIL), and/or a T cell comprising an antigen receptor reactive to a tumor-specific antigen.
  • TRM tissue resident memory T cell
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • TIL tumor-infiltrating lymphocyte
  • TIL tumor-infiltrating lymphocyte
  • the tumor-specific antigen is one or more of CD 19, CD20, BCMA, MUC1, PSA, CEA, HER1, HER2, TRP-2, EpCAM, GPC3, mesothelin l(MSLN), or EGFR.
  • the modified PBMC is generated by any one of: (a) introducing an agent that reduces Zscan20 expression or non- naturally occurring genetic modification that reduces functional ZSCAN20 into a PBMC, thereby generating the modified PBMC with reduced expression of Zscan20, reduced activity of ZSCAN20, or both; (b) introducing an agent that reduces Jdp2 expression or non-naturally occurring genetic modification that reduces functional JDP2 into a PBMC, thereby generating the modified PBMC with reduced expression of Jdp2, reduced activity of IDP2, or both; (c) introducing an agent that reduces Nfil3 expression or non-naturally occurring genetic modification that reduces functional NFIL3 into a PBMC, thereby generating the modified PBMC with reduced expression of Nfil3, reduced activity of NFIL3, or both; and/or (d) introducing an agent that reduces Znf324 expression or non-naturally occurring genetic modification that reduces functional ZNF324 into a PBMC, thereby generating the
  • the PBMC is a T cell.
  • the method includes incubating the modified PBMC with interleukin 2, (IL- 2), interleukin 7 (IL-7), interleukin 15 (IL-15), or a combination thereof.
  • the modified PBMC is reactive to a tumor-specific antigen, such as CD19, CD20, BCMA, MUC1, PSA, CEA, HER1, HER2, TRP-2, EpCAM, GPC3, mesothelin l(MSLN), and EGFR.
  • reduced activity of NFIL3, reduced expression of Znf324, and/or reduced activity of ZNF324 increases effector function of the T cell; and/or reduced expression of Zscan20, reduced activity of ZSCAN20, reduced expression of Jdp2, reduced activity of JDP2, reduced expression of Nfil3, reduced activity of NFIL3, reduced expression of Znf324, and/or reduced activity of ZNF324 reduces exhaustion of the T cell.
  • the method includes selecting the modified PBMC with reduced expression of Zscan20, reduced activity of ZSCAN20, or both; selecting the modified PBMC with reduced expression of Jdp2, reduced activity of JDP2, or both; selecting the modified PBMC with reduced expression of Nfil3, reduced activity of NFIL3, or both; selecting the modified PBMC with reduced expression of Znf324, reduced activity of ZNF324, or both; and/or introducing the selected modified PBMC into a subject.
  • the selecting step includes the use of flow cytometry, panning, or magnetic separation.
  • the subject has cancer
  • the method includes the step of selecting a subject who has cancer.
  • a pharmaceutical composition including the modified PBMC generated by the previous examples, and optionally a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is in an intravenous formulation.
  • the pharmaceutical composition further includes comprising one or more immune checkpoint blockade (ICB) agents.
  • the pharmaceutical composition further includes one or more antiviral agents.
  • the pharmaceutical composition further includes one or more anti-tumor agents, such as a therapeutic monoclonal antibody.
  • a method for treating cancer or a tumor in a subject including administering a therapeutically effective amount of a modified PBMC as described by the previous examples; or a therapeutically effective amount of the pharmaceutical composition described by the previous examples, to a subject having cancer or a tumor, treating the cancer or the tumor.
  • the modified PBMC is autologous or allogenic to the subject.
  • the method includes administering a therapeutically effective amount of 11-2, 11-7, and/or 11-15 to the subject, and/or treating the subject with one or more of surgery, radiation, chemotherapy, biologic therapy, or immunotherapy.
  • the method includes administering to the subject a therapeutically effective amount of one or more of: a T cell agonist antibody, an oncolytic virus, or an adoptive cell transfer (ACT) immunotherapy.
  • the method includes administering to the subject a therapeutically effective amount of immune checkpoint blockade (ICB) agent or immunostimulatory antibody.
  • the ICB agent comprises anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-LAG3 anti-GITR, anti-4-lBB, anti-CD40, and anti- 0X40, anti-TIGIT, anti- VISTA, anti-CD73, anti-CD39, anti-HVEM, anti-BTLA, anti-CD27, or a combination of two or more thereof.
  • the anti-PD-1 is nivolumab, pembrolizumab, pidilizumab, or cemiplimab.
  • the anti-CTLA-4 is ipilimumab or tremelimumab.
  • the modified PBMC is administered simultaneously with the ICB agent or the immunostimulatory antibody.
  • the ICB agent includes anti-PD-1, anti-PD-Ll, anti-CTLA-4, anti-LAG3 anti-GITR, anti-4-lBB, anti-CD40, and anti-OX40, anti-TIGIT, anti- VISTA, anti-CD73, anti-CD39, anti-HVEM, anti-BTLA, anti-CD27 and/or a combination of two or more of these ICB agents.
  • the RNAi specific for Zscan20, JDP2, Nfil3, and/or Znf324 consists of a sequence at least 90% complementary (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% 99% or 100% complementary) to a unique contiguous portion of Zscan20, JDP2, Nfil3, and/or Znf324 gene or transcript (such as a portion of SEQ ID NOs: 2, 4, 6, or 29).
  • the Cas nuclease (or a dead Cas nuclease) sequence is codon optimized for expression in a host cell.
  • gRNA molecules and Cas nucleases are expressed from a vector introduced into a host cell (e.g., PBMC, antigen presenting cell, B cell, dendritic cell, monocyte/macrophage, NK cell, T cell, CD8+ TRM T cell, tumor infiltrating lymphocyte, CAR T cell, exhausted T cell, terminally exhausted T cell, or a cell with an antigen receptor reactive to a tumor-specific antigen).
  • the gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 gene or transcript includes one having at least 90%, at least 95%, or at least 98% sequence identity to SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 (such as substitution of 1, 2, 3, 4, 5, or 6 nt).
  • Nucleic acids e.g., heterologous nucleic acids or isolated nucleic acid molecules, such as DNA, cDNA, RNA (e.g., mRNA)) encoding the RNAi, gRNAs, and/or Cas protein are also provided herein. Nucleic acids can readily be produced using the disclosed sequences provided herein, sequences available in the art, and the genetic code. In one example, nucleic acids are DNA. In one example, nucleic acids are RNA.
  • leucine can be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encoded by TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT or AAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encoded by TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG; glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TAT or TAC; and isoleucine can be encoded by ATT, ATC, or ATA.
  • a Cas nuclease (or dead nuclease) sequence is codon optimized for expression in a human PBMC (PBMC, antigen presenting cell, B cell, dendritic cell, monocyte/macrophage, NK cell, T cell, CD8+ TRM T cell, tumor infiltrating lymphocyte, CAR T cell, exhausted T cell, or a cell with an antigen receptor reactive to a tumor-specific antigen).
  • PBMC human PBMC
  • B cell antigen presenting cell
  • dendritic cell monocyte/macrophage
  • NK cell T cell
  • CD8+ TRM T cell tumor infiltrating lymphocyte
  • CAR T cell exhausted T cell
  • a cell with an antigen receptor reactive to a tumor-specific antigen a cell with an antigen receptor reactive to a tumor-specific antigen
  • the disclosed nucleic acids can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
  • a promoter can be operably linked to an RNAi, gRNA, or Cas nuclease (or dead nuclease) to drive its expression.
  • a vector encodes both a Cas nuclease (or dead nuclease) and a gRNA. Additional expression control sequences, such as one or more enhancers, transcription and/or translation terminators, and initiation sequences can also be included in the expression vector.
  • the disclosed nucleic acids are included in a viral vector.
  • Exemplary viral vectors that can be used include, but are not limited to, polyoma, SV40, adenovirus, vaccinia virus, adeno-associated virus (AAV), herpes viruses including HSV and EBV, Sindbis viruses, alphaviruses and retroviruses of avian, murine, and human origin.
  • Baculovirus (Autographa californica multinuclear polyhedrosis virus; AcMNPV) vectors can also be used.
  • Other suitable vectors include orthopox vectors, avipox vectors, fowlpox vectors, capripox vectors, suipox vectors, lentiviral vectors, alpha virus vectors, and poliovirus vectors.
  • poxvirus vectors such as vaccinia virus, fowlpox virus and a highly attenuated vaccinia virus (MVA), adenovirus, baculovirus and the like.
  • Pox viruses of use include orthopox, suipox, avipox, and capripox virus.
  • Orthopox include vaccinia, ectromelia, and raccoon pox.
  • One example of an orthopox of use is vaccinia.
  • Avipox includes fowlpox, canary pox and pigeon pox.
  • Capripox include goatpox and sheeppox.
  • the suipox is swinepox.
  • viral vectors that can be used include other DNA viruses such as herpes virus and adenoviruses, and RNA viruses such as retroviruses and polio.
  • the vector includes a selectable marker (such as an antibiotic resistance gene (e.g., puromycin) or a reporter gene (e.g., green fluorescent protein (GFP)).
  • a selectable marker and/or reporter is not included in the vector.
  • the disclosed nucleic acids can be introduced into a host cell by DNA transfer (e.g., oligonucleotides), or introduced and expressed in a suitable host cell (e.g., expression cassette or vector).
  • the expressed product is an RNA (e.g., siRNA or gRNA), in other examples, the expressed product is a protein (e.g., Cas9).
  • the cell may be prokaryotic or eukaryotic.
  • the host cell is a PBMC (e.g., B cell, monocyte/macrophage, dendritic cell, T cell). Methods of transient or stable transfer can be used.
  • Transient transfer indicates that the foreign nucleic acid is only present transiently (e.g., degraded after a period of time, cleared by the host cell, or otherwise not stably replicated). Stable transfer indicates that the foreign nucleic acids is continuously maintained in the host.
