Regulation of SUV39H1 expression by RNA

Abstract

The present invention provides compositions and methods for modulating the expression of SUV39H1 using inhibitory or activating polynucleotides based on the sequences of long non-coding RNA or short hairpin RNA (shRNA).

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compositions and methods for modulating the expression of SUV39H1 using RNA. [Background technology]

[0002] 2. Background of the Invention Long non-coding RNAs (lncRNAs) are regulators of gene expression and are involved in numerous cellular processes, and their expression has been found to be particularly tissue- or developmental stage-specific (Ahmad et al., 2021).

[0003] Small hairpin RNA (shRNA) is a sequence of RNA, typically about 80 base pairs long, that contains an internal hybridization region that creates a hairpin structure.shRNA molecules are processed in cells to form siRNA, which then knocks down gene expression.The advantage of shRNA is that it can be incorporated into plasmid vectors and integrated into genomic DNA for long-term or stable expression, and thus long-term knockdown of target mRNA.

[0004] Histone methyltransferases promote chromatin remodeling into euchromatin (open, actively transcribed chromatin) and heterochromatin (closed, inactive chromatin) through their post-translational modification of histone molecules at nucleosomes. Histone methyltransferases actively control gene expression and cell fate. SUV39H1 was one of the first methyltransferases identified (Aagaard et al., 1999). SUV39H1 has been shown to be involved in numerous developmental processes, especially in the immune system (Rao et al., 2017; Nicetto & Zaret, 2019; Allan et al., 2012; Pace et al., 2018).

[0005] Pace et al., 2018 and WO 2018 / 234370 disclose that inhibiting expression of SUV39H1 in T cells and NK cells enhances their memory potential and increases survival. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2018 / 234370 [Patent Document 2] International Publication No. 2019 / 157454 [Patent Document 3] International Publication No. 2021 / 043804 [Patent Document 4] International Publication No. 2022 / 189620 [Patent Document 5] International Publication No. 2022 / 189626 [Patent Document 6] International Publication No. 2022 / 189636 [Patent Document 7] International Publication No. 2017 / 062451 [Patent Document 8] International Publication No. 2017 / 180989 [Patent Document 9] International Publication No. 2014 / 055668 [Patent Document 10] International Publication No. 2018 / 132506 [Patent Document 11] International Publication No. 2019 / 133969 [Patent Document 12] International Publication No. 2018 / 067993 [Patent Document 13] International Publication No. 2000 / 014257 [Patent Document 14] International Publication No. 2013 / 126726 [Patent Document 15] International Publication No. 2012 / 129514 [Patent Document 16] International Publication No. 2014 / 031687 [Patent Document 17] International Publication No. 2013 / 166321 [Patent Document 18] International Publication No. 2013 / 071154 [Patent Document 19] International Publication No. 2013 / 123061 [Patent Document 20] US Patent Application Publication No. 2002131960 [Patent Document 21] US Patent Application Publication No. 2013287748 [Patent Document 22] U.S. Patent Application Publication No. 20130149337 [Patent Document 23] U.S. Patent No. 6,451,995 [Patent Document 24] U.S. Patent No. 7,446,190 [Patent Document 25] U.S. Patent No. 8,252,592 [Patent Document 26] U.S. Patent No. 8,339,645 [Patent Document 27] U.S. Patent No. 8,398,282 [Patent Document 28] U.S. Patent No. 7,446,179 [Patent Document 29] U.S. Patent No. 6,410,319 [Patent Document 30] U.S. Patent No. 7,070,995 [Patent Document 31] U.S. Patent No. 7,265,209 [Patent Document 32] U.S. Patent No. 7,354,762 [Patent Document 33] U.S. Patent No. 7,446,191 [Patent Document 34] U.S. Patent No. 8,324,353 [Patent Document 35] U.S. Patent No. 8,479,118 [Patent Document 36] European Patent Application Publication No. 2537416 [Patent Document 37] International Publication No. 2021 / 016174 [Patent Document 38] International Publication No. 2011 / 009173 [Patent Document 39] International Publication No. 2012 / 135854 [Patent Document 40] US Patent Application Publication No. 20140065708 [Patent Document 41] U.S. Patent No. 5,399,346 [Patent Document 42] US Patent Application Publication No. 2003 / 0170238 [Patent Document 43] U.S. Patent No. 4,690,915 [Patent Document 44] International Application No. PCT / EP2020 / 070845 [Non-patent literature]

[0007] [Non-Patent Document 1] genome-euro.ucsc.edu [Non-Patent Document 2] SMITH and WATERMAN, Ad. App. Math., vol. 2, p. 482, 1981 [Non-Patent Document 3] NEEDLEMAN and WUNSCH, J. Mol. Biol, vol. 48, p. 443, 1970 [Non-Patent Document 4] PEARSON and LIPMAN, Proc. Natl. Acad. Sci. USA, vol. 85, p. 2444, 1988. [Non-Patent Document 5] Edgar, Robert C, Nucleic Acids Research, vol.32, page 1792, 2004 [Non-Patent Document 6] Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol Biol. 2010;629:141~58 pages

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[0008] SUMMARY OF THE PRESENT APPLICATION The present disclosure provides compositions and methods for regulating the expression of SUV39H1 using RNA.

[0009] The present invention relates to a heterologous nucleic acid expressing an RNA comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell; a heterologous nucleic acid comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof capable of expressing an RNA capable of inhibiting the expression of SUV39H1 in a cell; The present invention relates to a cell comprising a first nucleic acid, which is a nucleic acid operably linked to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequence) that expresses an RNA comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell, and is operably linked to a heterologous expression control sequence; or a nucleic acid operably linked to a heterologous expression control sequence that is operably linked to a heterologous expression control sequence that expresses an RNA comprising the nucleotide sequence of [SEQ ID NO: 5] (genomic sequence encoding AF196970.3 (ENSG00000232828)) or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell, and is operably linked to a heterologous expression control sequence. The first nucleic acid may comprise any of the nucleotide sequences of SEQ ID NOs: 5 to 9 or a fragment thereof. The first nucleic acid is capable of expressing an RNA comprising the nucleobase sequence of [SEQ ID NO: 2] (lncRNA AF196970.3 exon 1) or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell. The first nucleic acid can express an RNA comprising the nucleic acid base sequence of [SEQ ID NO: 3] (lncRNA AF196970.3 exon 2) or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell. The first nucleic acid can express an RNA comprising the nucleic acid base sequence of [SEQ ID NO: 4] (lncRNA AF196970.3 exon 3) or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell.In any of the above-described embodiments, the first nucleic acid can express RNA of at least about 12-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 250-750, 500-750 or more bases in length.

[0010] The present invention also relates to a heterologous nucleic acid expressing an RNA comprising any one of the nucleotide sequences of SEQ ID NOs: 13-17 or 26-30, or a fragment or derivative thereof capable of inhibiting the expression of SUV39H1 in a cell; or a fragment thereof expressing an RNA capable of inhibiting the expression of SUV39H1 in a cell; a nucleic acid expressing an RNA comprising any one of the nucleotide sequences of SEQ ID NOs: 13-17 or 26-30, or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell, typically a nucleic acid operably linked to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequence). The nucleic acid may comprise any one of the nucleotide sequences of SEQ ID NOs: 13-17 or 26-30, or a fragment thereof.

[0011] The present invention also relates to a cell comprising a heterologous polynucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 32-36 and 45-49, or a fragment thereof capable of inhibiting expression of SUV39H1 in a cell. In some embodiments, the nucleic acid may be operably linked to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequence).

[0012] The cell may be a modified immune cell.

[0013] The cells can be any of the following cell types: T cells, CD4+ T cells, CD8+ T cells, CD4+ and CD8+ T cells, NK cells, Treg cells, Tm cells, memory stem cells (TSCM), TCM cells, TEM cells, monocytes, dendritic cells, or macrophages, T cell precursors, NK cell precursors, pluripotent stem cells, induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), adipose-derived stem cells (ADSCs), pluripotent stem cells of the myeloid or lymphoid lineages.

[0014] The cells may contain one or more, two or more, or three or more engineered receptors.

[0015] The cells may contain a second heterologous nucleic acid that expresses one or more engineered receptors. The engineered receptor comprises: a) an extracellular antigen-binding domain that specifically binds an antigen, optionally comprising an antibody heavy chain variable region and / or an antibody light chain variable region, which may optionally be bispecific or trispecific; b) a transmembrane domain, optionally comprising a fragment of the transmembrane domain of the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD34, CD137 or CD154, NKG2D, OX40, ICOS, 2B4, DAP10, DAP12, CD40; c) optionally one or more costimulatory domains from 4-1BB, CD28, ICOS, OX40, DAP10 or DAP12, 2B4, CD40, FCER1G; and d) The engineered receptor may comprise an intracellular signaling domain from CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, or CD66d, 2B4, or any fragment thereof. The engineered receptor may be a chimeric antigen receptor (CAR) comprising an extracellular antigen binding domain, optionally an scFv, optionally a transmembrane domain from CD28, CD8 or CD3ζ, optionally one or more costimulatory domains from 4-1BB, CD28, ICOS, OX40 or DAP10, and an intracellular signaling domain from CD3ζ, optionally with ITAM2 and ITAM3 inactivated.

[0016] The engineered receptor may also be a modified TCR.

[0017] The modified TCR may comprise (a) an extracellular domain comprising an antigen-binding fragment or CDRs of an antibody, preferably all three CDRs of the heavy chain variable region (VH) and / or the light chain variable region (VL), and (b) a native or variant constant region of an α, β, γ or δ chain. For example, the modified TCR may comprise one or more heterologous polypeptides, such as (a) an antibody VH or a fragment or variant having at least 90% sequence identity thereto, fused to TRBC1 (typically human or murine TRBC1) or TRBC2 (typically human or murine TRBC2), or a fragment or variant of TRBC1 or TRBC2 having at least 90% sequence identity thereto, and (b) an antibody VL or a fragment or variant having at least 90% sequence identity thereto, fused to TRAC (human or murine TRAC sequence), or a fragment or variant of TRAC having at least 90% sequence identity thereto. The modified TCR may optionally further comprise a native or variant CD3ζ polypeptide, for example a modified CD3ζ polypeptide in which one or two of the ITAM domains (e.g., ITAM2 and ITAM3) have been deleted.

[0018] Recombinant HLA-independent (or non-HLA-restricted) modified TCRs (referred to as "HI-TCRs") that bind to an antigen of interest in an HLA-independent manner are typically described in WO 2019 / 157454. Such HI-TCRs comprise (a) a heterologous antigen-binding domain, e.g., an antigen-binding fragment of an immunoglobulin variable region, that binds to an antigen in an HLA-independent manner; and (b) an antigen-binding chain that comprises a constant domain capable of associating with (and thus activating) a CD3ζ polypeptide. Preferably, the antigen-binding domain or fragment thereof comprises (i) an antibody heavy chain variable region (VH) and / or (ii) an antibody light chain variable region (VL). The constant domain of the TCR is, for example, a native or modified TRAC polypeptide (typically a human or murine TRAC sequence), or a native or modified TRBC polypeptide (typically a human or murine TRBC sequence). The constant domain of the TCR is, for example, a (human or murine) native TCR constant domain (α or β) or a fragment thereof. Unlike chimeric antigen receptors, which typically contain an intracellular signaling domain themselves, HI-TCRs do not directly generate an activation signal; rather, the antigen-binding chain associates with and consequently activates the CD3ζ polypeptide. Immune cells containing recombinant TCRs provide superior activity when the antigen has a low density (typically intermediate density) on the cell surface of less than about 10,000 molecules per cell, e.g., less than about 5,000, 4,000, 3,000, 2,000, 1,000, 500, 250, or 100 molecules per cell.

[0019] Thus, an engineered receptor may typically be a modified TCR comprising a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH); and a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL), wherein the first and second antigen-binding chains each comprise a TRAC polypeptide or a TRBC polypeptide, optionally, at least one of the TRAC polypeptide and the TRBC polypeptide is endogenous, and optionally one or both of the endogenous TRAC and TRBC polypeptides are inactivated.

[0020] The cells may further comprise a chimeric costimulatory receptor comprising (a) the extracellular and transmembrane domains of CD86, 41BBL, CD275, CD40L, OX40L, PD-1, TIGIT, 2B4, or NRP1, or a fragment or variant thereof, and (b) an intracellular costimulatory molecule of CD28, 4-1BB, OX40, ICOS, CD27, CD40, or CD2, or a fragment or variant thereof. In some embodiments, the chimeric costimulatory receptor comprises (a) the extracellular domain of a costimulatory ligand, optionally from CD80, (b) the transmembrane domain, optionally from CD80, and (c) the intracellular domain of a costimulatory molecule, optionally CD28, 4-1BB, OX40, ICOS, DAP10, CD27, CD40, NKGD2, or CD2, preferably 4-1BB.

[0021] The extracellular antigen-binding domains of a cell are approximately 1 × 10 -7 Below, about 5×10 -8 Below, approximately 1×10 -8 Below, about 5×10 -9 Below, approximately 1×10 -9 Below, about 5×10 -10 Below, approximately 1×10 -10 Below, about 5×10 -11 Below, approximately 1×10 -11 Below, about 5×10 -12 or less, or about 1 x 10 -12 The antigen can be bound with the following KD affinities (lower numbers indicate higher affinities): The antigen bound by the extracellular antigen-binding domain can have a low density on the cell surface of less than about 10,000 molecules, or less than about 5,000 molecules, or less than about 2,000 molecules per cell.

[0022] The extracellular antigen-binding domain is capable of binding to an antigen, for example:

[0023] Orphan tyrosine kinase receptor ROR1, tEGFR, Her2, p95HER2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, claudin 18.2, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, CD70, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, FcRH5, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, κ light chain, BCMA, Lewis Y, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen (PSMA), estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, MAGE A3, CE7, or Wilms tumor 1 (WT-1). In some embodiments, the antigen can be any of the tumor neoantigenic peptides disclosed in WO 2021 / 043804, WO 2022 / 189620, WO 2022 / 189626, WO 2022 / 189636, which are incorporated by reference in their entireties.