  • expression cassettes can contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator can be used.
  • a promoter such as the T7, trp, lac, or lamda promoters, a ribosome binding site, and preferably a transcription termination signal can be used.
  • control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, S V40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). Additional operational elements include, but are not limited to, leader sequence, termination codons, polyadenylation signals and any other sequences necessary for the appropriate transcription and subsequent translation of the nucleic acid sequence.
  • the disclosed nucleic acids or vectors can be introduced into the host cell by any suitable method (e.g., transformation).
  • transformation e.g., transformation
  • Numerous methods of transformation can be used, such as: chemical methods (e.g., calcium-phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), lipofection, nucleofection, receptor- mediated endocytosis (e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes), particle gun accelerator (gene gun), and by biological infection by viruses such as recombinant viruses (Wolff, J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)).
  • chemical methods e.g., calcium-phosphate transfection
  • physical methods e.g., electroporation, microinjection, particle bombardment
  • fusion e.g., liposomes
  • the infecting retrovirus particles are absorbed by the target cells, resulting in reverse transcription of the retroviral RNA genome and integration of the resulting provirus into the cellular DNA.
  • Successfully transformed cells can be selected by resistance to antibiotics conferred by genes contained in the vector, such as the amp, gpt, neo and hyg genes.
  • a disclosed nucleic acid e.g., gRNA
  • RNP ribonucleoprotein
  • RNPs can be introduced into a host cell by transformation, for example, by nucleofection.
  • Modifications can be made to the disclosed nucleic acids without diminishing biological activity of the encoded product.
  • modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein.
  • Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.
  • modified PBMCs have reduced expression of Zscan20 (and/or reduced activity of ZSCAN20) and reduced expression of Nfil3 (and/or reduced activity of NFIL3).
  • modified PBMCs have reduced expression of Zscan20 (and/or reduced activity of ZSCAN20), reduced expression of Jdp2 (and/or reduced activity of JDP2), and reduced expression of Nfil3 (and/or reduced activity of NFIL3).
  • modified PBMCs have reduced expression of Nfil3 (and/or reduced activity of NFIL3).
  • modified PBMCs have reduced expression of Jdp2 (and/or reduced activity of JDP2).
  • modified PBMCs have reduced expression of Nfil3 (and/or reduced activity of NFIL3) and reduced expression of Jdp2 (and/or reduced activity of JDP2). In some examples, modified PBMCs have reduced expression of Znf324 (and/or reduced activity of ZNF324).
  • expression of Zscan20, Jdp2, and/or Nfd3 is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or at least 100% relative to a suitable control (e.g., a PBMC prior to modification).
  • activity of ZSCAN20, JDP2, and/or NFIL3 is reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100% relative to a suitable control (e.g., a PBMC prior to modification).
  • Reducing activity includes reducing any measurable biological function of ZSCAN20, JDP2, and/or NFIL3, for example, reduced interaction by these TFs with the PBMC’s genome.
  • the modified PBMC with reduced expression of Zscan20, Jdp2, and/or Nfil3, reduced activity of ZSCAN20, JDP2, and/or NFIL3, or any combination thereof has increased effector activity (e.g. , anti-tumor) relative to a suitable control (e.g., unmodified PBMC).
  • a suitable control e.g., unmodified PBMC
  • the modified PBMC is a T cell, and the T cell has increased resistance to T cell exhaustion relative to a suitable control (e.g., unmodified PBMC).
  • the modified PBMC can further include additional modifications, for example, the PBMC can express or otherwise contain a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR engineered T cell receptor
  • the modified PBMC is a T cell, for example, a CD4+, a CD8+ or a CD3+ T cell.
  • the T cell can be reactive to a tumor-specific antigen, for example, CD 19, CD20, BCMA, MUC1, PSA, CEA, HERE HER2, TRP-2, EpCAM, GPC3, mesothelin l(MSLN), or EGFR.
  • the T cell is a tumor-infiltrating lymphocyte (TIL).
  • T cell is a therapeutic T cell, or will be used as a therapeutic T cell, for example, as an ACT therapy.
  • the T cell is an exhausted T cell (including terminally exhausted T cells).
  • Exhausted T cells are dysfunctional T cells characterized by a progressive loss of effector function (for example, loss of IL-2, TNF-a, and IFN-y production) and sustained expression of inhibitory receptors such as PD- 1 , T cell immunoglobulin domain and mucin domain-containing protein 3 (Tim-3), CTLA-4, lymphocyte-activation gene 3 (LAG-3), and CD160.
  • the exhausted T cell is a terminally exhausted T cell, which have high and persistent expression of programmed cell death 1 (PDl hl ) and are positive for T cell immunoglobulin and mucin domain-containing protein 3 (TIM3 + ).
  • the T cell is a tissue resident memory T cell.
  • the modified PBMC includes an agent that reduces Zscan20, Jdp2, Nfil3, and/or Znf324 expression, for example, one or more of the disclosed inhibitory RNA (RNAi) specific for gene or transcript, or one or more guide RNA (gRNAs) specific for Zscan20, Jdp2, Nfil3, and/or Zq/324gene or transcript (for example in combination with a Cas nuclease or dead Cas nuclease, such as an RNP).
  • RNAi inhibitory RNA
  • gRNAs guide RNA
  • the agent that reduces Zscan20, Jdp2, Nfil3, and/or Znf324 expression is one or more of the disclosed inhibitory RNA (RNAi), for example, a short hairpin RNA (shRNA), short interfering RNA (siRNA), microRNA (miRNA), or an antisense RNA specific to Zscan20, Jdp2, Nfil3, and/or Znf324.
  • RNAi inhibitory RNA
  • shRNA short hairpin RNA
  • siRNA short interfering RNA
  • miRNA microRNA
  • the RNAi is a shRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 gene or transcript
  • the siRNA is specific to a sequence comprising at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, 6, and/or 29.
  • the shRNA is specific to a sequence with at least 90% sequence identity to a unique, contiguous portion of SEQ ID NOs: 2, 4, 6, and/or 29.
  • the agent is a disclosed gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 gene or transcript
  • the gRNA is specific for a sequence with at 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 2, 4, 6, and/or 29.
  • the gRNA can be specific to a sequence with at least 90% sequence identity to SEQ ID NOs: 2, 4, 6, and/or 29.
  • the gRNA comprises a targeting sequence specific to Zscan20, Jdp2, Nfd3, and/or Znf324 gene or transcript, for example, by having a targeting sequence that is at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% complementary to a unique, contiguous portion of SEQ ID NOs: 2, 4, 6, and/or 29.
  • the gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 gene or transcript includes a contiguous sequence at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and/or 46.
  • the gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 gene or transcript includes SEQ ID NOs: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, and/or 46.
  • the modified PBMC includes a RNP complex that includes the disclosed gRNA and a Cas nuclease, such as Cas3, dCas3, Cas9, dCas9, Casl2, dCasl2, Casl3a, dCasl3a, Casl3b, dCasl3b, Casl3d, or dCasl3d.
  • a Cas nuclease such as Cas3, dCas3, Cas9, dCas9, Casl2, dCasl2, Casl3a, dCasl3a, Casl3b, dCasl3b, Casl3d, or dCasl3d.
  • the modified PBMC includes a heterologous nucleic acid molecule encoding one or more of the disclosed nucleic acids encoding the RNAi (e.g., shRNA, siRNA, antisense RNA) or gRNA.
  • RNAi or gRNA may be encoded as DNA (for example, encoded on a DNA vector), but expressed as RNA.
  • the heterologous nucleic acid molecule encodes the disclosed gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 and a Cas nuclease (or a dead Cas nuclease).
  • the Cas nuclease is a Cas9 nuclease.
  • the Cas nuclease is a Casl3d nuclease, or a Casl2 nuclease.
  • the modified PBMC includes the disclosed vector encoding the RNAi or gRNA.
  • the modified PBMC expresses the RNAi or gRNA.
  • a Cas nuclease e.g., Cas3, dCas3, Cas9, dCas9, Casl2, dCasl2, Casl3a, dCasl3a, Casl3b, dCasl3b, Cas 13d, or dCasl3d
  • the gRNA includes a spacer sequence and DR sequence (such as DR-spacer-DR-spacer) and the Cas nuclease is Cas 13d, and Zscan20, Jdp2, Nfil3, and/or Znf324 RNA is edited.
  • the gRNA includes a crRNA and tracrRNA (expressed either as two separate molecules, or as one fusion molecule, such as a sgRNA) and the Cas nuclease is Cas9.
  • the vector includes a cassette including two or more gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znj324 wherein the two or more gRNAs have the same or different targeting sequences (e.g., may target two different regions of Zscan20, Jdp2, Nfil3, and/or Znf324).
  • the vector includes gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324.
  • the vector includes multiple gRNA specific for any combination of Zscan20, Jdp2, Nfil3, and/or Znf324 (e.g., a vector with a first gRNA specific for ZscanZO and a second gRNA specific for N[U3).
  • Nucleic acids or vectors can be transiently or stably introduced into a PBMC (e.g., T cell).
  • the vector is stably introduced into the modified PBMC, thereby resulting in stable expression of the RNAi or gRNA in the modified PBMC.
  • the nucleic acid encoding the RNAi or gRNA is operably linked to a cell specific promoter (e.g., a T cell specific promoter such as GzmB promoter or CD4 promoter) in the vector.
  • a cell specific promoter e.g., a T cell specific promoter such as GzmB promoter or CD4 promoter
  • Expression of the RNAi or gRNA can be constitutive or inducible.
  • Exemplary promoters include NF AT, EFla, PGK, U6, or Hl.
  • gRNA is expressed from a U6 or Hl promoter.
  • a Cas nuclease (or dead Cas nuclease) is expressed
  • the modified PBMC includes a non-naturally occurring genetic modification that reduces an amount of functional ZSCAN20, JDP2, NFIL3, and/or ZNF324.