[0024] The cell may contain two engineered antigen receptors, each of which binds a different antigen.

[0025] The cells can be autologous or allogeneic.

[0026] SUV39H1 expression can be reduced or inhibited in cells by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

[0027] The cells may initially be isolated from a subject suffering from or at risk of suffering from cancer.

[0028] The present invention also relates to modified oligonucleotides comprising a nucleobase sequence from any of [SEQ ID NOs: 1-4] at least about 12 bases in length, the modified oligonucleotides comprising one or more of a modified backbone linkage, a modified sugar moiety, a modified phosphate moiety, a modified nucleobase, or a chemically conjugated moiety. The modified oligonucleotides can be at least about 12-50, 50-75, or 50-100 bases in length. The modified oligonucleotides can comprise modified backbone linkages comprising phosphorothioate, phosphonoacetate, thiophosphonoacetate, methylphosphonate, boranophosphate, or phosphorodithioate moieties. Modified oligonucleotides may include modified sugars that are modified to replace the 2'OH group with another group, optionally H, -OR, -R (where R can be, for example, an alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), halo, -F, -Br, -Cl, or -I, -SH, -SR (where R can be, for example, an alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or a sugar), -arabino, F-arabino, amino (where amino can be, for example, NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or an amino acid); or cyano (-CN); optionally a 2'-O-methoxy, a 2'-O-methoxyethyl modification, a 2'-fluoro, a 2'-deoxy, or a combination thereof.Modified oligonucleotides may contain one or more 5-methylcytosines, modified uridines, such as 5-(2-amino)propyluridine, and 5-bromouridine, modified adenosines and guanosines, such as those including modifications at the 8 position, such as 8-bromoguanosine, deazanucleotides, such as 7-deazaadenosine, or O- and N-alkylated nucleotides, such as N6-methyladenosine, or modified nucleotides that are polycyclic (e.g., tricyclic; and "unlocked" forms, such as glycol nucleic acids (GNAs) (e.g., R-GNAs or S-GNAs, in which the ribose is replaced by a glycol unit linked to a phosphodiester bond), or threose nucleic acid (TNA, in which the ribose is replaced by α-L-threofuranosyl-(3'→2')).

[0029] The present invention also relates to a nucleic acid operably linked to a heterologous expression control sequence, comprising (a) a nucleotide sequence encoding or expressing an RNA comprising the nucleic acid base sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or any one of the nucleotide sequences of SEQ ID NOs: 13-17 and 26-30, or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell, or (b) a nucleotide sequence of [SEQ ID NO: 5] (a genomic sequence encoding AF196970.3 (ENSG00000232828)) or any one of the nucleotide sequences of SEQ ID NOs: 32-36 and 45-49, or a fragment thereof capable of expressing an RNA capable of inhibiting the expression of SUV39H1 in a cell. The nucleic acid may comprise any one of the nucleotide sequences of SEQ ID NOs: 5-9, 32-36, 42-49, or a fragment or group of fragments thereof. The heterologous regulatory sequence may be a constitutive, inducible, or tissue-specific promoter, optionally EF1α, CMV, SFFV, hPGK, RPBSA, or CAG.

[0030] The present invention also relates to vectors comprising a nucleic acid as described herein and one or more additional expression control sequences, including vectors, viral vectors, optionally including adenoviruses, adeno-associated viruses (AAV), poxviruses, papillomaviruses, lentiviruses, retroviruses, herpesviruses, foamiviruses, Semliki Forest virus vectors, and pseudotyped viruses.

[0031] The present invention also relates to a delivery vehicle, optionally a liposome, a lipid-containing complex, a nanoparticle, a gold particle, or a polymer complex, comprising the above-mentioned nucleic acid or vector.

[0032] The present invention also relates to a method of generating a cell as described herein comprising at least the steps of: (a) introducing into a cell (i) a nucleic acid as described herein or (ii) a vector as described herein, and optionally (b) introducing into the cell a nucleic acid encoding an antigen-specific receptor.

[0033] The present invention also relates to a method of generating a cell as described herein, comprising at least the step of introducing into the cell a heterologous expression control sequence in a manner operably linked to an endogenous nucleic acid expressing an RNA comprising the nucleobase sequence of [SEQ ID NO: 1] (lncRNA AF196970.3) or a nucleotide sequence of any one of SEQ ID NOs: 13-17, 26-30, or a fragment thereof capable of inhibiting expression of SUV39H1 in the cell, optionally a genomic sequence encoding [SEQ ID NO: 5] (AF196970.3 (ENSG00000232828) or an allelic variant thereof, or a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 32-36 and 45-49.

[0034] The invention also relates to a method of using the cells as described herein to treat a disease comprising administering to a subject in need thereof an effective amount of the cells to treat the disease, optionally cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an allergic disease, wherein the cells express one or more antigen-specific receptors that bind to an antigen associated with the disease.

[0035] The present invention also relates to a method for treating a subject suffering from cancer, comprising administering to the subject: (1) a cell as described herein; and (2) a second cancer therapeutic agent. The second cancer therapeutic agent can be an immune checkpoint modulator, a cancer vaccine, a chemotherapeutic agent, or an anti-angiogen.

[0036] The present invention also relates to a method for treating a subject suffering from cancer, comprising administering to the subject (1) a cell as described herein, a T cell, a NK cell or a T cell precursor comprising a genetically engineered antigen receptor, wherein expression of the SUV39H1 gene is inhibited, and wherein inhibition of the SUV39H1 gene results in enhanced anti-cancer activity of the immune cell; and (2) an immune checkpoint modulator. The immune checkpoint modulator can be an inhibitor of PD1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, EP2 / 4 adenosine receptor, or A2AR. The immune checkpoint modulator can be an anti-PD-1 inhibitor or an anti-PDL-1 inhibitor. BRIEF DESCRIPTION OF THE DRAWINGS [Brief description of the drawings]

[0037] [Figure 1] Figure 1 shows the locus of the SUV39H1 gene, including the gene ENSG00000232828 that encodes (expresses) lncRNA AF196970.3, in the UCSC genome browser (genome-euro.ucsc.edu). [Diagram 2]Figure 2A shows lncRNA AF196970.3 including its three exons, Figure 2B shows the overlap in the genomic region and the shared nucleotides between lncRNA AF196970.3 exon 2 and SUV39H1 exon 3, and C) the overlap in the genomic region and the shared nucleotides between lncRNA AF196970.3 exon 3 and SUV39H1 exon 2. [Diagram 3] FIG. 3A shows bulk tissue gene expression for AF196970.3, FIG. 3B shows bulk tissue gene expression for SUV39H1, FIG. 3C shows single cell expression for AF196970.3 in different tissues, FIG. 3D shows single cell expression for SUV39H1 in different tissues, and FIG. 3E shows expression of the AF196970.3 transcript ENST00000416061.1 in different tissues. [Figure 4] Figures 4A, 4B, and 4C show expression levels of AF196970.3 and SUV39H1 in single-cell RNAseq-derived clusters from glioma, head and neck squamous cell carcinoma, and hepatocellular carcinoma, respectively. [Diagram 5] FIG. 5 shows a plasmid map representing a PiggyBac backbone containing a GFP-puromycin reporter and a lncRNA SUV39H1 exon sequence containing a CMV promoter (5A) or a lncRNA SUV39H1 exon sequence containing a hPKG promoter (5B). [Figure 6]Figure 6A shows a schematic diagram of the experimental procedure of Piggy Bac transfection in HEK293 FT cells using the two plasmid constructs of Figure 5. Figure 6B shows GFP expression levels by flow cytometry and comparison of untransfected cells with cells transfected with either the empty Piggy Bac construct or one of the lncRNA SUV39H1 Piggy Bac plasmids. Figure 6C shows the expression of SUV39H1 and actin by Western blotting in cells. Quantification of SUV SUV39H1 protein levels normalized to actin. Figure 6D illustrates a schematic diagram of the experimental procedure of lncRNA SUV39H1 expression in CD8+ T cells. [Figure 7] Figure 7A shows a representation of the shRNA target sequence on the SUV39H1 gene. Figure 7B shows the target and loop sequences of different shRNAs targeting SUV39H1. Figure 7C shows the plasmid map of the lentiviral construct used for shRNA expression in cells using the U6 promoter and EGFP reporter. [Figure 8] Figure 8A illustrates the experimental procedure for shRNA expression in HEK293 FT cells. Figure 8B shows GFP expression levels by flow cytometry: comparison of untransduced cells and cells transduced with either scrambled shRNA or one of five shRNAs against SUV39H1. Figure 8C shows SUV39H1 and actin expression by Western blotting in cells. [Figure 9]FIG. 9A shows an illustrated experimental procedure of shRNA expression in CD8+ T cells. FIG. 9B shows GFP expression levels by flow cytometry: comparison of untransduced cells with cells transduced with either scrambled shRNA or one of five shRNAs against SUV39H1. Results are shown for a representative donor. FIG. 9C shows SUV39H1 and actin expression by Western blotting in cells. Control cells in the absence (mock) or presence (gRNA SUV) of SUV39H1-targeting gRNA. FIG. 9D shows levels of H3K9 trimethylation (geometric mean fluorescence intensity) by flow cytometry in T cells. Results are shown for a representative donor. FIG. 9E-F shows flow cytometric expression of memory marker CD27 in T cells at day 7: comparison of scrambled sequence versus shRNA 1 for three donors (average of duplicate replicates). FIG. 9F shows flow cytometric expression of memory marker CD27 in T cells at day 14: comparison of shRNA 1 versus scrambled sequence for two donors (average of duplicate replicates). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] Detailed Description of the Invention SUV39H1 is an H3K9 histone methyltransferase that functions in silencing memory and stem cell programs during terminal differentiation of effector CD8+ T cells. Silencing SUV39H1 has in turn been shown to enhance long-term memory potential and increase survival. See human SUV39H1, UniProt accession number O43463.

[0039] The present disclosure relates to the identification of AF196970.3, an antisense lncRNA, which has a silencing function against SUV39H1 in human cells, inhibits SUV39H1 expression, and thus reduces the level of SUV39H1 protein in cells. AF196970.3 has an expression pattern very similar to that of SUV39H1. AF196970.3 is detected in many different cell types, with the highest levels in endothelial cells, fibroblasts, and myocytes, similar to SUV39H1. AF196970.3 is expressed in human tumor-infiltrating lymphocytes in at least three different types of cancer, including glioblastoma, head and neck squamous cell carcinoma, and hepatocellular carcinoma. AF196970.3 expression levels correlate with SUV39H1 levels, and are relatively high in proliferating T cells, similar to SUV39H1.

[0040] The present disclosure also relates to the identification of short hairpin RNAs comprising any one of the target sequences as set forth in any one of SEQ ID NOs: 13-17 (as described herein, shRNA1-5) or any one of the sequences in SEQ ID NOs: 26-30 (including the target sequence, loop sequence and guide sequence for forming the shRNA).

[0041] The present disclosure provides inhibitory polynucleotides and uses thereof. SUV39H1 expression in cells can be inhibited by increasing ectopic or endogenous expression of this antisense lncRNA sequence, or shRNA or similar polynucleotides based on this sequence as disclosed herein, variants and / or fragments thereof. Reduced SUV39H1 levels in cells affect the commitment of cells to terminal differentiation and / or prolong survival. This lncRNA sequence, or fragments or variants thereof, including shorter RNAs or similar polynucleotides based on the lncRNA sequence, and shRNAs as described herein, typically comprising one of the target sequences of SEQ ID NOs: 13-17, or comprising any one of SEQ ID NOs: 26-30, could function either as a local inhibitor of SUV39H1 transcription and / or via its overlapping antisense exon. In any of these embodiments, inhibition of SUV39H1 reduces SUV39H1 expression and / or activity by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% or more compared to wild-type cells in which SUV39H1 expression is not regulated. In some embodiments, RNA inhibitors as described herein reduce H3K9 trimethylation by at least about 20%, 30%, 40%, 50%, 60%, 70%, 75%, or 80% or more compared to wild-type cells in which SUV39H1 expression is not regulated (see also the Results section for exemplary assays).

[0042] The present disclosure also provides activating polynucleotides and their uses.SUV39H1 expression in cells is increased by inhibiting the expression or increasing the degradation of lncRNA sequence AF196970.3.RNA interference (RNAi), short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) can be used to inhibit the expression or decrease the degradation of RNA.

[0043] Immune cells, particularly T cells or NK cells, whose expression of SUV39H1 is inhibited may show enhanced central memory phenotype, enhanced survival rate and persistence after adoptive transfer, and reduced exhaustion.In particular, such cells accumulate and reprogram with increased efficiency into long-lived central memory T cells.Such cells are more efficient in inducing tumor cell rejection and show enhanced efficacy for treating cancer.

[0044] definition As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0045] Also, as used herein, "and / or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0046] The term "about" as used herein in reference to a measurable value, such as amount of a polypeptide, dosage, time, temperature, enzymatic activity or other biological activity, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated amount.

[0047] The term "antibody" herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, variable heavy (VH) regions capable of specific binding to an antigen, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses recombinant and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecifics, such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD, in some embodiments, an antibody comprises a heavy chain variable region and a light chain variable region.

[0048] By "antibody fragment" is meant a molecule other than an intact antibody that contains a portion of an intact antibody that binds to an antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; variable heavy (VH) regions, VHH antibodies, single-chain antibody molecules, e.g., scFv and single-domain VH monoclonal antibodies; and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment, e.g., scFv, that contains a variable heavy chain region and / or a variable light chain region.