  • Reducing functional ZSCAN20, JDP2, NFIL3, and/or ZNF324 includes genetic modifications that decrease Zscan20, Jdp2, Nfil3, and/or Znf324 expression (e.g., decreasing transcription or translation of Zscan20, Jdp2, Nfil3, and/or Znf324 gene or transcript) in the modified PBMC.
  • the genetic modification is a non-naturally occurring genetic modification of a Zscan20, Jdp2, Nfil3, and/or Znf324 gene.
  • the non-naturally occurring genetic modification is a modification that reduces an amount of functional ZSCAN20, JDP2, NFIL3, and/or ZNF324 in the modified PBMC.
  • the genetic modification can result in the production of dysfunctional ZSCAN20, JDP2, NFIL3, and/or ZNF324.
  • the genetic modification results in the production of unstable ZSCAN20, JDP2, NFIL3, and/or ZNF324, such that the accumulation of functional ZSCAN20, JDP2, NFIL3, and/or ZNF324 is reduced.
  • the genetic modification can be any non-naturally occurring modification that results in a decreased amount of ZSCAN20, JDP2, NFIL3, and/or ZNF324.
  • Non- limiting examples of genetic modifications include a point mutation, partial deletion, full deletion, or insertion.
  • PBMCs are also provided herein are methods of generating the disclosed modified PBMCs by introducing the non-naturally occurring genetic modification into a PBMC, thereby generating the modified PBMC with reduced expression of Zscan20, Jdp2, Nfil3, and/or Znf324 and/or reduced activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324.
  • the PBMC is obtained from a subject before introducing the non-naturally occurring genetic modification.
  • PBMCs can be harvested or isolated, for example, from a blood sample, such as a venous blood sample, from the subject.
  • PBMCs Several techniques for isolating PBMCs can be used, for example, density centrifugation (the Ficoll approach), isolation by cell preparation tubes (CPTs), or isolation by SepMateTM tubes.
  • CPTs cell preparation tubes
  • SepMateTM tubes isolation by SepMateTM tubes.
  • aphersis or leukapheresis is used to harvest PBMCs. Erythrocyte contamination can be evaluated, for example, by microscopic analysis of the sample.
  • Flow cytometry techniques ⁇ ?.g., FACS
  • FACS techniques can be used to assess the composition of the isolated PBMC populations, for example, to identify monocytes ( ⁇ ?.g., CD14), T cells (e.g., CD3, CD8, CD4), B cells (e.g., CD20), or NK cells (e.g., CD56).
  • FACS techniques can also be used to enrich or deplete a particular cell type from a PBMC (for example, enrich or deplete CD14,
  • the PBMC is harvested or isolated from a solid tissue sample, for example from a tumor.
  • Tumor samples can be surgically resected, enzymatically digested, and PBMCs can subsequently be isolated, for example via the methods of Donia et al., Characterization and Comparison of ‘Standard’ and ‘Young’ Tumour-Infiltrating Lymphocytes for Adoptive Cell Therapy at a Danish Translational Research Institution, Scand. J. Immuno. (2011) 75:157-67, incorporated by reference herein.
  • T cells are isolated from a PBMC sample, or the PBMC sample is enriched for T cells, for example, isolated or enriched for CD3 + or CD8 + T cells.
  • a sample is enriched by negative selection, for example, by selecting and removing unwanted cell types from a sample ( ⁇ ?.g., cell types other than T cells, and/or naive or memory T cells).
  • FACS is used to enrich for a particular PBMC, for example, to enrich for T cells (e.g., CD3 or CD8 positive T cells).
  • FACS can also be used to assess whether exhausted T cells, or specifically terminally exhausted T cells (PD-l hl , TIM3 + or PD-l hl LAG3 + or PD-l hl CD39 + ), are present in a PBMC sample, or sort a PBMC sample to enrich for exhausted T cells (including terminally exhausted T cells), or conversely remove exhausted T cells.
  • Antigen responsiveness of the PBMCs can be assessed, for example, by measuring release of cytokines, e.g. , IFNy, IL-10, IL-6, IL-8 and TNFa.
  • the PBMCs are obtained from a subject to be treated, such as a subject having cancer or one having a chronic viral infection.
  • the PBMCs are obtained from a donor subject, such as a subject who does not have cancer.
  • exhausted T cells are obtained from a tumor biopsy or sample (e.g., tumor infiltrating lymphocytes).
  • the agent, non-naturally occurring genetic modification, or inhibitor is introduced into a PBMC ex vivo.
  • such methods can further include selecting modified PBMCs having reduced expression of Zscan20, Jdp2, Nfil3, and/or Znf324, reduced activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324, or any combination of the aforementioned (such as purifying or isolating such cells away from cells not having reduced expression of Zscan20, Jdp2, Nfil3, and/or Znf324, and not having reduced activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324).
  • Such methods can also further include selecting modified PBMCs that are T cells, for example, T cells that are CD3+ or CD8+.
  • Exemplary selection methods include using flow cytometry, panning or magnetic separation.
  • the disclosed methods in some examples further include introducing the selected modified PBMCs having reduced expression of Zscan20, Jdp2, NJU3, and/or Znf324, reduced activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324, or both, into a subject, such as a subject with a cancer to be treated with the selected modified PBMCs having reduced expression of Zscan20, Jdp2, Nfil3, and/or Znf324, reduced activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324, or any combination the aforementioned.
  • the agent, non-naturally occurring genetic modification, or inhibitor is administered to the subject, and the agent, non-naturally occurring genetic modification, or inhibitor is introduced into a PBMC (e.g., T cells, CD8+ TRM, tumor infiltrating lymphocytes, CAR T cells, or exhausted T cells (including terminally exhausted T cells)) in vivo.
  • a PBMC e.g., T cells, CD8+ TRM, tumor infiltrating lymphocytes, CAR T cells, or exhausted T cells (including terminally exhausted T cells)
  • the method of generating the modified PBMC further includes selecting a PBMC or cell type (e.g., T cells, CD8+ TRM, tumor infiltrating lymphocytes, CAR T cells, or exhausted T cells (including terminally exhausted T cells)), for example, from a sample (e.g., tumor biopsy, blood, population of T cells) before introducing the inhibitor, agent, or non-naturally occurring genetic modification.
  • a PBMC or cell type e.g., T cells, CD8+ TRM, tumor infiltrating lymphocytes, CAR T cells, or exhausted T cells (including terminally exhausted T cells)
  • a sample e.g., tumor biopsy, blood, population of T cells
  • the selected PBMC is reactive to a tumorspecific antigen, for examples, one or more of: CD19, CD20, BCMA, MUC1, PSA, CEA, HER1, HER2, TRP-2, EpCAM, GPC3, mesothelin l(MSLN), or EGFR.
  • the selected PBMC is a T cell.
  • the T cell is CD8+ or CD3+.
  • the T cell is an adoptive cell transfer (ACT) therapy T cell, for example, the selected exhausted T cell can include a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR) specific for a tumor antigen.
  • the selected PBMC is a tumor-infiltrating lymphocyte (TIL).
  • the T cell is an exhausted T cell, such as a terminally exhausted T cell, which highly expresses programmed cell death 1 (PDl hl ) and is positive for T cell immunoglobulin and mucin domain-containing protein 3 (TIM3 + ).
  • the agent (RNAi or gRNA) is introduced, for example, by contacting a PBMC with the agent, thereby generating the modified PBMC.
  • the agent is introduced by transfecting or transforming a PBMC with the disclosed nucleic acid molecule encoding the inhibitor or agent or the vector encoding a disclosed nucleic acid molecule, thereby generating the modified PBMC.
  • Methods of transforming or transfecting a host cell are described herein, and can include: chemical methods (e.g., calcium-phosphate transfection), physical methods (e.g., electroporation, microinjection, particle bombardment), fusion (e.g., liposomes), nucleofection, receptor-mediated endocytosis (e.g., DNA-protein complexes, viral envelope/capsid- DNA complexes) and by biological infection by viruses, such as recombinant viruses.
  • the infecting retrovirus particles are absorbed by the target cells, resulting in reverse transcription of the retroviral RNA genome and integration of the resulting pro virus into the cellular DNA.
  • a ribonucleoprotein (RNP) complex including the gRNA and a Cas nuclease or dead nuclease (e.g., Cas3, Cas9, Casl2, or Casl3d) is directly introduced into the PBMC.
  • a Cas nuclease or dead nuclease e.g., Cas3, Cas9, Casl2, or Casl3d
  • the PBMC can be nucleofected with the RNP.
  • the PBMC is transfected with the RNP by electroporation (see e.g., Seki and Rutz, (2016) J Exp Med. 215(3): 985-997).
  • lipid-containing oligoaminoamides are used to as a carrier for intracellular delivery of the RNP complex (see e.g., Kuhn et al. (2020) Bioconjugate Chem. 31(3):729-742).
  • the introduced agent is shRNA, and the shRNA is introduced into the PBMC through infection with a viral vector encoding the shRNA. Introduction by a viral vector allows for stable integration of shRNA and long-term knockdown of the targeted gene.
  • the introduced agent is siRNA, and siRNA is introduced cytosolically into a host cell capable of transfection.
  • the non-naturally occurring genetic modification is introduced into the PBMC.
  • the genetic modification can be any non-naturally occurring modification that results in decreased expression of Zscan20. Jdp2, Nfil3, and/or Znf324 or reduced activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324.
  • Non- limiting examples of genetic modifications include a point mutation, partial deletion, full deletion, or insertion.
  • the genetic modification is induced by a targeted genome editing technique, such as CRISPR/Cas, zinc finger nuclease, or TALEN modification of a Zscan20, Jdp2, Nfil3, and/or Znf324 gene.
  • CRISPR/Cas CRISPR/Cas
  • zinc finger nuclease or TALEN modification of a Zscan20, Jdp2, Nfil3, and/or Znf324 gene.
  • the genetic modification reduces Zscan20, Jdp2, Nfil3, and/or Znf324 expression, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.
  • the genetic modification reduces ZSCAN20, JDP2, NFIL3, and/or ZNF324 activity, for example, by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%.
  • the agent that reduces Zscan20 expression or a non-naturally occurring genetic modification that reduces an amount of functional ZSCAN20 comprises a zinc finger nuclease (ZFN) or transcription activator- like effector nuclease (TALEN) specific for Zscan20.
  • the agent that reduces Jdp2 expression or a non-naturally occurring genetic modification that reduces an amount of functional JDP2 comprises a zinc finger nuclease (ZFN) or transcription activator-like effector nuclease (TALEN) specific for Jdp2.
  • the agent that reduces Nfil3 expression or a non-naturally occurring genetic modification that reduces an amount of functional NFIL3 comprises a zinc finger nuclease (ZFN) or transcription activatorlike effector nuclease (TALEN) specific for Nfd3.
  • the agent that reduces Znf324 expression or a non-naturally occurring genetic modification that reduces an amount of functional ZNF324 comprises a zinc finger nuclease (ZFN) or transcription activator- like effector nuclease (TALEN) specific for Znf324.
  • the modified PBMC is incubated with at least one cytokine selected from the group consisting of interleukin 2 (IL-2), interleukin 7 (IL-7), interleukin 15 (IL-15), TGF- P, and retinoic acid TGF-P, and retinoic acid.
  • IL-2 interleukin 2
  • IL-7 interleukin 7
  • IL-15 interleukin 15
  • TGF- P retinoic acid
  • retinoic acid TGF-P, and retinoic acid.
  • introducing the non-naturally occurring genetic modification reduces activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more relative to a suitable control.
  • Reducing activity includes reducing any measurable biological function of ZSCAN20, JDP2, and/or NFIL3, and/or ZNF324, for example, reducing the interaction between ZSCAN20, JDP2, and/or NFIL3, and/or ZNF324 and the genome.
  • decreasing expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 in a PBMC increases effector function, reduces exhaustion, increases resistance to exhaustion, or combinations thereof.
  • the PBMC is a T cell, and decreasing expression of Zscan20, Jdp2, Nfil3, Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 in the PBMC increases effector function of the T cell, reduces exhaustion of the T cell, or causes the T cell to express at least one of Itgae, It gal, Runx3, Cxcr3, Prdml, Notch2, Tcf7, Cxcr5, 117 r, Id3, or Cd69 and/or causes reduced expression of Slprl, Klf2, Klf3, Pdcdl, Tox, Entpdl, Cxcr6, Eomes, Tbx21, Tigit, Cd38, Lag3, Cx3crl, CdlOl, or Havcr2 by the T cell.
  • decreasing expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 in a PBMC causes CD69 and/or CD 103 to be present on the cell’s surface.
  • the PBMC is a T cell and decreasing expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 in the PBMC increases resistance to T cell exhaustion.
  • the disclosed modified PBMCs such as modified T cells, do not become exhausted (e.g., do not become PDl hl and TIM3 + ).
  • the disclosed modified PBMCs such as modified T cells, become exhausted at a slower rate, for example the number of days to progress to an exhausted cell (e.g., PDl hl and TIM3 + ) is increased by at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or at least 99%, for example relative to a PBMC/T cell with native ZSCAN20, JDP2, NFIL3, and/or ZNF324 expression/activity.
  • an exhausted cell e.g., PDl hl and TIM3 +
  • the disclosed modified PBMCs results in a population of modified PBMCs, such as modified T cells, with fewer exhausted cells (e.g., PDl hl and TIM3 + ), such as a reduction of at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, or at least 99%, for example relative to a PBMC/T cell with native ZSCAN20, JDP2, NFIL3 and/or ZNF324 expression/activity.
  • exhausted cells e.g., PDl hl and TIM3 +
  • the pharmaceutical composition includes (1) one or more of: the disclosed RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, one or more gRNAs specific for Zscan20, Jdp2, Nfd3, and/or Znf324, the nucleic acid or vector encoding the RNAi or gRNA, the inhibitor (e.g., ZSCAN20, JDP2, NFIL3, and/or ZNF324 inhibitor), or the modified PBMC; and (2) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes a modified PBMC and a pharmaceutically acceptable carrier, such as water or saline.
  • the pharmaceutical composition includes (1) one or more of: the RNAi specific to ZscanZO, Jdp2, Nfil3, and/or Znf324, the gRNAs specific for Zscan20, Jdp2, Nfil3, and/or Znf324, the nucleic acid or vector encoding the RNAi or gRNA, the Zscan20, Jdp2, Nfil3, and/or Znf324 inhibitor, or the modified PBMC; (2) a cancer immunotherapy; and (3) a pharmaceutically acceptable carrier.
  • the cancer immunotherapy is an ACT therapy (e.g., CAR-T, TCR, TIL), a monoclonal antibody (e.g., anti-PD-1, anti-EGFR, anti- CTLA4), a T cell agonist antibody, or an oncolytic virus.
  • the cancer immunotherapy includes one or more ICB agents.
  • the pharmaceutical composition includes the modified PBMC, an antibody cancer immunotherapy, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes: one or more of the RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, the gRNAs specific for Zscan20, Jdp2, Nfil3, and/or Znf324, the nucleic acid or vector encoding the RNAi or gRNA; an ACT immunotherapy (e.g., CAR-T, TCR, TIL); and a pharmaceutically acceptable carrier.
  • an ACT immunotherapy e.g., CAR-T, TCR, TIL
  • the pharmaceutical composition includes: one or more of the RNAi specific to Nfil3, the gRNAs specific for Nfil3, the nucleic acid or vector encoding the RNAi or gRNA; one or more ICB agents (e.g., anti-PDl, anti-PD-Ll, anti-CTLA4); and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes: one or more of the RNAi specific to Znf324, the gRNAs specific for Znf324, the nucleic acid or vector encoding the RNAi or gRNA; one or more ICB agents (e.g., anti-PDl, anti-PD-Ll, anti-CTLA4); and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition includes (1) one or more of: the RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, the gRNAs specific for Zscan20, Jdp2, NfilS, and/or Znf324, the nucleic acid or vector encoding the RNAi or gRNA, the Zscan20, Jdp2, Nfil3, and/or ZnJ 324 inhibitor, or the modified PBMC; (2) an antiviral agent; and (3) a pharmaceutically acceptable carrier.
  • the antiviral agent is aciclovir, ganciclovir, zidovudine, interferon alpha, or another direct acting antiviral agents.
  • compositions for treating cancer, a tumor, and/or a viral infection (such as a chronic viral infection) in a subject by administering an effective amount of a disclosed composition (the RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, the gRNA specific to Zscan20, Jclp2, Nfil3, and/or Znf324 and a Cas nuclease or dead Cas nuclease (which may be administered as an RNP complex), a nucleic acid or vector encoding the RNAi or gRNA (wherein in some examples the vector also expresses and a Cas nuclease or Cas dead nuclease), the modified PBMC, or the pharmaceutical composition disclosed herein (hereinafter collectively referred to as “composition”)), to the subject, thereby treating the cancer, tumor, or vims.
  • a disclosed composition the RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, the
  • the administered composition is an effective amount of the modified PBMCs disclosed herein.
  • PBMCs are removed from the subject and modified as disclosed herein ex vivo, then the modified cells are introduced into the subject.
  • PBMCs are modified in vivo, for example by introducing a therapeutic molecule provided herein (e.g., RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 ⁇ into the subject.
  • a therapeutic molecule provided herein (e.g., RNAi specific to Zscan20, Jdp2, Nfil3, and/or Znf324, gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 ⁇ into the subject.
  • the administered composition includes one or more antiviral agents, such as aciclovir, ganciclovir, zidovudine, interferon alpha, and/or direct acting antiviral agents.
  • the administered composition includes one or more ICB agents.
  • the method is a method of increasing a response to immunotherapy in a subject and the composition is the disclosed vector encoding the RNAi or gRNA.
  • the subject has a tumor or cancer.
  • the subject has a solid tumor or cancer, such as breast carcinomas (e.g. lobular and duct carcinomas, such as a triple negative breast cancer), sarcomas, carcinomas of the lung (e.g., non-small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of the lung, colorectal adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma and mucinous cystadenocarcinoma), ovarian germ cell tumors, testicular carcinomas and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including, for instance, transitional cell carcinoma, adenocarcinoma, and squamous carcinoma), renal cell a
  • breast carcinomas
  • the subject has a liquid tumor or cancer, such as a lymphatic, white blood cell, or other type of leukemia.
  • the tumor treated is a tumor of the blood, such as a leukemia (for example acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), chronic myelogenous leukemia (CML), hairy cell leukemia (HCL), T-cell pro lymphocytic leukemia (T-PLL), large granular lymphocytic leukemia, and adult T-cell leukemia), a lymphoma (such as Hodgkin’s lymphoma or nonHodgkin’s lymphoma), or a myeloma.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • AML acute myelogenous leukemia
  • CML chronic myelogenous leukemia
  • HCL hairy cell leukemia
  • the subject has leukemia, colorectal cancer, cervical cancer, lung cancer, bladder cancer, head and neck cancer, pancreatic cancer, glioblastoma, head and neck squamous cell carcinoma, ovarian cancer, uterine cancer, prostate cancer, breast cancer, melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, sarcomas, or adrenal carcinoma.
  • the subject has melanoma.
  • the subject has an acute or chronic leukemia, Hodgkin or Non-Hodgkin lymphoma, myeloma, gastric cancer, esophageal cancer, colorectal cancer, hepatocellular carcinoma or other liver cancer, cholangiocellular carcinoma, melanoma, cervical cancer, uterine cancer, lung cancer, ovarian cancer, bladder cancer, urothelial cancer, breast cancer, pancreatic cancer, renal cell carcinoma, prostate cancer, testicular cancer, glioblastoma, nephroblastoma, neuroblastoma, neuroendocrine cancer, pheochromocytoma, sarcoma, thyroid cancer, laryngeal cancer or head and neck cancer.