[0049] "Inactivation" or "disruption" of a gene refers to a change in the sequence of genomic DNA that causes a reduction or elimination of gene expression or causes the expression of a non-functional gene product. Exemplary methods include gene silencing, knockdown, knockout, and / or gene disruption techniques, such as gene editing, for example, via introduction of truncations and / or homologous recombination. Examples of such gene disruptions are insertion of a gene or a portion of a gene, including deletion of the entire gene, frameshift and missense mutations, deletions, knock-ins, and knock-outs. Such disruptions can occur in the coding region, for example, in one or more exons, for example, by insertion of a stop codon, resulting in the inability to produce a full-length product, a functional product, or any product. Such disruptions can also occur by disruption in the promoter or enhancer or other regions that affect activation of transcription to prevent transcription of the gene. Gene disruptions include gene targeting, including targeted gene inactivation by homologous recombination.

[0050] As used herein, "inhibition" of a gene product means a reduction in its activity and / or gene expression by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more compared to the uninhibited or suppressed wild-type activity or expression level.

[0051] As used herein, "express" or "expression" means that a gene sequence is transcribed and, optionally, translated. If the gene expresses a non-coding RNA, expression will typically result in the RNA after transcription and, optionally, splicing. If the gene is a coding sequence, expression will typically result in the production of a polypeptide after transcription and translation.

[0052] As used herein, "expression control sequence" refers to a nucleotide sequence that influences the transcription, RNA processing, RNA stability, or translation of an associated nucleotide sequence. Examples include, but are not limited to, promoters, enhancers, introns, translation leader sequences, polyadenylation signal sequences, transcription initiation factors, and transcriptional and / or translational termination regions (i.e., termination regions).

[0053] As used herein, a "fragment" refers to a portion of a referenced sequence (polynucleotide or polypeptide) having a length of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the full-length sequence.

[0054] As used herein, "heterologous" refers to a polynucleotide or polypeptide that contains sequences that are not found in the same relationship to each other in nature. For example, a heterologous sequence is either derived from another species, or derived from the same species or organism, but modified from either its native form or the form predominantly expressed in the cell. Thus, a heterologous polynucleotide includes a nucleotide sequence that is derived from and inserted into the same naturally occurring, original cell type, but that is present in a non-native state, e.g., in a different copy number, and / or is under the control of different regulatory sequences than those found in nature, and / or is located in a different location (adjacent to a different nucleotide sequence) than the location in which it is originally located.

[0055] As used herein, "nucleic acid," "nucleotide sequence," and "oligonucleotide" or "polynucleotide" are used interchangeably and include both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA or RNA, and chimeras of RNA and DNA. The terms polynucleotide, nucleotide sequence, or nucleic acid refer to a chain of nucleotides, regardless of the length of the chain. A nucleic acid can be double-stranded or single-stranded. If single-stranded, the nucleic acid can be the sense strand or the antisense strand. A nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids with altered base-pairing abilities or increased resistance to nucleases. The disclosure further provides nucleic acids that are complements (which can be either full complements or partial complements) of the nucleic acids, nucleotide sequences, or polynucleotides described herein. Modified bases (modified nucleobases), such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others, can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications, such as modifications to the phosphodiester backbone or the 2'-hydroxyl in the ribose sugar group of RNA, can also be made.

[0056] As used herein, "operably linked" means that an element, such as an expression control sequence, is configured to perform its normal function on a nucleotide sequence of interest. For example, a promoter operably linked to a nucleotide sequence of interest is capable of influencing the expression of the nucleotide sequence of interest. An expression control sequence need not be contiguous with the nucleotide sequence of interest, so long as it functions to direct its expression.

[0057] As used herein, the expression "percentage of identity" between two sequences refers to the percentage of identical bases or amino acids between the two sequences being compared, obtained using the best alignment of the sequences, and this percentage is purely statistical, and the differences between these two sequences are spread randomly throughout the two sequences. Bases are considered to be complementary when hybridizing under standard conditions. For example, modified nucleobases can be aligned in a similar manner to the bases that they mimic hybridization patterns. As used herein, "best alignment" or "optimal alignment" refers to the alignment in which the determined percentage of identity (see above) is the highest. Sequence comparison between two nucleic acid sequences (also referred to herein as nucleotide sequences or nucleobase sequences) is usually achieved by comparing these sequences that have been aligned beforehand according to best alignment. This comparison is performed on a comparison segment to identify and compare local regions of similarity. The best sequence alignment for the comparison can be performed manually, or by using the global homology algorithm developed by SMITH and WATERMAN (Ad. App. Math., vol. 2, p. 482, 1981), by using the local homology algorithm developed by NEEDLEMAN and WUNSCH (J. Mol. Biol, vol. 48, p. 443, 1970), by using the similarity method developed by PEARSON and LIPMAN (Proc. Natl. Acad. Sci. USA, vol. 85, p. 2444, 1988), by using computer software with such algorithms (GAP, BESTFIT, BLAST P, BLAST N, FASTA, TFASTA in the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI, USA), by using the MUSCLE multiple alignment algorithm (Edgar, Robert C, Nucleic Acids This can be achieved by using the method described in "Research, vol. 32, p. 1792, 2004."To obtain the best local alignment, preferably, BLAST software can be used. The identity percentage between two sequences is determined by comparing the two sequences that are optimally aligned, and the sequence may contain additions or deletions to the reference sequence to obtain the optimal alignment between the two sequences. The identity percentage is calculated by determining the number of identical positions between the two sequences and dividing this number by the total number of positions compared, and multiplying the result by 100 to obtain the identity percentage between the two sequences.

[0058] As used herein, "treatment" or "treating" includes the application of the disclosed cells or compositions comprising cells to a patient in need thereof for the purpose of curing, relieving, alleviating, altering, correcting, ameliorating, improving, or affecting a disease, such as cancer, or any of the symptoms of a disease (e.g., cancer). In particular, the term "treat" or "treatment" refers to reducing or alleviating at least one adverse clinical symptom associated with the disease. With reference to cancer treatment, the term "treat" or "treatment" also refers to slowing or reversing the progression of neoplastic uncontrolled cell proliferation, i.e., shrinking an existing tumor and / or halting tumor growth. The term "treat" or "treatment" also refers to inducing apoptosis in cancer or tumor cells in a subject.

[0059] As used herein, a "variant" refers to a variant that is a sequence that is at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, It refers to a sequence (polynucleotide or polypeptide) that has mutations (deletions, substitutions or insertions) that are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a reference sequence over a region of 600, 700, 800, 900, 1000, or 1100 nucleotides or amino acids. With respect to polynucleotide sequences, variants also encompass polynucleotides that hybridize under stringent conditions to the reference sequence or its complement.

[0060] As used herein, a "vector" is any nucleic acid molecule for transfer into or expression of a nucleic acid in a cell. The term "vector" includes both viral and non-viral (e.g., plasmid) nucleic acid molecules for introducing a nucleic acid into a cell in vitro, ex vivo, and / or in vivo. A vector may include expression control sequences, restriction sites, and / or selection markers. A "recombinant" vector refers to a vector that includes one or more heterologous nucleotide sequences.

[0061] Inhibitory polynucleotides that inhibit SUV39H1 expression The locus of SUV39H1 is located on the X chromosome (p11.23, positions 48695554-48709016 in the GRCh38.p13 assembly). At the same locus, in a retrograde orientation, is the unannotated gene ENSG00000232828, located at positions 48698963-48737163 (Figure 1). Both genes have annotated promoter regions.

[0062] AF196970.3 (SEQ ID NO: 1) is the predicted RNA sequence expressed from ENSG00000232828 (SEQ ID NO: 5) after transcription and splicing. SEQ ID NO: 1 is a 925 base sequence containing three exons (Figure 2A). AF196970.3 exon 1 (SEQ ID NO: 2) is 125 bases long and has no significant complementarity to the SUV39H1 gene. AF196970.3 exon 2 (SEQ ID NO: 3) is 600 bases long and is retrograde to a substantial portion of exon 3 of the SUV39H1 gene with 100% similarity (Figure 2B). AF196970.3 exon 3 (SEQ ID NO: 4) is 200 bases long and is retrograde to a portion of exon 2 of the SUV39H1 gene (42.5% similarity), as well as to a portion of the adjacent intron (Figure 2C).

[0063] Inhibitory polynucleotides of the present disclosure for use in the cells and methods of the present disclosure include AF196970.3 (SEQ ID NO: 1) or fragments or variants thereof. Such fragments include fragments of any of SEQ ID NOs: 1-4 of about 12-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 250-750, 500-750 or more bases in length. Variants include polynucleotides in which SEQ ID NO:1 or the above-mentioned fragments of SEQ ID NO:1 have been mutated, including by deletion, substitution, modification (including chemical modification), or insertion, or chemically modified polynucleotides. Such variants can have a nucleobase sequence that is at least about 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to any of SEQ ID NOs:1-4 or any fragment thereof, for example, a fragment of SEQ ID NO:1 of the above-mentioned length. Variants include, for example, any of the above-mentioned fragments of SEQ ID NO:1 linked to additional nucleotides at either the 5' or 3' end or both. Variants also include RNAs produced by alternative splicing of ENSG00000232828 (SEQ ID NO:5) or RNAs produced by allelic variants of ENSG00000232828 (SEQ ID NO:5).

[0064] Inhibitory RNA as disclosed herein also includes shRNA as described herein.The principles and design of shRNA are described, for example, in Moore CB, Guthrie EH, Huang MT, Taxman DJ. Short hairpin RNA (shRNA): design, delivery, and assessment of gene knockdown. Methods Mol Biol. 2010;629:141-58, but also see Taxman DJ. siRNA and shRNA design. In: Helliwell TDC, editor. RNA Interference Methods for Plants and Animals. Vol.10. CABI;Oxfordshire, UK:2009.228-253.The mechanism of RNAi is based on the sequence-specific degradation of host mRNA via cytoplasmic delivery of double-stranded RNA (dsRNA) that is identical to the target sequence. shRNAs may be transfected as plasmid vectors encoding shRNAs transcribed by heterologous promoters, but can also be delivered to mammalian cells via infection of cells with virally generated vectors. shRNAs are capable of DNA integration and typically consist of two complementary 19-22 bp RNA sequences linked by a short loop of 4-11 nt similar to the hairpins found in naturally occurring miRNAs. Exemplary loop sequences are also described herein, although any variation thereof based on standard knowledge of the skilled artisan can be used (see also the above references for designing shRNAs). Typically, shRNAs comprise or are encoded by a nucleotide sequence comprising one of the sequences of SEQ ID NOs: 13-17 and 26-30, and SEQ ID NOs: 32-36 and 45-49, respectively.

[0065] Chemical modifications include one or more of a modified backbone linkage, a modified sugar moiety, a modified phosphate moiety, a modified nucleobase, or a chemically conjugated moiety. Such chemical modifications can be present throughout the polynucleotide or in an alternating pattern. Chemical modifications can be present at the 5'-end or 3'-end or both, for example, 5-10 bases at the 5'-end and / or 3'-end include one or more of a modified backbone linkage, a modified sugar moiety, a modified phosphate moiety, a modified nucleobase, or a chemically conjugated moiety.

[0066] Such inhibitory polynucleotides can be delivered directly as heterologous RNA or heterologous polynucleotides, e.g., chemically modified polynucleotides modified to increase their serum half-life and / or affinity. Alternatively, such inhibitory polynucleotides can be ectopically expressed from DNA or vectors expressing such inhibitory polynucleotides. In yet another alternative, endogenous expression of such inhibitory polynucleotides can be upregulated, e.g., by inserting an expression control sequence, e.g., a constitutive, inducible, strong or tissue-specific promoter.

[0067] Activating polynucleotides that increase SUV39H1 expression Activating polynucleotides include agents known in the art for inhibiting expression or increasing degradation of the lncRNA sequence, including RNA interference (RNAi), short hairpin RNA (shRNA), antisense oligonucleotides (ASO), or ribozymes, each of which comprises a segment complementary to the lncRNA sequence. Also contemplated are ZF or ZFN, TALE or TALEN, or CRISPR systems, such as CRISPR-Cas9 or CRISPR-Cas13, each of which comprises a guide RNA or a gene encoding or expressing the lncRNA, each of which comprises a segment complementary to the lncRNA. The complementary region can be at least about 12-20, 12-18, or 15-18 bases in length.

[0068] Typically, after transfection of siRNA or expression of shRNA, RNAi triggers degradation of target RNA molecules via direct complementarity mediated by RNA-induced silencing complexes. An alternative to RNAi for lncRNA degradation is ASO (Figure 2B). ASOs are 15-20 nucleotide single-stranded DNA oligomers that are typically chemically modified to increase knockdown efficacy and reduce in vivo toxicity. In particular, 2'-MOE and LNA gapmer modifications have been shown to increase affinity for target RNA transcripts and confer resistance to nucleases, thereby allowing these modified ASOs to have half-lives of days to weeks in vivo. ASOs hybridize to target RNA transcripts via complementarity and induce RNase H-mediated degradation of the target transcripts.

[0069] CRISPR-Cas13 can also efficiently cleave RNA targets when provided with sgRNAs complementary to the target RNA. Cas13 has been used to knock down lncRNAs in mammalian cells. CRISPRi or zinc finger (ZF) or TALE proteins, in which dCas9, TALE and / or ZF are directly or indirectly linked to repressors and / or inhibitors, can also suppress the expression of RNA. Finally, the expression of lncRNAs can be eliminated by deleting or replacing all or part of the coding gene, for example, through CRISPR-Cas9, ZF nuclease (ZFN) or TALE nuclease (TALEN) gene editing. See Liu and Lim, EMBO Reports (2018) 19:e46955.