  • the subject has a viral infection, such as a chronic viral infection, such as an infection caused by: adenovirus (Ad), a herpes simplex virus (HSV, type 1 and 2), a hepatitis B virus (HBV), a hepatitis C virus (HCV), a hepatitis D virus (HDV), a hepatitis E virus (HEV), a vesicular stomatitis virus (VSV), a human immunodeficiency virus (HIV), an influenza virus, a varicella zoster virus (VZV), a human papillomavirus (HPV), an Epstein-Barr virus (EBV), a cytomegalovirus (CMV), a human herpesvirus (HHV-6, HHV-7), a human T-cell leukemia virus (HTLV-1, HTLV-2), IC virus, BK virus, an enterovirus, a parvovirus, a paramyxovirus (Ad
  • the subject is receiving, has received, or will receive immunotherapy, for example, one or more ICB agent targeting PD-1, PD-L1, CTLA-4, LAG3 GITR, 4-1BB, CD40, CD40L, and 0X40, TIGIT, VISTA, CD73, CD39, HVEM, BTLA, CD27, CDK4, CDK6, or any combination of two or more of thereof.
  • immunotherapy for example, one or more ICB agent targeting PD-1, PD-L1, CTLA-4, LAG3 GITR, 4-1BB, CD40, CD40L, and 0X40, TIGIT, VISTA, CD73, CD39, HVEM, BTLA, CD27, CDK4, CDK6, or any combination of two or more of thereof.
  • Exemplary checkpoint inhibitors include ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, palbociclib, ribociclib, abemaciclib, pidilizumab, cosibelimab, envafolimab, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, MEDI-4737, and tremelimumab.
  • the effective amount of the composition is an amount that increases a response of the subject to an immunotherapy (e.g., a checkpoint inhibitor or ACT); for example, an amount that when administered with the immunotherapy, is more effective at treating cancer or a tumor relative to administration of the immunotherapy (or composition) alone.
  • the effective amount is an amount that is synergistic when administered with an immunotherapy, for example, an amount that synergistically prevents, treats, reduces, and/or ameliorates one or more sign or symptom of cancer.
  • the effective amount of the composition is an amount sufficient to prevent, treat, reduce, and/or ameliorate one or more signs or symptoms of cancer in the subject. For example, an amount sufficient to reduce tumor size or tumor load in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, as compared to a baseline measurement for the same subject, or a suitable control. In some examples, the effective amount is an amount sufficient to inhibit or slow metastasis in the subject.
  • the effective amount is an amount that increases life expectancy of the subject, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% as compared to a baseline measurement for the same subject, or a suitable control.
  • the effective amount is an amount that increases life expectancy of the subject, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 400%, or more.
  • the effective amount is an amount sufficient to reduce tumor density in the subject, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% as compared to a baseline measurement for the same subject or other suitable control.
  • suitable controls include untreated subjects or subjects not receiving the composition (e.g., subjects receiving other agents or alternative therapies).
  • the effective amount is an amount sufficient to target and eliminate tumor cells, for example, eliminate at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or even 100%, relative to a suitable control.
  • the effective amount of the composition is an amount sufficient to prevent, treat, reduce, and/or ameliorate one or more signs or symptoms of viral infection in the subject, for example, an amount sufficient to reduce viral load by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, as compared to a baseline measurement for the same subject, or a suitable control.
  • the effective amount is an amount sufficient to inhibit or slow viral replication in the subject for example, an amount sufficient to inhibit or slow viral replication by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, as compared to a baseline measurement for the same subject, or a suitable control.
  • the effective amount is an amount that increases life expectancy of the subject, for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 400%, or more.
  • an effective amount is an amount sufficient to increase T cell counts in an HIV infected subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100%, as compared to a baseline measurement for the same subject, or a suitable control.
  • the method reduces expression of Zscan20, Jdp2, and/or Njil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 in a target tissue or cell in the subject, for example, in a PBMC, T cell, or exhausted T cell (including terminally exhausted T cells)).
  • expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 is decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% relative to a suitable control ( ⁇ ?.g., an untreated subject or a baseline reading of the same subject prior to treatment).
  • the method reduces protein levels of ZSCAN20, JDP2, NFIL3, and/or ZNF324 (or functional ZSCAN20, JDP2, NFIL3, and/or ZNF324), for example, by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% relative to a suitable control ( ⁇ ?.g., an untreated subject or a baseline reading of the same subject prior to treatment).
  • the method reduces expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or accumulation of mRNA transcripts by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% relative to a suitable control (e.g., an untreated subject or a baseline reading of the same subject prior to treatment).
  • a suitable control e.g., an untreated subject or a baseline reading of the same subject prior to treatment.
  • decreasing expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 increases T cell effector function or decreases T cell exhaustion.
  • decreasing expression of Zscan20, Jdp2, Nfil3, and/or Znf324 or activity of ZSCAN20, JDP2, NFIL3, and/or ZNF324 reduces (including prevents or inhibits) T cell exhaustion or increases resistance to (including prevents or inhibits) T cell exhaustion.
  • increasing T cell response or reducing T cell exhaustion in a subject increases response to an immunotherapy in the subject.
  • the method includes administering to the subject the modified PBMC and a pharmaceutically acceptable carrier.
  • the composition includes about 10 4 to 10 12 of the modified PBMCs (for example, about 10 4 - 10 8 cells, about 10 6 -l 0 8 cells, about 10 6 -l 0 12 cells, about 10 8 -10 12 cells, or about 10 9 -l O 10 cell).
  • the composition may be prepared such that about 10 4 to IO 10 modified PBMCs (e.g., about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or IO 10 cells/kg) are administered to a subject.
  • the composition includes at least 10 4 , 10 3 , 10 6 , 10 7 , 10 8 , 10 9 , or IO 10 modified PBMCs.
  • about 10 s - 10 10 modified PBMCs are administered to the subject.
  • An appropriate dose can be determined by a treating clinician based on factors such as the subject, the cancer being treated, treatment history, tumor load and type, clinical stage and grade of the disease, viral load, overall health of the subject, and other factors.
  • non-modified lymphocytes are depleted in the subject prior to administering the disclosed composition.
  • the subject is also administered one or more cytokine(s) (such as IL-2, IL-7, IL-15, IL-21, and/or IL-12), for example, to support survival and/or growth of the disclosed modified PBMCs and/or an additional ACT therapy administered in combination, in the subject.
  • cytokine(s) such as IL-2, IL-7, IL-15, IL-21, and/or IL-12
  • at least one of IL-2, IL-7, and IL- 15 is also administered to the subject.
  • the cytokine(s) are administered before, after, or substantially simultaneously with the composition.
  • At least one cytokine (e.g., IL-2, IL-7, and/or IL- 15) is administered simultaneously, for example, with the composition.
  • the modified PBMC is reactive to a tumor- specific antigen in the subject having cancer.
  • the antigen is one or more of: CD19, CD20, BCMA, MUC1 , PSA, CEA, HER1, HER2, TRP-2, EpCAM, GPC3, mesothelin l(MSLN), or EGFR.
  • compositions can be local or systemic.
  • routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
  • the agent is injected or infused into a tumor, or close to a tumor (local administration), or administered to the peritoneal cavity.
  • Appropriate routes of administration can be determined by a treating clinician based on factors such as the subject, the condition being treated, and other factors.
  • compositions can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently.
  • a treating clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors.
  • the subject having cancer receives a treatment in addition to the composition, such as one or more of surgery, radiation, chemotherapy, biologic therapy, immunotherapy, or other therapeutic.
  • chemotherapeutic agents include (but are not limited to) alkylating agents, such as nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine); antimetabolites such as folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine; or natural products, for example vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as
  • Additional agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide); hormones and antagonists, such as adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone proprionate and fluoxymesterone).
  • platinum coordination complexes such as cis-diamine-dichloroplatinum II, also known as cisplatin
  • adriamycin examples include adriamycin, melphalan (Alkeran®) Ara-C (cytarabine), carmustine, busulfan, lomustine, carboplatinum, cisplatinum, cyclophosphamide (Cytoxan®), daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as docetaxel), vinblastine, vincristine, VP- 16, while newer drugs include gemcitabine (Gemzar®), trastuzumab (Herceptin®), irinotecan (CPT-11), leustatin, navelbine, rituximab (Rituxan®) imatinib (STI-571),
  • the subject treated is administered an additional therapeutic, such as a monoclonal antibody cancer immunotherapy (e.g. , anti-CTLA-4, anti-PDl, or anti-PDLl), a T cell agonist antibody, an oncolytic virus, an adoptive cell transfer (ACT) therapy, or any combination of two or more thereof.
  • a monoclonal antibody cancer immunotherapy e.g. , anti-CTLA-4, anti-PDl, or anti-PDLl
  • T cell agonist antibody e.g., anti-CTLA-4, anti-PDl, or anti-PDLl
  • ACT adoptive cell transfer
  • the administration of an additional therapeutic may be before, after, or substantially simultaneously with the administration of the disclosed composition.
  • the additional therapeutic is a cell cycle or checkpoint inhibitor.
  • the checkpoint inhibitor targets PD-1, PD-L1, CTLA-4, CDK4, and/or CDK6.
  • Exemplary inhibitors include ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, palbociclib, ribociclib, and abemaciclib.
  • the subject treated is also administered an ACT therapy, for example, a chimeric antigen receptor (CAR)-expressing T cell, engineered TCR T cell, or a tumor-infiltrating lymphocyte (TIL).
  • an ACT therapy for example, a chimeric antigen receptor (CAR)-expressing T cell, engineered TCR T cell, or a tumor-infiltrating lymphocyte (TIL).
  • the subject is administered an effective amount of the composition and the ACT therapy, and an effective amount of the composition is an amount that increases effectiveness of the ACT (e.g., increases elimination of cancerous cells relative to ACT therapy alone).
  • the additional therapeutic may be administered substantially simultaneously with the disclosed composition.
  • the additional therapeutic is administered prior to administering the composition, for example, at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 12 days, at least 14 days, at least three weeks, at least four weeks, at least one month, or more prior.
  • Multiple doses of the additional therapeutic can be administered to a subject, for example, administered twice daily, once daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently.
  • a treating clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, tumor load and type, clinical stage and grade of the disease and overall health of the subject, and other factors.
  • compositions and kits that can be used with the disclosed methods.
  • the composition or kit includes one or more of the RNAi specific to Zscan20. Jdp2, Nfil3, and/or Znf324, the gRNA specific to Zscan20, Jdp2, Nfil3, and/or Znf324, a nucleic acid or vector encoding the RNAi or gRNA, and the modified PBMC, for example with a pharmaceutically acceptable carrier.
  • the kit includes one or more gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324 and a Cas nuclease or Cas dead nuclease (which may be an RNP complex).
  • the kit includes a vector encoding one or more gRNA specific for Zscan20, Jdp2, Nfil3, and/or Znf324, which can further encode a Cas nuclease or Cas dead nuclease.
  • the kit includes the disclosed modified PBMCs.
  • the kit includes ICB agents, optionally in a separate container.
  • the ICB agents target PD-1, PD-L1, CTLA-4, LAG3 GITR, 4-1BB, CD40, CD40L, and 0X40, TIGIT, VISTA, CD73, CD39, HVEM, BTLA, CD27, CDK4, and/or CDK6.
  • the kit includes other anti-tumor agents, such as a chemotherapeutic agent, optionally in a separate container.
  • the kit includes anti-viral agents, optionally in a separate container.
  • the anti-viral agents could include small molecules that inhibit virus replication, and/or antibodies which neutralize a virus.
  • the kit can include additional reagents, such as one or more of anti-CD3, anti-CD28, IL-2, and IL-15.
  • the reagents are present in separate containers.
  • anti-CD3 and anti-CD28 are in the same container, and may be present, for example, on a bead.
  • the kit further includes one or more of a transfection reagent, culture medium, antibiotic, cytokines (e.g., IL-2, IL-15, and IL-7), optionally wherein such reagents are present in separate containers.
  • the kit or composition includes media in which the PBMCs can be cultured or expanded ex vivo, such as AIM V® media.
  • This example illustrates the materials and methods of the following examples.
  • the analyzed 42 CD8+ T cell samples were collected from ten datasets including this study (FIG. 3A). In total, 139 experiments including 64 ATAC-seq and 75 RNA-seq were harnessed to generate the paired samples and used as input of the Taiji pipeline.
  • the CD8+ T cell samples span nine subtypes: naive cells (Naive), terminal effector cells (TE), memory precursor cells (MP), tissue-resident memory cells (TRM), effector memory cells (TEM), central memory cells (TCM), progenitor exhausted cells (TexProg), intermediate exhausted cells (TexEff-like) and terminal exhausted cells (TexTerm).
  • Taiji vl.1.0 with default parameters was used for the integrative analysis of RNA-seq and ATAC-seq data (Find the page for Taiji at the github website, under Taiji-pipeline / Taiji).
  • the motif file was downloaded directly from the CIS-BP database (33). There were 871 mouse motifs in the analysis.
  • samples were divided into two groups: target group and background group.
  • Target group included all the samples belonging to the cell type of interest and the background group comprised the remaining samples.
  • the normality test using Shapiro-Wilk’s method was used to determine whether the two groups were normally distributed and it was found that the PageRank scores of most samples (90%) follow log-normal distribution. Based on lognormality assumption, an unpaired t-test was used to calculate the P-value. A P-value cutoff of 0.05 and log2 fold change cutoff of 0.5 were used for calling lineage-specific TFs.
  • Taiji-reprogram framework was used(22) to identify differentiation-step-specific TFs, first PageRank ratios were calculated between target and source cell types. Ratio. abs was defined as the reciprocal of ratio if the ratio was smaller than 1 , or otherwise, as the ratio itself. A higher PageRank ratio, abs represents that the TF behaves quite differently between target and source. The top 30 TFs were selected based on PageRank score ratio. abs as candidate TFs. Then, the product of PageRank score ratio, abs of three candidate TFs for all combinations was calculated. All the products were transformed to z-scores and p-value of 0.001 was used as cut-off to select candidate recipes. Finally the TFs were ranked based on the frequency of candidate TFs in all the candidate recipes.
  • DBPNet (49), which is a framework to identify cooperations between DNA- binding proteins using Chromatin immunoprecipitation followed by sequencing (ChlP-seq) and Hi- C data
  • the TF interaction network was constructed based on Taiji’s output, which is TF-regulatee network.
  • Texierm samples’ network was combined by taking the mean value of edge weight for each TF-regulatee pair.
  • nxn correlation matrix (n is the total TF number) was calculated by taking account of the Spearman’s correlation of edge weight for each TF-regulatee pair.
  • R package “huge” (50) was used to build graphical model and to construct the graph.
  • the Graphical lasso algorithm and the shrunken ECDF (empirical cumulative distribution function) estimator were employed, using a lasso penalty X equal to 0.2 to control the regularization. This value was chosen because around 5% of TF-TF pairs have a correlation score > 0.2.
  • ECDF empirical cumulative distribution function
  • a null model was generated by random shuffling the edge weight of TF-regulatee pair across TFs.
  • the chosen cutoff identifies zero interaction, suggesting that the method with cutoff equal to 0.2 has a very low false discovery rate.
  • Communities were detected using Leiden algorithm (51) with modularity as objective function and resolution as 1.8. Network visualization was performed by igraph with Fruchterman-Reingold layout algorithm (52).
  • C57BL/6/J mice were purchased from Jackson Laboratories. P14 mice (Pircher et al., 1987) mice have been previously described. Cas9 P14 mice were generated by crossing P14 mice with B6(C)-Gt(ROSA)26Soreml.l(CAG-cas9*,-EGFP)Rsky/J (Jackson Laboratories). Animals were housed in specific-pathogen-free facilities at the Salk Institute and all experimental studies were approved and performed in accordance with guidelines and regulations implemented by the Salk Institute Animal Care and Use Committee. Mice were infected with 2xl0 5 PFU LCMV- Armstrong by intraperitoneal injection or 2x106 PFU LCMV-Clonel3 by retro-orbital injection under anesthesia.
  • Spleens were mechanically dissociated with ImL syringe plungers over a 70um nylon strainer. Spleens were incubated in ammonium chloride potassium (ACK) buffer for 5 minutes.
  • ACK ammonium chloride potassium
  • Peyer's patches were first removed by dissection. Intestines were longitudinally cut and then cut into 1cm pieces and washed in PBS. Pieces were incubated in 30mL HBSS with 10% FBS, lOmM HEPES, and ImM dithioerythritol with vigorous shaking at 37C for 30 minutes. Supernatants were collected, washed, and further isolated using a 40/67% discontinuous percoll density centrifugation for 20 minutes at room temp with no brakes.
  • B16-gp33 melanoma cell line was cultured in DMEM (Invitrogen) with 10% fetal bovine serum, 1% penicillin-streptomycin and 250 pg/ml G418 (Invitrogen #10131027). All the tumor cell lines were used for experiments when in exponential growth phase.
  • P14 splenocytes were activated in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillin-streptomycin, 2mM L-glutamine, 0.1 mg/ml gp33, BME and 10 U/ml IL-2.
  • tumor-bearing mice were treated with anti-PDl antibody (200 pg per injection, clone GK1.5, BioXcell) twice per week from day 7 post tumor implantation. All experiments were conducted according to the Salk Institute Animal Care and Use Committee.
  • Tumors were minced into small pieces in RPMI containing 2% FBS, DNase I (0.5 pg/ml, Sigma-Aldrich), and collagenase (0.5 mg/ml, Sigma-Aldrich) and kept for digestion for 30 min at 37°C, followed by filtration with 70 pm cell strainers (VWR). Filtered cells were incubated with ACK lysis buffer (Invitrogen) to lyse red blood cells, mixed with excessive RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillin-streptomycin, and centrifuged at 400g for 5 min to obtain single-cell suspension.
  • ACK lysis buffer Invitrogen
  • RPMI 1640 medium Invitrogen
  • gRNA retrovirus vector over-expression 293T cells were transfected with Eco-helper and MSCV gRNA vectors. 48 hr and 72 hr later, supernatant containing retroviral particles was ready for transduction. P14 donor splenocytes were in vitro activated by 0.1 mg/ml gp33 and 10 U/ml IL-2 at 37°C for 24h, then spin-transduced (1500 g) with fresh RV supernatant from 293T cells for 90 min at 30°C in the presence of 5 pg/ml polybrene.
  • Both single cell suspensions were incubated with Fc receptor-blocking anti-CD 16/32 (BioLegend) on ice for 10 min before staining.
  • Cell suspensions were first stained with Red Dead Cell Stain Kit (ThermoFisher) for 10 min on ice. Surface proteins were then stained in FACS buffer (PBS containing 2% FBS and 0.1% sodium azide) for 30 min at 4°C.
  • FACS buffer PBS containing 2% FBS and 0.1% sodium azide
  • Cells were processed for surface marker staining as described above.
  • intracellular cytokine staining cells were fixed in BD Cytofix/Cytoperm (BD #554714) for 30 min at 4 °C, then washed with lx Permeabilization buffer (Invitrogen #00-8333-56).
  • lx Permeabilization buffer Invitrogen #00-8333-56.
  • transcription factor staining cells were fixed in Foxp3 / Transcription Factor Fixation/Permeabilization buffer (Invitrogen #00- 5521-00) for 30 min at 4 °C, then washed with lx Permeabilization buffer. Cells were then stained with intraceulluar antibodies for 30 min at 4 °C.