[0070] Expression of Inhibitory or Activating Polynucleotides The present disclosure also provides a nucleic acid that encodes or expresses (e.g., is a template for the production of) AF196970.3 (SEQ ID NO:1), or any one of SEQ ID NOs:13-17 and 26-30, or a fragment or variant thereof capable of inhibiting expression of SUV39H1 in a cell. In some embodiments, the nucleic acid is heterologous to the cell and operably linked to an expression control sequence. In other embodiments, the nucleic acid is native to the cell but operably linked to a heterologous expression control sequence. Examples of such nucleic acids include ENSG00000232828 (SEQ ID NO:5) or SEQ ID NO:6, or the cDNA of SEQ ID NOs:32-36 and 45-49, or a fragment or variant thereof. Other examples include nucleic acids encoding any of exons 1-3 (SEQ ID NOs:2-4), or a fragment or variant thereof. Still other examples of such nucleic acids include any of the cDNAs of SEQ ID NOs:6-9, 32-36 and 45-49, or a fragment or variant thereof. Such a nucleic acid can be part of a plasmid or vector or a transposase system.

[0071] In some embodiments, expression of an endogenous nucleic acid encoding or expressing AF196970.3 (SEQ ID NO: 1) or any one of the shRNAs as described herein (SEQ ID NOs: 13-17 or 26-30), or a fragment or variant thereof capable of inhibiting expression of SUV39H1, is upregulated in a cell, e.g., by operably linking the endogenous nucleic acid to a heterologous expression control sequence (e.g., a promoter, enhancer, other regulatory sequence). In some embodiments, expression of an endogenous gene or an allelic variant thereof, e.g., ENSG00000232828 (SEQ ID NO: 5), is upregulated in a cell, e.g., by operably linking the endogenous nucleic acid to a heterologous expression control sequence.

[0072] Means and vectors for expressing polynucleotides, such as RNA, are well known in the art and commercially available.Known vectors include viral vectors and pseudotyped viral vectors, such as retrovirus, lentivirus, adenovirus, adeno-associated (AAV), alphavirus, vaccinia virus, poxvirus, EBV, and herpes simplex virus papillomavirus, foamy virus, or Semliki Forest virus vectors, or transposase systems, such as Sleeping Beauty transposase vector.Non-viral systems for delivery of naked plasmids to cells include lipids, cationic lipid complexes, liposomes, nanoparticles, gold particles, or polymer complexes, polylysine conjugates, synthetic polyamino polymers, and artificial viral envelopes.

[0073] To express small non-coding RNAs, such as shRNAs, polymerase III promoters are commonly used, examples of which include the U6, H1, or 7SK promoters.

[0074] Polymerase II promoters are commonly used to express long RNAs, such as mRNAs or long non-coding RNAs. Examples include CMV, EF-1a, hPGK, and RPBSA. CAG promoters have been used to overexpress lncRNAs. Yin et al., Cell Stem Cell. 2015 May 7;16(5):504-16. Inducible promoters driven by signals from activated T cells include the nuclear factor of activated T cells (NFAT) promoter. Other promoters for T cell expression of RNA include the CIFT chimeric promoter (containing a portion of the cytomegalovirus (CMV) enhancer, the core interferon gamma (IFN-γ) promoter, and the T-lymphotropic virus long terminal repeat (TLTR)), the endogenous TRAC promoter, or the TRBC promoter. Inducible, constitutive, or tissue-specific promoters are contemplated.

[0075] Viral vectors have been used to overexpress lncRNAs, see Yang et al. (2013) Mol Cell 49:1083-1096; Lu et al. (2018) Mol Ther Nucleic Acids 10:387-397. In addition, lncRNAs have been expressed from lentivectors and the SB transposase system. See, e.g., Zhang et al., Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon. J Nanobiotechnol 19, 303 (2021).

[0076] A CRISPR-Cas9-based activation system has also been used to upregulate endogenous expression of lncRNAs. Rankin et al., Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR. J Vis Exp.2019;(145):10.3791 / 59233.

[0077] In some embodiments, dsRNA, for example RNAi, is produced in cells by a vector that comprises an expression control sequence operably linked to a nucleotide sequence that expresses dsRNA (is a template for one or both strands).In further embodiments, a promoter can be adjacent to either end of the template nucleotide sequence, where the promoter drives the expression of each individual DNA strand, thereby producing two complementary (or substantially complementary) RNAs that hybridize to form dsRNA.In other embodiments, dsRNA is produced in cells by a vector that expresses shRNA, and the shRNA is processed to form interfering dsRNA.

[0078] Delivery of nucleic acids or polynucleotides Art-recognized techniques for introducing foreign nucleic acids (e.g., DNA and RNA) into host cells include calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, DNA-loaded liposomes, lipofectamine-DNA complexes, cell sonication, gene bombardment using high-velocity microprojectiles, biolistics, and viral-mediated transfection. The composition containing the nucleic acid may also contain transfection-facilitating agents, including surfactants, quinone analogs, vesicles, such as squalene and squalene, hyaluronic acid, lipids, liposomes, lecithin liposomes, calcium ions, viral proteins, polyanions, polycations, including poly-L-glutamate, or nanoparticles, gold particles, or other known agents. Delivery vehicles include liposomes, lipid-containing complexes, nanoparticles, gold particles, or polymer complexes.

[0079] Lipid materials have been used to create lipid nanoparticles (LNPs) based on ionizable cationic lipids that exhibit a cationic charge at the reduced pH of late endosomes, thereby inducing endosomal escape due to tertiary amines in their structure. These LNPs have been used, for example, to deliver RNA interference (RNAi) components, as well as genetic constructs or CRISPR-Cas systems. See, for example, Wilbie et al., Acc Chem Res; 52(6):1555-1564, 2019. Wang et al., Proc Natl Acad Sci USA.; 113(11):2868-2873, 2016, describe the use of biodegradable cationic LNPs. Chang et al., Acc.Chem.Res., 52, 665-675, 2019, describe the use of ionizable lipids in conjunction with cholesterol, DSPC, and PEGylated lipids to generate LNPs.

[0080] Polymer-based particles can be used for gene construct delivery in a similar manner to lipids. A number of materials have been used for delivery of nucleic acids. For example, cationic polymers such as polyethyleneimine (PEI) can be complexed to nucleic acids and can induce endosomal uptake and release similar to cationic lipids. Poly(amide-amine) (PAMAM) dendrimer structures can also be used for transfection. These particles consist of a core from which polymers branch. They present cationic primary amines on their surface, which can be complexed to nucleic acids. Zinc-based networks that help crosslink imidazoles have been used as a delivery method relying on the low pH of late endosomes, which upon uptake results in cationic charges due to dissociation of the zeolitic imidazole framework (ZIF), followed by release of the component into the cytosol. Colloidal gold nanoparticles have also been used. See Wilbie et al., supra.

[0081] Generation and Chemical Modification of Inhibitory or Activating Polynucleotides In vitro transcribed, chemically synthesized, or partially chemically synthesized RNA can be delivered. For example, RNA can be in vitro transcribed and chemically linked at the 5' and / or 3' ends to chemically synthesized RNA. Direct injection or transfection of in vitro transcribed lncRNA has been performed to demonstrate lncRNA function. Ulitsky et al. (2011) Cell, 147(7):1537-1550.

[0082] Chemical modifications to oligonucleotides (also referred to as polynucleotides) can improve their resistance to degradation, thereby increasing their half-life and / or increasing their affinity to complementary polynucleotides. Modified oligonucleotides (e.g., RNA oligonucleotides) contain chemical modifications including one or more of modified backbone linkages, modified phosphate moieties, modified sugar moieties, modified nucleobases, or chemically conjugated moieties, or any combination thereof. 1, 2, 3, 4, 5, 10, 15, 20 or more of the same type of modification, optionally combined with 1, 2, 3, 4, 5, 10, 15, 20 or more of another type of modification or pattern of modification are contemplated. Patterns include alternating modifications throughout the oligonucleotide, such as 2'-fluoro or 2'-methoxy, or terminal modifications, such as 1, 2, 3, 4, 5 or more of the bases, sugars, or linkages at the 5' and / or 3' ends of the oligonucleotide are modified.

[0083] Examples of modified backbone linkages include phosphorothioate, phosphothioate (PhTx) group or phosphonoacetate, thiophosphonoacetate, methylphosphonate, boranophosphate, or phosphorodithioate.Other internucleotide bridging modified phosphates can be used, such as methylphosphonothioate, phosphoromorpholidate, phosphoropiperazidate, and phosphoramidate.For example, all or every other of the internucleotide bridging phosphate residues can be modified as described.

[0084] Examples of modified sugar moieties include replacement of the 2'OH group with deoxyribose or another group. The majority of sugar analogue modifications are at the 2' position, although other positions, including the 4' position, can be modified. Examples of replacement groups include H, -OR, -R (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), lower alkyl moiety (e.g., C1-C4, straight or branched chain, saturated or unsaturated alkyl, e.g., methyl, ethyl, ethenyl, propyl, 1-propenyl, 2-propenyl, and isopropyl), halo, -F, -Br, -Cl, or -I, -SH, -SR (where R can be, for example, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar), -arabino, F-arabino, amino (amino can be, for example, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). Specific examples include 2'-fluoro sugar, 2'-O-methyl sugar, 2'-O-methoxyethyl sugar, or locked nucleic acid (LNA) nucleotide. Specific examples of modified 2'-sugars include 2'-F or 2'-O-methyl adenosine (A), 2'-F or 2'-O-methyl cytidine (C), 2'-F or 2'-O-methyl uridine (U), 2'-F or 2'-O-methyl thymidine (T), 2'-F or 2'-O-methyl guanosine (G), 2'-O-methoxyethyl-5-methyl uridine (Teo), 2'-O-methoxyethyl adenosine (Aeo), 2'-O-methoxyethyl-5-methyl cytidine (m5Ceo), and any combination thereof. For example, all or every other nucleotide can be modified as described.

[0085] Examples of modified nucleobases include, but are not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methyl Aminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueuosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxocine, pseudouracil, queuosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w, and 2,6-diaminopurine.

[0086] Polynucleotides can also be stabilized by complexing to lipids or liposomes. In some embodiments, the liposome comprises l,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). In certain embodiments, the lipid particle comprises cholesterol, l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), PEG-cDMA or PEG-cDSA, and 1,2-dilinoleyloxy-3-(N,N-dimethyl)aminopropane (DLinDMA).

[0087] Cells with regulated SUV39H1 expression The cells according to the present disclosure show modulation, preferably inhibition, of SUV39H1 expression. The cells are typically mammalian cells or cell lines, such as mouse, rat, pig, non-human primate, or preferably human. Such cells include cells derived from blood, bone marrow, lymph, or lymphoid organs (particularly the thymus), and are preferably cells of the immune system (i.e., immune cells), such as cells of innate or adaptive immunity, such as monocytes, macrophages, dendritic cells, or lymphocytes, typically myeloid or lymphoid cells, including T cells and / or NK cells. The immune cells or precursors thereof also preferably comprise one or more, or two or more, or three or more antigen-specific receptors (CARs and / or TCRs) as described herein, and optionally one or more costimulatory receptors. Among the antigen-specific receptors according to the present disclosure are recombinant modified T cell receptors (TCRs) and components thereof, as well as functional non-TCR antigen-specific receptors, such as chimeric antigen receptors (CARs).

[0088] The cells according to the present disclosure may also be immune cell precursors, such as lymphoid progenitors and more preferably T cell precursors. Examples of T cell precursors include pluripotent stem cells (PSCs), induced pluripotent stem cells (iPSCs), hematopoietic stem cells (HSCs), human embryonic stem cells (ESCs), adipose-derived stem cells (ADSCs), multipotent progenitor cells (MPPs); lymphoid-primed multipotent progenitors (LMPPs); common lymphoid progenitors (CLPs); lymphoid progenitors (LPs); thymic colonized progenitors (TSPs); or early thymic progenitors (ETPs). Hematopoietic stem and progenitor cells can be obtained, for example, from umbilical cord blood or from peripheral blood, and may be, for example, peripheral blood-derived CD34+ cells after mobilization treatment with granulocyte colony-stimulating factor (G-CSF). T cell precursors typically express a set of consensus markers, including CD44, CD117, CD135, and / or Sca-1.

[0089] In some embodiments, the cells include one or more subsets of T cells or other cell types, e.g., the entire T cell population, CD4+ and / or CD8+ T cells, and subpopulations thereof, e.g., those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and / or persistence, antigen specificity, type of antigen-specific receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. In some embodiments, the cells include myeloid derived cells, e.g., dendritic cells, monocytes, or macrophages.

[0090] Among the subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. Specifically contemplated herein are TEFF cells, which have stem / memory properties and higher reconstitution potential due to inhibition of SUV39H1, as well as TN cells, TSCM, TCM, TEM cells and combinations thereof.

[0091] In some embodiments, one or more of the T cell populations are enriched for or depleted of cells that are positive for or express high levels of one or more particular markers, e.g., surface markers, or that are negative for or express relatively low levels of one or more markers. In some cases, such markers are absent or expressed at relatively low levels on certain populations of T cells (e.g., non-memory cells) but present or expressed at relatively high levels on certain other populations of T cells (e.g., memory cells). In one embodiment, cells (e.g., CD8+ cells or T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive for or express high surface levels of CD117, CD135, CD45RO, CCR7, CD28, CD27, CD44, CD127, and / or CD62L, and / or are depleted (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched or depleted for cells that are positive for or express high surface levels of CD122, CD95, CD25, CD27, and / or IL7-Ra (CD127). In some examples, CD8+ T cells are enriched for cells that are positive for CD45RO (or negative for CD45RA) and positive for CD62L. The subset of cells that are CCR7+, CD45RO+, CD27+, CD62L+ cells constitutes the central memory cell subset.

[0092] For example, in accordance with the present disclosure, the cells may comprise a CD4+ T cell population and / or a CD8+ T cell subpopulation, e.g., a subpopulation enriched for central memory (TCM) cells. Alternatively, the cells may be other types of lymphocytes, including natural killer (NK) cells, mucosal-associated invariant T (MAIT) cells, innate lymphoid cells (ILCs), and B cells.