  • anti-CD8a 53-6.7
  • anti-PD-1 29F.1A12
  • anti-CX3CRl SA011F11
  • anti-SLAMF6 13G3
  • anti-CD38 90
  • anti-CD39 24DMS1
  • anti-CDlOl MoushilOl
  • anti-KRLGl 2F1
  • anti-CD69 H1.2F3
  • anti-CD103 M290
  • anti-CD62L MEL-14
  • anti-Tim3 RMT3-23
  • anti-Ly5.1 A20
  • anti-Ly5.2 104
  • anti- IFN-y XMG1.2
  • anti-TNF-a MP6-XT22
  • anti-GZMB GB11
  • ATAC-seq was performed as previously described (Corces et al., 2017). Briefly, 5,000- 50,000 viable cells were washed with cold PBS, collected by centrifugation, then lysed in resuspension buffer (RSB) (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgC12) supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin. Samples were incubated on ice for 3 min, then washed out with 1 ml RSB containing 0.1% Tween-20.
  • RSB resuspension buffer
  • Nuclei were pelleted by centrifugation at 500g for 10 min at 4°C then resuspended in 50 ul transposition mix (25ul 2x TD buffer, 2.5 ul transposase (100 nM final), 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul H2O) and incubated at 37°C for 30 min in a thermomixer with 1000 RPM mixing.
  • DNA was purified using a Qiagen MinElute PCR cleanup kit, then PCR amplified using indexed oligos. The optimal number of amplification cycles for each sample was determined by qPCR. Libraries were size selected using AmpureXP beads and sequenced using an Illumina NextSeq500 for 75bp paired-end reads.
  • Paired-end 42-bp, or paired-end 75 -bp reads were aligned to the M. musculus mm 10 genome using BWA (53, 54) with parameters “bwa mem -M -k 32”.
  • ATAC-seq peaks were called using MACS2 (55) program using parameters “callpeaks -qvalue 5.0e-2 -shift -100 -extsize 200”.
  • Differentially accessible regions were identified using DESeq2 (56). Batch effect was removed using limma (57). Heatmap visualization of ATAC-seq data was performed using pheatmap.
  • Seurat s module score feature was used to score each cluster based on its per cell expression of TFs.
  • This example illustrates identification of transcription factors (TFs) by dissecting the transcriptional networks and assessing the genome-wide influences of TFs.
  • the global influence of a regulator in the cell is conveyed through its regulatory effect on the target genes, which is propagated over the genetic network.
  • a given regulator's activity is affected by its own expression level and post-translational modification as well as other mechanisms such as the presence of collaborative co-factors and target accessibility. Therefore, the expression level of a regulator such as a TF is not always correlated with its activity (25).
  • many methods have been proposed to infer the activity of regulators using statistical or machine-learning approaches. For instance, Schacht et al. (25) developed a statistical model to estimate the regulatory activity of TFs using their cumulative effects on their target genes.
  • TF activity TFA
  • SCENIC TF activity constructs the genetic network by predicting each gene’s expression using the TF’s expression levels and finding the most predictive TFs.
  • Maslova et al. introduced AI-TAC (28) to predict ATAC-seq signals and identified the most enriched motifs when evaluating the TF importance. Although these methods were able to predict the local activity of a TF, i.e., the expression level of their direct target genes, measuring the system-wide influence of a given TF is not their focus.
  • Taiji builds a genetic network by integrating transcriptomic and open chromatin data, upon which it assesses the global rather than local importance of regulators using the Personalized PageRank algorithm. This feature makes Taiji robust and suitable for integrating multiomics data from noisy genomic measurements. In fact, Taiji clearly outperforms the motif enrichment analysis and the TFA approach (15, 21, 31).
  • This example illustrates the use of a transcription and epigenetic atlas to generate a transcription factor catalog that specifies heterogeneous T cell state.
  • Taiji integrates aforementioned multiomics data to build gene regulatory networks in which each node is a gene and an edge represents a regulatory interaction. Taiji first scans each open chromatin region, presumably active promoter or enhancer, to identify putative TF binding sites using motifs documented in the CIS-BP database (33). These TFs are then linked to their target genes predicted by EpiTensor (34).
  • the node weights were determined by the z scores of gene expression levels, allocating higher ranks to the TFs that regulate more differentially expressed genes.
  • the edge weights were set to be proportional to expression levels and binding strength of TFs and open chromatin intensity (FIG. 1 A).
  • the average number of nodes and edges of the genetic networks of the landscape of the CD8 + T cell states were 18,041 and 1,613,381, respectively, including 871 (4.83%) TF nodes. On average, each TF regulated 1852 genes, and each gene was regulated by 20 TFs.
  • TFs were identified that have high PageRank scores suggesting high activity in nine states of CD8 + T cell (FIG. IB, C). Interestingly 124 out of the 273 TFs are identified as potentially important TFs for more than one cell type (FIG. 1C, D). These TFs, called “multi-taskers”, include many well-known regulators. For instance, TCF7, widely known as a proliferation-related protein (3, 10), is identified as Naive-, MP-, and Texprog-driving TF. All three cell states are multipotent and have high proliferative capacity. T-bet (encoded by Tbx21) is predicted to be highly active in both MP, and TE.
  • T-bet is reported to program cytotoxic T cell terminal differentiation in response to LCMV viral infection in cooperation with another TF ZEB2 (35).
  • MP and TE shared, not only T-bet, but an additional 21 TFs that program early activation (30% of TE or MP TFs) (FIG. ID).
  • Another large number of multi-taskers are observed in exhaustion-associated states, Texprog, TexEff- iike, and TexTerm such as Vax2, Batf, Irf8, Statl, Nfatcl, and Jdp2 (Fig. ID).
  • Texierm also shared 11 multi-taskers with TRM.
  • IE describes single-tasker TFs in TRM and Tex Term (FIG. IE). These single-taskers include a multitude of TFs such as Zscan20, Zbtb49, Arid3a, and Bhlhe41 that have not yet been reported before. Their study should improve the understanding of TF mechanisms underlying T cell state differentiation.
  • This example illustrates how global analysis of the atlas identifies core TF networks that program context-dependent T cell differentiation.
  • naive CD8 + T cells differentiate to early effector cells followed by the differentiation into various CD8 + T cell states in a parallel manner (1).
  • naive cells become either TE or MP.
  • MP can further become diverse types of memory cells including TRM (FIG. 14A).
  • naive cells generate a counterpart of MP, Texpr Og , which further differentiate into Tex Term (FIG. 14B).
  • PageRank ratios between target and source cell types were calculated.
  • the TF members of the cluster show higher expression in CD8+ T cells from chronic LCMV infection compared to acute LCMV infection (FIG. 1G).
  • the TFs are enriched in gene sets associated with cell fate commitment, PD-1 pathway, chronic inflammation/infection (FIG. 1H).
  • This transcriptional wave analysis also reports distinct clusters TFs bifurcating T cell differentiation in acute vs chronic, resident vs circulating, and MP vs TE and their associated biological pathways (FIGs 16-18). These analysis implicate the importance of differential combinations of TFs and connectivity to single taskers in specifically program each cell state.
  • This example illustrates how integrative analysis of TF networks reveals core biological circuits to drive given cell states.
  • T cell therapy can be achieved by programming T cells to avoid dysfunctional TexTerm to favor functional effector state without compromising immunological memory potential. It was sought to identify TFs to perturb that are not only highly active in TexTerm but also the core member of TF communities that cooperate with many other TFs to control biological circuits driving terminal exhaustion and/or suppressing effector function.
  • the intricate network of TFs in TexTerm states was constructed (FIG. 19A) and 11 communities were discovered (FIG. 19B).
  • a list of genes was curated (hereafter referred to as regulatees) controlled by TF members of each community. Different communities have distinct and shared regulatee pools (FIG. 19C).
  • Regulatees of Texierm TFs communities are commonly associated with biological circuits such as: negative regulation of immune system process, covalent chromatin modification, histone modification, dephosphorylation, FoxO signaling pathway, HBV, PD1-PDL1 pathway, and mitochondria organization (FIG. 19D, F) of which the associations to T cell exhaustion have previously been reported (7, 8, 41, 42).
  • the network analysis also reveals connection between TexTerm TFs and pathways that might otherwise have been unappreciated such as cellular catabolic process, GTPase activities, and response to hypoxia/oxidative stress, which provide interesting circuits for future study.
  • FIG. 19C In accordance with the interaction intensities (proximity between communities) between TF communities (FIG. 19B), there are two big groups of communities that shares a large number of regulatees within each group (FIG. 19C).
  • the first group has community (Cm.)Bhlhe40, Cm.Pbx3, Cm.Nfil3.
  • the regulatees of this group are highly enriched in biological pathways associated with epigenetic regulation such as chromatin assembly, methylation, and demethylation (FIG. 2D, F).
  • the second community group has Cm.Prdml, Cm.Nfatcl, Cm.Hicl, and Cm.Nr4a2 of which regulatees are enriched in calcium ion transport, calcium homeostasis, TNF signaling, regulation of oxidative stress, and phosphates activities (Fig. 2D,F).
  • Cm.Prdml shares 86.7% of regulatees and large amount of pathways with other communities (FIG. 19C) implicating importance of Cm.Prdml in orchestrating Texierm biological circuits.
  • Prdml a member of the central community in Texierm
  • Nfil3 which has not yet been reported on its role in CD8 + T cell differentiation
  • Prdml and Nfil3 show clearly different networks in Texierm and TRM states (FIG. 19B; fig. S6; fig. S7).
  • Prdml is found in the community of Zscan20, Irf8, and Gfil in Texierm state but Prdml is neighboring with Nr4al, Irf4, and Hsf2 in TRM state (FIGs. 19B, 20, and 21).