[0093] The cells include primary cells that are directly isolated from a biological sample obtained from a subject and optionally frozen. In some embodiments, the subject is a subject who needs and / or will receive cell therapy (adoptive cell therapy). In relation to a subject to be treated with cell therapy, the cells can be allogeneic and / or autologous. In autologous immune cell therapy, immune cells are collected from a patient, modified as described herein, and returned to the patient. In allogeneic immune cell therapy, immune cells are collected from a healthy donor rather than the patient, modified as described herein, and administered to the patient. Typically, they are HLA-matched to reduce the possibility of rejection by the host. Immune cells can also include modifications to reduce immunogenicity, such as disruption or removal of HLA class I molecules, HLA-A locus, and / or beta-2 microglobulin (B2M).

[0094] Universal "off-the-shelf" immune cells typically contain modifications designed to reduce graft-versus-host disease, such as disruption or deletion of endogenous TCR. The TRAC locus is a common target for removing or disrupting endogenous TCR expression, since a single gene encodes the alpha chain (TRAC) rather than two genes encoding the beta chain (TRBC).

[0095] Samples include tissue samples from tissues or organs, or fluid samples, such as blood, plasma, serum, cerebrospinal fluid, or synovial fluid. Samples can be obtained directly from a subject or can result from one or more processing steps, such as separation, centrifugation, genetic manipulation (e.g., transduction with a viral vector), washing, and / or incubation. Blood or blood-derived samples can be derived from apheresis or leukapheresis products. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, bone marrow-derived cells, and / or cells derived therefrom.

[0096] Methods for generating cells Methods of generating cells of the present disclosure having regulated SUV39H1 expression are provided herein. For cells having inhibited SUV39H1 expression, such methods include introducing into such cells one or more, or two or more, or three or more of the inhibitory polynucleotides disclosed herein. More specifically, such methods include introducing into the cells a polynucleotide comprising the nucleobase sequence of AF196970.3 (SEQ ID NO: 1) or a fragment or variant thereof capable of inhibiting expression of SUV39H1 in the cells, as described herein, in an amount and under conditions effective to increase SUV39H1 expression. Other examples include polynucleotides comprising the nucleobase sequence of any of exons 1-3 (SEQ ID NOs: 2-4), or a fragment or variant thereof capable of inhibiting expression of SUV39H1 in the cells. Such polynucleotides can be chemically modified to reduce degradation and / or increase affinity.

[0097] Such methods also include introducing into the cell a nucleic acid, plasmid, vector or transposase system, preferably operably linked to an expression control sequence, encoding or expressing AF196970.3 (SEQ ID NO: 1) or an shRNA as described herein (including one of the sequences of SEQ ID NOs: 13-17 or 26-30 as described herein) or a fragment or variant thereof capable of inhibiting expression of SUV39H1 in the cell, in an amount and under conditions effective to increase SUV39H1 expression. Examples of such nucleic acids include ENSG00000232828 (SEQ ID NO: 5) or cDNAs of SEQ ID NOs: 6, 32-36 and 45-49 or fragments or variants thereof. Other examples include nucleic acids encoding any of exons 1-3 (SEQ ID NOs: 2-4) or fragments or variants thereof. Yet other examples of such nucleic acids include any of the cDNAs of SEQ ID NOs: 6-9 or fragments or variants thereof.

[0098] Such methods also include upregulating endogenous expression of a nucleic acid encoding or expressing AF196970.3 (SEQ ID NO: 1) or an shRNA as described herein (including any one of SEQ ID NOs: 13-17 and 26-30 as described herein) or a fragment or variant thereof capable of inhibiting expression of SUV39H1 in the cell by inserting a heterologous expression control sequence (e.g., a constitutive, inducible or tissue-specific promoter, enhancer, other regulatory sequence) operably linked to the nucleic acid into the genome of the cell. In some embodiments, expression of an endogenous gene or an allelic variant thereof, such as ENSG00000232828 (SEQ ID NO: 5), is upregulated in the cell.

[0099] Heterologous expression control sequences can be inserted into cells by homologous recombination with a donor template after cleavage using, for example, known nuclease systems, such as ZFNs, TALENs, or CRISPR systems (e.g., CRISPR-Cas9) including guide RNA.

[0100] For cells having increased SUV39H1 expression, such methods include introducing into such cells one or more, or two or more, or three or more of the activating polynucleotides described herein, such as RNAi, shRNA, ASO, ribozyme, or ZFN or TALEN or CRISPR system including guide RNA, in an amount and under conditions effective to increase SUV39H1 expression.

[0101] Antigen-specific receptors The cells of the present disclosure having regulated SUV39H1 expression include immune cells expressing one or more, or two or more, or three or more antigen-specific receptors on their surface, and optionally one or more costimulatory receptors. Antigen-specific receptors include recombinant or modified T cell receptors (TCRs) and components thereof, and / or chimeric antigen receptors (CARs). For example, at least two CARs, at least two TCRs, or at least one CAR with at least one TCR are contemplated. The antigen-specific receptors can bind to the same or different antigens. In some embodiments, the two or more antigen-specific receptors have different signaling domains. In some embodiments, the cells include an antigen-specific receptor that includes an activating signaling domain and an antigen-specific receptor that includes an inhibitory signaling domain. Typically, such antigen-specific receptors are present in a range of about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 It binds to the target antigen with a Kd binding affinity less than or equal to M (a lower number indicates a higher binding affinity).

[0102] Thus, the cell may comprise one or more nucleic acids encoding one or more antigen-specific receptors, optionally operably linked to heterologous regulatory control sequences. Typically, the nucleic acids are heterologous (i.e., not normally found in, for example, the cell being engineered and / or in the organism from which such cells are derived). In some embodiments, the nucleic acids are not naturally occurring, including chimeric combinations of nucleic acids encoding various domains from multiple different cell types. The nucleic acids and their regulatory control sequences are typically heterologous. For example, the nucleic acid encoding the antigen-specific receptor can be heterologous to the immune cell and operably linked to an endogenous promoter of the T cell receptor, such that its expression is under the control of the endogenous promoter. In some embodiments, the nucleic acid encoding the CAR is operably linked to an endogenous TRAC promoter.

[0103] Immune cells, especially when xenogeneic, can be engineered to reduce graft-versus-host disease such that the cells contain an inactivated (e.g., disrupted or deleted) endogenous TCR. The TRAC locus is a typical target for reducing TCR receptor expression because a single gene encodes the alpha chain (TRAC) rather than two genes encoding a beta chain. Thus, a nucleic acid encoding an antigen-specific receptor (e.g., CAR or TCR) can be integrated into the TRAC locus at a position that significantly reduces expression of a functional TCR alpha chain, preferably in the 5' region of the first exon (SEQ ID NO: 3). See, e.g., Jantz et al., WO 2017 / 062451; Sadelain et al., WO 2017 / 180989; Torikai et al., Blood, 119(2):5697-705 (2012); Eyquem et al., Nature. 2 Mar. 2017; 543(7643):113-117. Expression of endogenous TCR alpha can be reduced by at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, and 99%. In such embodiments, expression of a nucleic acid encoding an antigen-specific receptor is optionally under the control of the endogenous TCR alpha promoter.

[0104] Chimeric antigen receptors (CARs) In some embodiments, the engineered antigen-specific receptor comprises a chimeric antigen receptor (CAR), including an activating or stimulatory CAR, a costimulatory CAR (see WO 2014 / 055668), and / or an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)).

[0105] Chimeric antigen receptors (CARs) (also known as chimeric immune receptors, chimeric T cell receptors, artificial T cell receptors) are engineered antigen-specific receptors that transfer any specificity to immune effector cells (T cells). Typically, these receptors are used to transfer the specificity of monoclonal antibodies to T cells, with the transfer of their coding sequences facilitated by retroviral vectors.

[0106] CARs generally comprise an extracellular antigen (or ligand) binding domain linked, in some embodiments, via a linker and / or a transmembrane domain, to one or more intracellular signaling components. Such molecules typically mimic or approximate the signaling through a natural antigen receptor, through such a receptor in combination with a costimulatory receptor, and / or through a costimulatory receptor alone.

[0107] A CAR may comprise (a) an extracellular antigen-binding domain, (b) a transmembrane domain, (c) optionally a costimulatory domain, and (d) an intracellular signaling domain.

[0108] In some embodiments, a CAR is constructed with specificity for a particular antigen (or marker or ligand), e.g., an antigen expressed in a particular cell type of a subject targeted by adoptive cell therapy, e.g., a cancer marker. A CAR typically comprises, in its extracellular portion, one or more antigen-binding molecules, e.g., one or more antigen-binding fragments, domains, or portions of an antibody, typically one or more antibody variable domains. For example, the extracellular antigen-binding domain may comprise a light chain variable domain or fragment thereof and / or a heavy chain variable domain or fragment thereof, typically as an scFv. In some embodiments, a CAR comprises an antibody heavy chain variable domain or fragment thereof that specifically binds to an antigen.

[0109] Moieties used to bind antigens include any of three general categories: single chain antibody fragments (scFv) derived from antibodies, Fabs selected from libraries, or natural ligands that engage their cognate receptors (for first generation CARs). Successful examples in each of these categories are described, inter alia, in Sadelain M, Brentjens R, Riviere I. The basic principles of chimeric antigen receptor (CAR) design. Cancer discovery. 2013;3(4):388-398 (see especially table 1) and are included in this disclosure.

[0110] The antibodies include chimeric, humanized or human antibodies and are further affinity matured and selected as described above. Chimeric or humanized scFvs derived from rodent immunoglobulins (e.g., mouse, rat) are commonly used because they are easily derived from well-characterized monoclonal antibodies. Humanized antibodies contain CDR regions derived from rodent sequences. Typically, rodent CDRs are grafted into a human framework, and some of the human framework residues can be mutated back to the original rodent framework residues to preserve affinity, and / or one or several of the CDR residues can be mutated to increase affinity. Fully human antibodies do not have murine sequences and are typically generated through phage display technology of human antibody libraries, or immunization of transgenic mice whose native immunoglobin loci have been replaced with segments of human immunoglobulin loci. Variants of antibodies can be generated that have one or more amino acid substitutions, insertions, or deletions in the native amino acid sequence, where the antibody retains or substantially retains its specific binding function. Conservative substitutions of amino acids are well known and are described above. Further variants with improved affinity for the antigen can also be generated.

[0111] In some embodiments, the modified TCR or CAR comprises an antibody fragment or antigen-binding fragment (e.g., an scFv, or a variable heavy (VH) or variable light (VL) region or one, two, or three CDRs of such a VH and / or VL) that specifically recognizes an intracellular antigen, e.g., a tumor-associated antigen, presented on the cell surface as an MHC-peptide complex. In general, a CAR containing an antibody or antigen-binding fragment that exhibits TCR-like specificity for a peptide-MHC complex may also be referred to as a TCR-like CAR.

[0112] The transmembrane domain in some embodiments is derived from either natural or synthetic sources. The transmembrane domain can be derived from the same receptor as the intracellular signaling domain or from a different receptor. The transmembrane region can be derived from (i.e., at least includes the transmembrane region of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, ICOS or GITR, or NKG2D, OX40, 2B4, DAP10, DAP12, or CD40. For T cells, CD8, CD28, CD3ε may be preferred. For NK cells, NKG2D, DAP10, DAP12 may be preferred. In some embodiments, the transmembrane domain is derived from CD28, CD8, or CD3ζ. In some cases, the transmembrane domain is selected to minimize interaction with other members of the receptor complex, or is modified by amino acid substitutions to avoid binding of such domains to transmembrane domains of the same or different surface membrane proteins.

[0113] In some embodiments, a short oligo- or polypeptide linker, e.g., a linker between 2-10 amino acids in length, is present and forms the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR.

[0114] CARs generally contain at least one intracellular signaling component(s). First generation CARs typically had an intracellular domain derived from the CD3 zeta chain, which is the main transmitter of signals from endogenous TCR. Second generation CARs typically further contain intracellular signaling domains derived from various costimulatory protein receptors on the cytoplasmic tail of the CAR to provide additional signals to T cells. Costimulatory domains include domains derived from human CD28, 4-1BB (CD137), ICOS, CD27, OX40 (CD134), DAP10, DAP12, 2B4, CD40, FCER1G or GITR (AITR). For T cells, CD28, CD27, 4-1BB (CD137), ICOS may be preferred. For NK cells, DAP10, DAP12, 2B4 may be preferred. Combinations of two costimulatory domains are contemplated, such as CD28 and 4-1BB, or CD28 and OX40. Third generation CARs combine multiple signaling domains, such as CD3ζ-CD28-4-1BB or CD3ζ-CD28-OX40, to enhance efficacy.

[0115] T cell activation, in some embodiments, is described as being mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that function in an antigen-independent manner to provide a secondary or costimulatory signal (secondary cytoplasmic signaling sequences). In some embodiments, a CAR comprises one or both of such signaling components.

[0116] In some aspects, the CAR comprises a primary cytoplasmic signaling sequence that regulates the primary activation of the TCR complex in either a stimulatory or inhibitory manner. A primary cytoplasmic signaling sequence that functions in a stimulatory manner may comprise a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM. Examples of ITAMs that comprise a primary cytoplasmic signaling sequence include those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR comprises a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3zeta. The CAR may also comprise a signaling domain and / or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB (CD137), ICOS, CD27, OX40 (CD134), DAP10, DAP12, 2B4, CD40, FCER1G, or GITR (AITR). In some embodiments, the same CAR contains both an activating component and a costimulatory component; alternatively, the activation domain is provided by one CAR while the costimulatory component is provided by another CAR that recognizes a different antigen.