  • the TF-TF interaction analysis of Prdml and Nfil3 reveals that ⁇ 50 % of interaction partners are distinct between two states (FIG. 21). This result indicates that even multi-taskers can be connected to distinct TF neighbors in different states.
  • This example illustrates use of regulatee analysis and in vivo validation to reveal new TFs programming Tex Teim .
  • Cm. Prdml is the hub of regulating biological circuits for overall TexTerm.
  • Cm. Bhlhe40 is the center of group 1 and particularly associated with cell cycle, demethylation, and chromatin reassembly.
  • Zscan20 was selected from Cm.
  • Prdml and Jdp2 from Cm. Bhlhe40 that have the most significant p- value activity score within the community and no activity in TRM (FIG. IE).
  • FRM TRM
  • new TF, Nfil3 and well-studied Prdml were tested.
  • Zscan20 The expression of Zscan20 in human T cells was first reported by Thiesen in 1990 (43). However, the role of the TFs has not yet been studied. According to the Taiji analysis, Zscan20 is exclusively active in terminal exhaustion.
  • the analysis of Zscan20 TF network (FIG. 22) implies its interaction with exhaustion-associated TFs that are already published such as Nfatcl, Nr4al, Nr4a2, and Ir/8 (44-47).
  • the predicted regulatees are specifically upregulated in Texterm state and associated with gene sets that are downregulated in KLRG1 high effector and stimulated T cells, DNA repair, T cell activation, lymphocyte differentiation, PDL- 1 expression and PD- 1 checkpoint pathway in cancer and covalent chromatin modification (FIG. 23).
  • Pan-exhaustion specific TF, Jdp2 is suggested to control transcription via direct regulation of the modification of histones and the assembly of chromatin or heterodimerization with a component of the AP-1 complex (48).
  • Taiji analysis indicates that Jdp2 regulatees suppress the effector- associated genes as they are enriched in naive cells compared to effector cells and biological pathways such as negative regulation of phosphorylation and dephosphorylation (FIG. 24). Therefore, depletion of Jdp2 possibly prevents dysfunctional exhaustion states and restores effector function.
  • This example illustrates how loss of new Tex Tam regulators Jdp2 and Zscan20 contribute to better effector function.
  • KOs contain chronic LCMV infection better then control (FIG. 27 A, B), express higher levels of KLRG1 and CX3CR1, effector- associated differentiation marker (FIG. 27C, D), and release more cytotoxic cytokines (FIG. 27E).
  • This example illustrates disruption of Zscan20 to improve tumor control and the synergy of disrupting Zscan20 with ICB.
  • TexTerm-specific TF-deficiency confers enhanced tumor control.
  • Texprog cells respond to ICB and become more effector-like, states which confer anti-tumor immunity, loss of Zscan20, exclusively deterring Texierm differentiation, will produce more beneficial cell states and generate synergy with immune checkpoint therapy.
  • gZscan20 RV transduced Cas9 P14 cells were transferred into mice with established melanoma tumors expressing GP33-41 (FIG. 27F).
  • Zscan20-deficiency enhanced tumor control in both anti-PDl and control treatment conditions (FIG. 27G-I).
  • This differential tumor growth is associated with the prevention of terminal exhaustion and promotion of Texprog population with the loss of Zscan20 (FIG. 27J-L).
  • Jdp2 KO also improves tumor suppression when it is combined with ICB (FIG. 29A).
  • ICB ICB
  • Jdp2 is likely to be less specific to Tex Term
  • KO can alter the properties of Texpr Og and TexEff-iike in the tumor, possibly making the beneficial effect of KO moderate.
  • TRM and Tex Term multitasker, A/z73-deficiency does not improve tumor control (FIG. 30B) and reduce TRM number (FIG.
  • This precise pipeline of identification of cell state- specifying TFs can be easily adapted in cell state programming of other types of cells to improve the effectiveness of cell therapy.
  • FIG. 31 showcases the systematic validation of cell state selectivity for transcription factors (TFs) using in vivo CRISPR-associated single-cell RNA sequencing.
  • Retroviral tandem gRNA vectors were designed to express two gRNAs.
  • a library of gRNAs was designed to target various TFs, and the resulting perturbations were analyzed for their impact on T cell exhaustion.
  • the results validate bioinformatics -powered TF activity prediction and some TF knockouts lead to the upregulation of genes associated with effector and memory functions.
  • FIG. 32 shows that that knockout of TEX-specific TFs did not hinder the formation of tissue-resident memory T cells (TRMs), indicating selective roles for these TFs in T cell state regulation and demonstrating the accuracy of TF activity prediction.
  • TRMs tissue-resident memory T cells
  • Example 11 Disruption of TEX single-tasker TFs rewire terminal exhaustion and enhance virus and tumor control
  • GSEA Gene set enrichment analysis of TEX-driving transcription factor (TF) knockouts suggests that such perturbations reduce cellular exhaustion and enhance effector functions.
  • TF TEX-driving transcription factor
  • mice and Infections C57BL/6/J mice were purchased from Jackson Laboratories. P14 mice (Pircher et al., 1987) mice have been previously described. Cas9 P14 mice were generated by crossing P14 mice with B6(C)-Gt(ROSA)26Soreml.l(CAG-cas9*,-EGFP)Rsky/J (Jackson Laboratories). Animals were housed in specific -pathogen-free facilities. Mice were infected with 2xl0 5 PFU LCMV- Armstrong by intraperitoneal injection or 2xl0 6 PFU LCMV-Clonel3 by retro- orbital injection under anesthesia.
  • Retrovirus transduction and adoptive transfer For gRNA retrovirus vector over-expression, 293T cells are transfected with Eco-helper and MSCV gRNA vectors. 48 hr and 72 hr later, the supernatant containing retroviral particles was ready for transduction. P14 donor CD8+ T cells are in vitro activated in anti-CD3 and anti-CD28 antibody-coated plate with 100 U/ml hIL-2 at 37°C for 24h, then spin-transduced (1500 g) with fresh RV supernatant from 293T cells for 90 min at 30°C in the presence of 4 ug/ml polybrene. For FIGs.
  • FIGs. 33C-33E right after viral transduction 2 2.5 x 10 A 4 Cas9 expressing gp33-specific P14 TCR transgenic CD8+ T cells were transferred into C57BL/6 mice (retro orbital) that were infected with LCMV one day prior.
  • FIGs. 33F-33M right after viral transduction 1 x 10 A 6 Cas9 expressing gp33-specific P14 TCR transgenic CD8+ T cells were transferred into C57BL/6 mice (retroorbital) that were implanted with 5 x 10 A 5 Bl 6- gp33 cells 7-8 days prior.
  • B16-gp33 melanoma cell line was cultured in DMEM (Invitrogen) with 10% fetal bovine serum, 1% penicillin-streptomycin and 250 pg/ml G418 (Invitrogen #10131027). All the tumor cell lines were used for experiments when in exponential growth phase.
  • P14 splenocytes were activated in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum and 1% penicillin-streptomycin, 2mM L- glutamine, 0.1 mg/ml gp33, BME and 10 U/ml IL-2.
  • tumor-bearing mice were treated with anti-PDl antibody (200 pg per injection, clone RMP1-14, BioXcell) twice per week from day 7 post tumor implantation. References
  • TRM Tissue Resident Memory T

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Abstract

Les données épigénomiques et transcriptomiques ont été utilisées pour identifier les facteurs de transcription (FT) qui définissent les différents états des lymphocytes T CD8+. Les lymphocytes T peuvent être programmés pour éviter un état d'épuisement terminal (TexTerm), un état dysfonctionnel des lymphocytes T que l'on retrouve souvent dans les tumeurs ou les infections chroniques, et pour favoriser un état d'effecteur cytotoxique. TexTerm présente une grande similitude avec les états des T mémoire résidant dans les tissus bénéfiques (TRM) quant à leur emplacement et à leur profil de transcription. Zscan20, un nouveau TF, <i /> s'est avéré actif dans TexTerm. Sa perturbation a empêché la différenciation de TexTerm in vivo, mais pas celle de TRM. La perturbation de Zscan20 programme les lymphocytes T dans un état de type effecteur qui confère un pouvoir de lutte contre les tumeurs et les virus et génère une synergie avec la thérapie des points de contrôle immunitaire. Jdp2, Nfil3 et Znf324 ont également été identifiés comme des conducteurs de TexTerm.
PCT/US2023/086236 2022-12-30 2023-12-28 Facteurs de transcription régulant la différenciation et la perturbation des lymphocytes t dans le cadre de la lutte contre les tumeurs et les virus Ceased WO2024145478A1 (fr)

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Citations (2)

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WO2006063252A2 (fr) * 2004-12-09 2006-06-15 Alnylam Pharmaceuticals, Inc. Compositions et methodes pour induire une reponse immunitaire chez un mammifere et methodes pour eviter une reponse immunitaire dirigee contre des agents oligonucleotidiques, notamment des arn interferents courts
WO2019173636A1 (fr) * 2018-03-07 2019-09-12 Poseida Therapeutics, Inc. Compositions de cartyrin et méthodes d'utilisation

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006063252A2 (fr) * 2004-12-09 2006-06-15 Alnylam Pharmaceuticals, Inc. Compositions et methodes pour induire une reponse immunitaire chez un mammifere et methodes pour eviter une reponse immunitaire dirigee contre des agents oligonucleotidiques, notamment des arn interferents courts
WO2019173636A1 (fr) * 2018-03-07 2019-09-12 Poseida Therapeutics, Inc. Compositions de cartyrin et méthodes d'utilisation

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GUMBER DIANA, WANG LEO D.: "Improving CAR-T immunotherapy: Overcoming the challenges of T cell exhaustion", EBIOMEDICINE, ELSEVIER BV, NL, vol. 77, 1 March 2022 (2022-03-01), NL , pages 103941, XP055949035, ISSN: 2352-3964, DOI: 10.1016/j.ebiom.2022.103941 *
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