[0117] The intracellular signaling domain may be derived from an intracellular component of the TCR complex, e.g., a TCR CD3 chain, e.g., CD3ζ chain, which mediates T cell activation and cytotoxicity. Alternative intracellular signaling domains include FcεRIγ. The intracellular signaling domain may comprise a modified CD3ζ polypeptide lacking one or two of its three immunoreceptor tyrosine-based activation motifs (ITAMs), the ITAMs being ITAM1, ITAM2 and ITAM3 (numbered from N-terminus to C-terminus). The intracellular signaling region of CD3ζ is residues 22-164 of SEQ ID NO: 10. ITAM1 is located at about amino acid residues 61-89, ITAM2 is located at about amino acid residues 100-128, and ITAM3 is located at about residues 131-159. Thus, the modified CD3ζ polypeptide can have any one of ITAM1, ITAM2, and ITAM3 inactivated, e.g., disrupted or deleted. Alternatively, the modified CD3ζ polypeptide can have any two ITAMs inactivated, for example, ITAM2 and ITAM3, or ITAM1 and ITAM2. Preferably, ITAM3 is inactivated, for example, deleted. More preferably, ITAM2 and ITAM3 are inactivated, for example, deleted, leaving ITAM1. For example, one modified CD3ζ polypeptide retains only ITAM1, and the remaining CD3ζ domain is deleted (residues 90-164). As another example, ITAM1 is replaced with the amino acid sequence of ITAM3, and the remaining CD3ζ domain is deleted (residues 90-164). See, e.g., Bridgeman et al., Clin. Exp. Immunol. 175(2):258-67 (2014); Zhao et al., J. Immunol. 183(9):5563-74 (2009); Maus et al., WO 2018 / 132506; Sadelain et al., WO 2019 / 133969; Feucht et al., Nat Med. 25(l):82-88 (2019).

[0118] Thus, in some aspects, the antigen-binding molecule is linked to one or more cell signaling modules. In some embodiments, the cell signaling modules include a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. The CAR can also further include one or more additional molecules, such as a portion of Fc receptor g, CD8, CD4, CD25, or CD16.

[0119] In some embodiments, upon ligation of the CAR, the cytoplasmic or intracellular signaling domain of the CAR activates at least one of the normal effector functions or responses of the corresponding non-engineered immune cell (typically a T cell). For example, the CAR can induce a T cell function, such as cytolytic or T helper activity, cytokine secretion, or other elements.

[0120] CARs or other antigen-specific receptors can also be inhibitory CARs (e.g., iCARs) and contain intracellular components that dampen or suppress a response, e.g., an immune response. Examples of such intracellular signaling components are those found on immune checkpoint molecules, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, or EP2 / 4 adenosine receptors, including A2AR. In some aspects, engineered cells contain inhibitory CARs that contain or are derived from the signaling domains of such inhibitory molecules to function to dampen the response of the cell. Such CARs are used, for example, to reduce the possibility of off-target effects when the activating receptor, e.g., the antigen recognized by the CAR, is also expressed or can be expressed on the surface of normal cells.

[0121] TCR In some embodiments, the antigen-specific receptor includes recombinant modified T cell receptors (TCRs) and / or TCRs cloned from naturally occurring T cells. Nucleic acids encoding TCRs can be obtained from a variety of sources, for example, by polymerase chain reaction (PCR) amplification of naturally occurring TCR DNA sequences, followed by expression of antibody variable regions, followed by selection for specific binding to antigen. In some embodiments, the TCRs are obtained from T cells isolated from a patient or from cultured T cell hybridomas. In some embodiments, TCR clones against target antigens have been generated in transgenic mice engineered with human immune system genes (e.g., human leukocyte antigen system, or HLA). See, e.g., tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate TCRs against a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354).

[0122] "T cell receptor" or "TCR" refers to a molecule that comprises variable alpha and beta chains (also known as TCRalpha and TCRbeta, respectively) or variable gamma and delta chains (also known as TCRgamma and TCRdelta, respectively) and is capable of specifically binding to an antigenic peptide bound to an MHC receptor. In some embodiments, the antigen-binding domain of a TCR comprises approximately 1×10 -7 Below, about 5×10 -8 Below, approximately 1×10 -8 Below, about 5×10 -9 Below, approximately 1×10 -9 Below, about 5×10 -10 Below, approximately 1×10 -10 Below, about 5×10 -11 Below, approximately 1×10 -11 Below, about 5×10-12 or less, or about 1 x 10 -12 It binds to its target antigen with the following KD affinity (lower numbers indicate higher affinity). In some embodiments, the TCR is in αβ form. Typically, TCRs present in αβ and γδ forms are generally structurally similar, but the T cells expressing them may have different anatomical localizations or functions. TCRs can be found on the surface of cells or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes), where they are broadly responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR can also include a constant domain, a transmembrane domain, and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Edition, Current Biology Publications, Inc., 4:33, 1997). For example, in some aspects, each chain of the TCR may possess one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminus. In some embodiments, the TCR associates with the invariant protein of the CD3 complex, which is involved in mediating signal transduction. Unless otherwise specified, the term "TCR" should be understood to encompass functional TCR fragments thereof. The term also encompasses intact or full-length modified TCRs, including TCRs in αβ or γδ forms. The term "TCR" also encompasses TCRs that have been modified to include the VH and / or VL of an antibody.

[0123] Thus, for purposes herein, reference to a TCR includes any modified TCR or functional fragment thereof, e.g., an antigen-binding portion of a TCR that binds to an MHC molecule, i.e., a specific antigenic peptide bound in an MHC-peptide complex. An "antigen-binding portion" or "antigen-binding fragment" of a TCR, which may be used interchangeably, refers to a molecule that includes a portion of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) to which the full-length TCR binds. In some cases, the antigen-binding portion includes the variable domain of the TCR, e.g., the variable α and β chains of the TCR, sufficient to form a binding site for binding to a specific MHC-peptide complex, e.g., typically where each chain includes three complementarity determining regions.

[0124] In some embodiments, the variable domains of the TCR chains associate to form loops, or complementarity determining regions (CDRs) analogous to immunoglobulins, that confer antigen recognition and determine peptide specificity by forming the binding site of the TCR molecule. Typically, as in immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l. Acad. Sci. USA 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the main CDR responsible for recognition of processed antigens, although CDR1 of the α chain has also been shown to interact with the N-terminal portion of antigenic peptides, while CDR1 of the β chain interacts with the C-terminal portion of peptides. CDR2 is believed to recognize an MHC molecule. In some embodiments, the variable region of the β chain may include an additional hypervariable (HV4) region.

[0125] In some embodiments, the TCR chain comprises a constant domain. For example, similar to an immunoglobulin, the extracellular portion of the TCR chain (e.g., α chain, β chain) may comprise two immunoglobulin domains: a variable domain at the N-terminus (e.g., Vα or Vβ; typically amino acids 1-116 according to Kabat numbering Kabat et al., Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and one constant domain adjacent to the cell membrane (e.g., α chain constant domain or Cα or TRAC, typically amino acids 117-259 according to Kabat, β chain constant domain or Cβ or TRBC, typically amino acids 117-295 according to Kabat). For example, in some cases, the extracellular portion of the TCR formed by the two chains comprises two membrane-proximal constant domains and two membrane-distal variable domains that include the CDRs. The constant domain of the TCR domain contains a short linking sequence in which cysteine ​​residues form disulfide bonds, thereby creating a link between the two chains. In some embodiments, the TCR can have an additional cysteine ​​residue in each of the α and β chains, such that the TCR contains two disulfide bonds in the constant domain.

[0126] In some embodiments, the TCR chain may include a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain includes a cytoplasmic tail. In some cases, the structure allows the TCR to associate with other molecules, such as CD3. For example, a TCR that includes a constant domain along with a transmembrane region can anchor the protein in the cell membrane and associate with the invariant subunit of the CD3 signaling apparatus or complex.

[0127] Generally, CD3 is a multiprotein complex that may possess three different chains (gamma (γ), delta (δ), and epsilon (ε)) and a zeta chain. For example, in mammals, the complex may comprise a homodimer of CD3γ, CD3δ, two CD3ε, and CD3ζ chains. The CD3γ chains are highly related cell surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, a feature that allows these chains to associate with the positively charged T cell receptor chains and play a role in the propagation of signals from the TCR to the cell. The intracellular tails of the CD3γ, CD3δ, and CD3ε chains each contain a single conserved motif known as an immunoreceptor tyrosine-based activation motif or ITAM, while each CD3ζ chain has three ITAMs. Generally, ITAMs are involved in the signaling capacity of the TCR complex. The CD3 gamma, delta, epsilon and zeta chains together form what is known as the T cell receptor complex.

[0128] The modified TCR of the present disclosure may comprise a heterologous antigen-binding domain and a native TCR constant domain (α or β) or a fragment thereof, where the modified TCR is capable of activating a CD3ζ polypeptide. An exemplary modified TCR, described herein as HI-TCR or HIT-CAR, comprises (a) a first antigen-binding chain comprising an antigen-binding fragment of an antibody heavy chain variable region (VH); and (b) a second antigen-binding chain comprising an antigen-binding fragment of an antibody light chain variable region (VL); where the first and second antigen-binding chains each comprise a native or variant TRAC (constant region) or a fragment thereof, or a native or variant TRBC (constant region) or a fragment thereof. In some embodiments, at least one of the TRAC and TRBC polypeptides, typically the TRAC polypeptide, is endogenous, and optionally one or both of the endogenous TRAC and TRBC polypeptides are inactivated.

[0129] In some designs, the HI-TCR comprises (a) a chimeric TCR α chain comprising a VH or a fragment thereof fused to a native or variant TRAC or a fragment thereof, optionally with amino acids of the VH (or TRAC) removed, and (b) a chimeric TCR β chain comprising a VL or a fragment thereof fused to a native or variant TRBC or a fragment thereof, optionally with amino acids of the VL (or TRBC) removed. In other designs, the HI-TCR comprises (a) a chimeric TCR α chain comprising a VL or a fragment thereof fused to a native or variant TRAC or a fragment thereof, optionally with amino acids of the VL (or TRAC) removed, and (b) a chimeric TCR β chain comprising a VH or a fragment thereof fused to a native or variant TRBC or a fragment thereof, optionally with amino acids of the VH (or TRBC) removed. In yet another design, the HI-TCR comprises only a VH or a fragment thereof, optionally fused to a native or variant TRAC or a fragment thereof, or fused to a native or variant TRBC or a fragment thereof, with amino acids of the VH (or TRAC or TRBC) removed. HI-TCRs (HIT-CARs) are described in WO 2019 / 157454, which is incorporated herein by reference in its entirety. Yet other modified TCRs are disclosed in WO 2018 / 067993, which is incorporated herein by reference in its entirety, and in Baeuerle et al., Synthetic TRuC receptors engaging the complete T cell receptor for potent anti-tumor or response. Nat Commun 10, 2087 (2019), which are incorporated herein by reference in their entirety. For example, any one or more of the α, β, γ or ε chains, or two or more, can be fused to an antibody variable region, e.g., VH and / or VL, e.g., an scFv.

[0130] In some embodiments, a nucleic acid encoding a heterologous antigen-binding domain (e.g., VH or a variant or fragment thereof, or VL or a variant or fragment thereof) is inserted into the endogenous TRAC locus and / or TRBC locus of an immune cell. Optionally, a nucleic acid encoding a chimeric TCR α (or β) chain is operably linked to an endogenous promoter of the T cell receptor such that its expression is under the control of the endogenous promoter. Insertion of a nucleic acid sequence can also inactivate or disrupt endogenous expression of a TCR, including a native TCR α chain and / or a native TCR β chain. Insertion of a nucleic acid sequence may reduce endogenous TCR expression by at least about 75%, 80%, 85%, 90% or 95%.

[0131] Immune cells comprising recombinant TCRs typically provide superior activity when the antigen has a low density on the cell surface, such as less than about 10,000 molecules per cell, e.g., less than about 5,000, 4,000, 3,000, 2,000, 1,000, 500, 250, or 100 molecules per cell. In some embodiments, the antigen is expressed by the target cell at a low density, e.g., less than about 6,000 molecules of target antigen per cell. In some embodiments, the antigen is expressed at a density of less than about 5,000 molecules, less than about 4,000 molecules, less than about 3,000 molecules, less than about 2,000 molecules, less than about 1,000 molecules, or less than about 500 molecules of target antigen per cell. In some embodiments, the antigen is expressed at a density of less than about 2,000 molecules of target antigen per cell, e.g., less than about 1,800, less than about 1,600, less than about 1,400, less than about 1,200, less than about 1,000, less than about 800, less than about 600, less than about 400, less than about 200, or less than about 100, etc. In some embodiments, the antigen is expressed at a density of less than about 1,000 molecules of target antigen per cell, e.g., less than about 900, less than about 800, less than about 700, less than about 600, less than about 500, less than about 400, less than about 300, less than about 200, or less than about 100, etc. In some embodiments, the antigen is expressed at a density ranging from about 5,000 to about 100 molecules of target antigen per cell, e.g., about 5,000 to about 1,000 molecules, about 4,000 to about 2,000 molecules, about 3,000 to about 2,000 molecules, about 4,000 to about 3,000 molecules, about 3,000 to about 1,000 molecules, about 2,000 to about 1,000 molecules, about 1,000 to about 500 molecules, about 500 to about 100 molecules of target antigen per cell, etc. In some embodiments, recombinant TCR T cell therapy targets antigens that are expressed at a lower density compared to the density in wild type cells.

[0132] Other examples of antigen-specific receptors, including CARs and recombinant modified TCRs, and methods for engineering and introducing receptors into cells are described in, for example, WO 2000 / 014257, WO 2013 / 126726, WO 2012 / 129514, WO 2014 / 031687, WO 2013 / 166321, WO 2013 / 071154, WO 2013 / 123061, U.S. Patent Application Publication Nos. 2002131960, 2013287748, and 20130149337. Nos. 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, and European Patent Application Publication No. 2 537 416, and / or those described in Sadelain et al., Cancer Discov. April 2013;3(4):388-398; Davila et al., (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., October 2012;24(5):633-39; Wu et al., Cancer, March 2012,18(2):160-75. In some embodiments, antigen-specific receptors include CARs as described in U.S. Pat. No. 7,446,190 and those described in WO 2014 / 055668 A1.

[0133] Costimulatory Receptors The cells of the present disclosure comprising altered SUV39H1 expression may further comprise at least one or at least two exogenous costimulatory ligands, including CD80, CD86, 4-1BBL, CD275, CD40L, OX40L, or any combination thereof. In some embodiments, the costimulatory ligand is CD80 or 4-1BBL.

[0134] In some embodiments, the cells comprise at least one or at least two costimulatory receptors. Such costimulatory receptors include chimeric receptors comprising a costimulatory ligand fused to at least one or at least two costimulatory molecules. Costimulatory ligands include CD80, CD86, 4-1BBL, CD275, CD40L, OX40L, or any combination thereof. In some embodiments, the costimulatory ligand is CD80 or 4-1BBL. Exemplary costimulatory molecules are CD28, 4-1BB, OX40, ICOS, DAP-10, CD27, CD40, NKG2D, CD2, or any combination thereof. In some embodiments, the chimeric receptor comprises a first costimulatory molecule that is 4-1BB and a second costimulatory molecule that is CD28.

[0135] In some embodiments, the cells comprise a costimulatory receptor comprising the extracellular domain of CD80, the transmembrane domain of CD80, and the intracellular 4-1BB domain. Exemplary costimulatory ligands, molecules and receptors (or fusion polypeptides) are described in WO2021 / 016174, which is incorporated by reference in its entirety.

[0136] The cells of the present disclosure comprising regulated SUV39H1 expression may also comprise a T cell specific interferon, e.g., a BiTE, or a bispecific antibody that binds not only a desired antigen but also an activating T cell antigen, e.g., CD3ε. In some embodiments, the BiTE comprises an antigen binding domain, e.g., an scFv, linked to a T cell recognition domain, e.g., a heavy chain variable domain and / or a light chain variable domain of an anti-CD3 antibody.

[0137] antigen Antigens include those associated with proliferative, neoplastic, and malignant diseases and disorders, particularly cancer. Infectious diseases and autoimmune, inflammatory, or allergic diseases are also contemplated.

[0138] The cancer may be a solid cancer or a "liquid tumor", such as a cancer affecting the blood, bone marrow and lymphatic system, also known as a tumor of the hematopoietic and lymphatic tissues, including, among others, leukemia and lymphoma, such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia (CLL), including various lymphomas, such as mantle cell lymphoma, non-Hodgkin's lymphoma (NHL), adenoma, squamous cell carcinoma, laryngeal carcinoma, gallbladder cholangiocarcinoma, and cancer of the retina, such as retinoblastoma.

[0139] Solid cancers include, inter alia, cancers affecting one of the organs selected from the group consisting of colon, rectum, skin, endometrium, lung (including non-small cell lung cancer), uterus, bone (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma), liver, kidney, esophagus, stomach, bladder, pancreas, cervix, brain (e.g., meningioma, glioblastoma, low-grade astrocytoma, oligodendroglioma, pituitary tumor, schwannoma, and metastatic brain cancer), ovary, breast, head and neck region, testis, prostate, and thyroid.

[0140] The cancer includes cancers affecting the blood, bone marrow and lymphatic system as described above. In some embodiments, the cancer is or is associated with multiple myeloma. Antigens associated with multiple myeloma include CD38, CD138, and / or CS-1. Other exemplary multiple myeloma antigens include CD56, TIM-3, CD33, CD123, and / or CD44.

[0141] Diseases also include infectious diseases or conditions, including, but not limited to, viral, retroviral, bacterial, protozoan or parasitic infections, HIV immunodeficiency, cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus, BK polyomavirus, etc. Viral antigens include antigens of HIV, HCV, HBV.

[0142] In some embodiments, the extracellular antigen binding domain binds to any of the tumor neoantigenic peptides disclosed in WO2021 / 043804, which is incorporated by reference in its entirety. For example, the antigen binding domain binds to a neoantigenic peptide that includes at least 8, 9, 10, 11, or 12 amino acids encoded by a portion of the open reading frame (ORF) of any of the peptides of SEQ ID NOs: 1-117 or any of the fusion transcript sequences of any one of SEQ ID NOs: 118-17492 of WO2021 / 043804.

[0143] Diseases also include autoimmune or inflammatory diseases or conditions, such as arthritis, e.g., rheumatoid arthritis (RA), type I diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Graves' disease, Crohn's disease, multiple sclerosis, asthma, and / or transplant-associated diseases or conditions. In such situations, the T regulatory cell can be a cell in which SUV39H1 is inhibited.

[0144] In some embodiments, the antigen is a polypeptide. In some embodiments, the antigen is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on disease or condition cells, e.g., tumor or pathogenic cells, when compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or expressed on engineered cells. In some such embodiments, multiple targeting and / or gene disruption approaches as provided herein are used to improve specificity and / or efficacy.

[0145] In some embodiments, the antigen is a universal tumor antigen. The term "universal tumor antigen" refers to an immunogenic molecule, e.g., a protein, that is generally expressed at a higher level in tumor cells than in non-tumor cells and is also expressed in tumors of different origins. In some embodiments, the universal tumor antigen is expressed in more than 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90% or more of human cancers. In some embodiments, the universal tumor antigen is expressed in at least three, at least four, at least five, at least six, at least seven, at least eight or more different types of tumors. In some cases, the universal tumor antigen may be expressed in non-tumor cells, e.g., normal cells, but at a lower level than expressed in tumor cells. In some cases, the universal tumor antigen is not expressed at all in non-tumor cells, e.g., not expressed in normal cells. Exemplary universal tumor antigens include, for example, human telomerase reverse transcriptase (hTERT), survivin, mouse double minute 2 homolog (MDM2), cytochrome P450 1 B1 (CYP1 B), HER2 / neu, p95HER2, Wilms tumor gene 1 (WT1), livin, alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), mucin 16 (MUC16), MUC1, prostate specific membrane antigen (PSMA), p53, or cyclin (D1). Peptide epitopes of tumor antigens, including universal tumor antigens, are known in the art, and in some aspects can be used to generate MHC-restricted antigen-specific receptors, such as TCRs or TCR-like CARs (see, for example, WO 2011 / 009173 or WO 2012 / 135854 and U.S. Patent Application Publication No. 20140065708).

[0146] In some embodiments, the cancer is accompanied by or associated with overexpression of HER2 or p95HER2. p95HER2 is a constitutively active C-terminal fragment of HER2 generated by alternative initiation of translation at methionine 611 of the transcript encoding the full-length HER2 receptor. The amino acid sequence of p95HER2 is shown in SEQ ID NO:11, and the amino acid sequence of the extracellular domain is SEQ ID NO:12.

[0147] HER2 or p95HER2 has been reported to be overexpressed in breast cancer, as well as gastric, gastroesophageal, esophageal, ovarian, endometrial, cervical, colon, bladder, lung, and head and neck cancers. Patients with cancers that express the p95HER2 fragment have a higher likelihood of developing metastases and a poorer prognosis than those who predominantly express the complete form of HER2. Saez et al., Clinical Cancer Research, 12:424-431 (2006).

[0148] Other antigens include orphan tyrosine kinase receptor ROR1, tEGFR, Her2, p95HER2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, claudin 18.2, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, CD70, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, FcRH5 fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-α, IL-13R-α2, kdr, kappa light chain, Lewis Y, L1-cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp1OO, carcinoembryonic antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, p95HER2, estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, and MAGE A3, CE7, Wilms' tumor 1 (WT-1), cyclins, e.g., cyclin A1 (CCNA1), and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[0149] In some embodiments, recombinant nucleic acids encoding antigen receptors as previously described are transferred into T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, recombinant nucleic acids are transferred into T cells via transposition (see, e.g., Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2, e74; and Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection; tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0150] Particularly useful vectors for generating targeting constructs that provide transgene vectorization for homologous recombination mediated targeting include, but are not limited to, recombinant adeno-associated virus (rAAV), recombinant non-integrating lentivirus (rNILV), recombinant non-integrating gamma-retrovirus (rNIgRV), single-stranded DNA (linear or circular), and the like.Such vectors can be used to introduce transgenes into the immune cells of the invention by creating targeting constructs (see, e.g., Miller, Hum. Gene Ther. 1 (1):5-14 (1990); Friedman, Science 244:1275-1281 (1989); Eglitis et al., BioTechniques 6:608-614 (1988); Tolstoshev et al., Current Opin. Biotechnol. 1:55-61 (1990); Sharp, Lancet 337:1277-1278 (1991); Cornetta et al., Prog. Nucleic Acid Res. Mol. Biol. 36:311-322 (1989); Anderson, Science 226:401-409 (1984); Moen, Blood Cells 17:407-416 (1991); Miller et al., Biotechnology 7:980-990 (1989); Le Gal La Salle et al., Science 259:988-990 (1993); and Johnson, Chest 107:77S-83S (1995); Rosenberg et al., N. Engl. J. Med. 323:370 (1990); Anderson et al., U.S. Patent No. 5,399,346; Scholler et al., Sci. 117(1): pp. 72-82 (2011); Reviere et al., Proc. Natl. Acad. Sci. USA 92:6733-6737 (1995); Wang et al., Gene Therapy 15:1454-1459 (2008).

[0151] In some embodiments, the exogenous nucleic acid or targeting construct comprises a 5' homology arm and a 3' homology arm to facilitate recombination of the nucleic acid sequence into the cell genome at the nuclease cleavage site.

[0152] In some embodiments, exogenous nucleic acid can be introduced into cells using a single-stranded DNA template. The single-stranded DNA may comprise the exogenous nucleic acid, and in a preferred embodiment, may comprise 5' and 3' homology arms to facilitate insertion of the nucleic acid sequence into the nuclease cleavage site by homologous recombination. The single-stranded DNA may further comprise a 5' AAV inverted terminal repeat (ITR) sequence 5' upstream of the 5' homology arm and a 3' AAV ITR sequence 3' downstream of the 3' homology arm. In other specific embodiments, the targeting construct comprises, in order from 5' to 3': a first viral sequence, a left homology arm, a nucleic acid sequence encoding elements creating a polycistronic expression cassette (e.g., various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, pestivirus IRES, aphthovirus IRES, picomavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and a cleavable linker (e.g., a 2A peptide, e.g., P2A, T2A, E2A, and F2A peptide), preferably a cleavable linker), a transgene, a polyadenylation sequence, a right homology arm, and a second viral sequence. In a preferred embodiment, the targeting construct comprises, in order from 5' to 3': a first viral sequence, a left homology arm, a nucleic acid sequence encoding a self-cleaving linker (e.g., porcine teschovirus 2A), a nucleic acid sequence encoding a CAR or modified TCR (e.g., Hi-CTR), a polyadenylation sequence, a right homology arm, and a second viral sequence. Another suitable targeting construct may comprise sequences from an integration-deficient lentivirus (see, e.g., Wanisch et al., Mol. Ther. 17(8):1316-1332 (2009)).

[0153] In some embodiments, the viral nucleic acid sequence comprises an integration-defective lentivirus sequence. It is understood that any suitable targeting construct compatible with the homologous recombination system utilized can be used. The AAV nucleic acid sequence that functions as part of the targeting construct can be packaged into some natural or recombinant AAV capsids or particles. In certain embodiments, the AAV particle is AAV6. In certain embodiments, the AAV2-based targeting construct is delivered to the target cell using an AAV6 viral particle. In certain embodiments, the AAV sequence is AAV2, AAV5 or AAV6 sequence.

[0154] In some embodiments, the gene encoding the exogenous nucleic acid sequence of the present invention can be introduced into cells by transfection using linearized DNA template. In some cases, the plasmid DNA encoding the exogenous nucleic acid sequence can contain nuclease cleavage sites (e.g., class II, II, V or VI Cas nuclease) on both sides of the left homology arm, thereby allowing the circular plasmid DNA to be linearized and the precise in-frame integration of the exogenous DNA without the backbone vector sequence (see, for example, Hisano Y, Sakuma T, Nakade S, et al., Precise in-frame integration of exogenous DNA mediated by CRISPR / Cas9 system in zebrafish. Sci Rep. 2015;5:8841).

[0155] In some embodiments, the vector incorporates an endogenous promoter, e.g., a TCR promoter. Such vectors could provide expression in a manner similar to that provided by an endogenous promoter, e.g., a TCR promoter. Such vectors could be useful, for example, when the site of integration does not provide efficient expression of the transgene, or when disruption of the endogenous gene controlled by the endogenous promoter would be detrimental to the T cell or result in a reduction in its effectiveness in T cell therapy. In preferred embodiments, such vectors could be useful, for example, when the site of integration does not provide efficient expression of the nucleic acid sequence encoding the CAR or modified TCR. The promoter could be an inducible promoter or a constitutive promoter. Expression of the nucleic acid sequence under the control of the endogenous or vector-associated promoter occurs under conditions suitable for the cell to express the nucleic acid, such as, for example, under growth conditions, or in the presence of an inducer with an inducible promoter. Such conditions are well understood by those skilled in the art.

[0156] The targeting construct can optionally be designed to include elements that create a polycistronic expression cassette immediately upstream of the nucleic acid sequence encoding the transgene, including but not limited to various viral and non-viral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, picomavirus IRES, poliovirus IRES, and encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). In a preferred embodiment, the targeting construct can optionally be designed to include a cleavable linkage (e.g., P2A, T2A, etc.) sequence immediately upstream of the nucleic acid sequence encoding the therapeutic protein (e.g., an engineered antigen receptor). P2A and T2A are self-cleaving peptide sequences that can be used for bicistronic or multicistronic expression of protein sequences (see Szymczak et al., Expert Opin. Biol. Therapy 5(5):627-638 (2005)).

[0157] Well-suited AAV constructs for HIT expression in immunoresponsive cells according to the present application are described, for example, in Mansilla-Soto, J., Eyquem, J., Haubner, S. et al., HLA-independent T cell receptors for targeting tumors with low antigen density. Nat Med 28, 345-352 (2022) and have been used in the results included herein, particularly for in vivo experiments.

[0158] Exemplary well-suited constructs typically include a TRBC or TRAC sequence (which may be a native or modified TRBC or TRAC sequence, including a murine sequence as described herein), a cleavable linker sequence (as defined above, but e.g., a 2A sequence), a TRAC or TRBC sequence (which may be a native or modified TRBC or TRAC sequence, including a murine sequence as described herein). The TRBC and / or TRAC sequence is typically fused (preferably 5') to a sequence encoding an antibody fragment (e.g., VH, VH, scFv, single domain antibody, VHH, etc.) as described above. In some embodiments, a booster (co-stimulatory ligand) sequence is included in the construct, such that in a preferred embodiment, the construct further includes a cleavable linker sequence (e.g., a 2A sequence) and a booster (co-stimulatory ligand) sequence. Typically, the TRAC or TRBC sequence at the 3' end of the construct is fused to a cleavable linker, which is also fused to a booster (costimulatory ligand and / or costimulatory receptor CCR) sequence (see Figure 11, and Figures 29-30). The booster (costimulatory ligand and / or costimulatory receptor CCR) sequence can be any one as described herein, and in particular can be a CD80 sequence or a CD80_4-1BB sequence as described herein (see, for example, SEQ ID NOs: 32-33 and 52-53).

[0159] If desired, the targeting construct can be designed to optionally include a reporter, e.g., a reporter protein that provides identification of transduced cells. Exemplary reporter proteins include, but are not limited to, fluorescent proteins, e.g., mCherry, green fluorescent protein (GFP), blue fluorescent proteins, e.g., EBFP, EBFP2, Azurite, and mKalamal, cyan fluorescent proteins, e.g., ECFP, Cerulean, and CyPet, and yellow fluorescent proteins, e.g., YFP, Citrine, Venus, and YPet. Typically, the targeting construct includes a polyadenylation (poly A) sequence 3' of the transgene. In a preferred embodiment, the targeting construct includes a polyadenylation (poly A) sequence 3' of the nucleic acid sequence encoding the CAR and / or modified TCR (e.g., Hi)-TCR).

[0160] therapeutic use The cells, modified oligonucleotides, nucleic acids, or vectors of the present disclosure can be used in adoptive cell therapy, particularly adoptive T cell therapy or adoptive NK cell therapy. In some embodiments, the use is in the treatment of cancer in a subject in need thereof, but also includes the treatment of infectious diseases and autoimmune, inflammatory, or allergic diseases. In some embodiments, the subject has cancer or is at risk of having cancer. The modified oligonucleotides, nucleic acids, or vectors of the present disclosure are optionally in a delivery vehicle or composition as disclosed herein, including liposomes, lipid-containing complexes, nanoparticles, gold particles, or polymer complexes. Such compositions may further include stabilizing or transfection-facilitating agents, such as surfactants, quinone analogs, vesicles, such as squalene and squalene, hyaluronic acid, lipids, liposomes, lecithin liposomes, calcium ions, viral proteins, polyanions, polycations, including poly-L-glutamate, or nanoparticles, gold particles, or other known agents.

[0161] In such methods, a subject is administered one or more cells, modified oligonucleotides, nucleic acids, or vectors described herein to a subject in need thereof in an amount effective to treat the disease or disorder. For example, cells expressing one or more antigen-specific receptors are administered in a dose effective to treat the disease or disorder associated with the antigen. Treatment of any of the diseases listed above under the "Antigen" section is contemplated.

[0162] In some embodiments, immune cells expressing an antigen receptor (e.g., Hi T cell antigen receptor) as described herein can be used to treat patients with a median of less than about 6,000 molecules of target antigen per cell. In some embodiments, the antigen is expressed at a density (typically a median) of less than about 5,000, less than about 4,000, less than about 3,000, less than about 2,000, less than about 1,000, or less than about 500 molecules of target antigen per cell. In some embodiments, the antigen is expressed at a density (typically a median) of less than about 2,000 molecules of target antigen per cell, e.g., less than about 1,800, less than about 1,600, less than about 1,400, less than about 1,200, less than about 1,000, less than about 800, less than about 600, less than about 400, less than about 200, or less than about 100 molecules of target antigen. In some embodiments, the antigen is expressed at a density of less than about 1,000 molecules of target antigen per cell, e.g., less than about 900 molecules, less than about 800 molecules, less than about 700 molecules, less than about 600 molecules, less than about 500 molecules, less than about 400 molecules, less than about 300 molecules, less than about 200 molecules, or less than about 100 molecules of target antigen, etc. In some embodiments, the antigen is expressed at a density of about 5,000 to about 100 molecules of target antigen per cell, e.g., about 5,000 to about 1,000 molecules, about 4,000 to about 2,000 molecules, about 3,000 to about 2,000 molecules, about 4,000 to about 3,000 molecules, about 3,000 to about 1,000 molecules, about 2,000 to about 1,000 molecules, about 1,000 to about 500 molecules, about 500 to about 100 molecules of target antigen, etc. Quantification of target antigen density per cell can be achieved as described in Jasper, GA, Arun, I., Venzon, D., Kreitman, RJ, Wayne, AS, Yuan, CM, Marti, GE, & Stetler-Stevenson, M. (2011). Variables affecting the quantitation of CD22 in neoplastic B cells. Cytometry. Part B, Clinical cytometry, 80(2), 83-90.

[0163] The cells can be administered at a particular dose. For example, immune cells (e.g., T cells or NK cells) in which SUV39H1 is inhibited can be administered at a dose of about 10 8 Less than 5 x 10 cells 7 Less than 10 cells 7 Less than 5 x 10 cells 6 Less than 10 cells 6 Less than 5 x 10 cells 5 Less than or about 10 cells 5 A dose of less than 10 cells can be administered to an adult. A dose for a pediatric patient can be about 100 times lower. In an alternative embodiment, any of the immune cells (e.g., T cells) described herein can be administered at a dose of less than about 10 5 ~about 10 9 cells, or about 10 5 ~about 10 8 cells, or about 10 5 ~about 10 7 cells, or about 10 6 ~about 10 8 A range of doses of cells can be administered to a patient.

[0164] The subject (i.e., patient) is a mammal, typically a primate, e.g., a human. In some embodiments, the primate is a monkey or ape. The subject can be male or female and of any suitable age, including infants, juveniles, adolescents, adults, and geriatric subjects. In some embodiments, the subject is a non-primate mammal, e.g., a rodent. In some examples, the patient or subject is a validated animal model for disease, adoptive cell therapy, and / or to assess toxicity outcomes, e.g., cytokine release syndrome (CRS). In some embodiments, the subject has cancer, is at risk of having cancer, or is in remission from cancer.

[0165] In some embodiments, the cells or compositions are administered to a subject, e.g., a subject having or at risk for cancer or any one of the diseases as mentioned above. In some aspects, the methods thus treat the disease or condition, e.g., ameliorate one or more symptoms thereof, e.g., for cancer, by reducing tumor burden in a cancer that expresses an antigen recognized by the engineered cells.

[0166] Methods for administration of cells for adoptive cell therapy are known and can be used in connection with the provided methods and compositions. For example, methods of adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003 / 0170238 to Gruenberg et al.; U.S. Patent No. 4,690,915 to Rosenberg et al.; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85. See, for example, Themeli et al. (2013) Nat Biotechnol. 31 (10):928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1):84-9; Davila et al. (2013) PLoS ONE 8(4):e61338.

[0167] Administration of at least one cell according to the present disclosure to a subject in need thereof can be combined with one or more additional therapeutic agents, or in conjunction with another therapeutic intervention, either simultaneously or sequentially in any order. In some contexts, the cells are co-administered with another therapy in sufficient temporal proximity such that the cell population enhances the effect of the one or more additional therapeutic agents, or vice versa. In some embodiments, the cell population is administered prior to the one or more additional therapeutic agents. In some embodiments, the cell population is administered after the one or more additional therapeutic agents.

[0168] With respect to cancer treatment, combination cancer therapies include, but are not limited to, cancer chemotherapeutic agents, cytotoxic agents, hormones, anti-angiogens, radiolabeled compounds, immunotherapy, surgery, cryotherapy, and / or radiation therapy.

[0169] Conventional cancer chemotherapeutic agents include alkylating agents, antimetabolites, anthracyclines, topoisomerase inhibitors, microtubule inhibitors, and B-raf enzyme inhibitors.

[0170] Alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, ifosfamide, melphalan, and chlorambucil), ethyleneamine and methyleneamine derivatives (e.g., altretamine, thiotepa), alkylsulfonates (e.g., busulfan), nitrosoureas (e.g., carmustine, lomustine, estramustine), triazenes (e.g., dacarbazine, procarbazine, temozolomide), and platinum-containing antineoplastic agents (e.g., cisplatin, carboplatin, oxaliplatin).

[0171] Antimetabolites include 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda®), cytarabine (Ara-C®), floxuridine, fludarabine, gemcitabine (Gemzar®), hydroxyurea, methotrexate, and pemetrexed (Alimta®).

[0172] Anthracyclines include daunorubicin, doxorubicin (Adriamycin®), epirubicin. Idarubicin. Other antitumor antibiotics include actinomycin-D, bleomycin, mitomycin-C, mitoxantrone.

[0173] Topoisomerase inhibitors include topotecan, irinotecan (CPT-11), etoposide (VP-16), teniposide, or mitoxantrone.

[0174] Microtubule inhibitors include estramustine, ixabepilone, taxanes (eg, paclitaxel, docetaxel, and cabazitaxel), and vinca alkaloids (eg, vinblastine, vincristine, vinorelbine, vindesine, and vinflunine).

[0175] B-raf enzyme inhibitors include vemurafenib (Zelboraf), dabrafenib (Tafinlar), and encorafenib (Viraftovi).

[0176] Immunotherapies include, but are not limited to, immune checkpoint modulators (i.e., inhibitors and / or agonists), cytokines, immunomodulatory monoclonal antibodies, and cancer vaccines.

[0177] Preferably, administration of cells in adoptive T cell therapy according to the present disclosure is combined with administration of an immune checkpoint modulator. Examples include inhibitors of EP2 / 4 adenosine receptors, including PD-1, CTLA4, LAG3, BTLA, OX2R, TIM-3, TIGIT, LAIR-1, PGE2 receptor, and / or A2AR (e.g., antibodies that specifically bind to and inhibit the activity of these). Preferably, the immune checkpoint modulator comprises an anti-PD-1 and / or anti-PDL-1 inhibitor (e.g., anti-PD-1 and / or anti-PDL-1 antibody).

[0178] The present disclosure also relates to the use of a composition comprising a cell as described herein for the manufacture of a medicament for treating cancer, an infectious disease or condition, an autoimmune disease or condition, or an inflammatory disease or condition in a subject. EXAMPLES

[0179] Example 1 Expression of lncRNA AF196970.3 in human tissues The expression of lncRNA AF196970.3 was investigated in various human tissues. AF196970.3 is expressed in many different healthy tissues, with relatively high expression in the cervix, brain, ovary, and uterus (Figure 3A), in part similar to SUV39H1 expression (Figure 3B). AF196970.3 is also detected in many different cell types, with the highest levels in endothelial cells, fibroblasts, and myocytes (Figure 3B), in part similar to SUV39H1 (Figure 3D). Only one AF196970.3 transcript (ENST00000416061.1) was found to be expressed in the GTEx project (Figure 3C).

[0180] The expression of lncRNA AF196970.3 was investigated in human T cells, specifically in the context of cancer. AF196970.3 was found to be expressed in tumor-infiltrating lymphocytes in three different projects: glioma (Wang et al., 2020) (Figure 4A), head and neck squamous cell carcinoma (Cillo et al., 2020) (Figure 4B), and hepatocellular carcinoma (Zhang et al., 2019) (Figure 4C). The expression level of AF196970.3 correlated with SUV39H1 levels and was relatively high in proliferating T cells (Figure 4B, Figure 4C).

[0181] Primers were designed to measure AF196970.3 expression by RT-PCR in freshly isolated human T cells from PBMCs and tumor-derived T cells, both activated or not in vitro. Expression patterns were detected.

[0182] Example 2 Overexpression of lncRNA AF196970.3 in HEK293 cells Piggy Bac scaffolds with GFP-puromycin reporter containing lncRNA SUV39H1 exon sequences with CMV or hPKG promoters are illustrated in Figure 5a-b, respectively. lncRNA AF196970.3 was overexpressed in HEK293 FT cells to evaluate its effect on SUV39H1 expression and activity.

[0183] Briefly, on day 0, HEK293 cells were cultured at 5.10 5 Cells were plated on 100 wells of 1000 cells. On day 1, cells were transfected with Piggy Bac (PB) constructs and Super PiggyBac transposase expression vector (using Purefection transfection reagent). On day 4, medium was changed to remove residual reagents. From day 7, transfected cells were selected with 1 pg / mL puromycin. On day 23, cells were harvested and analyzed for GFP and SUV39H1 expression. Figure 6b shows GFP expression levels by flow cytometry and a comparison of untransfected cells with cells transfected with either the Piggy Bac empty construct or one of the lncRNA SUV39H1 Piggy Bac plasmids as referenced above. Figure 6d shows quantification of SUV SUV39H1 protein levels normalized to actin. The results show that at D23 post-transfection, SUV39H1 protein levels are reduced for both lncRNA constructs when compared to the PB empty vector.

[0184] Example 3 Effect of lncRNA AF196970.3 overexpression on human T cells AF196970.3 is cloned into a lncRNA expression vector containing PB-CAG-BGHpA (Addgene #92161) (Yin et al., 2015, Cell Stem Cell). Alternative vector systems include the ELECTS transposon system (see Zhang et al., Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon. J Nanobiotechnol. 19, 303 (2021)).

[0185] The experimental procedure for lncRNA Suv39h1 (AF196970.3) overexpression in CD8+ T cells is described in Figure 6d. Briefly, T cell activation is performed on day 0. IL-2 is added on day 2. On day 3, cells are nucleofected with Piggy Bac (PB) constructs and Super PiggyBac transposase expression vector. Cas9 ribonucleolytic particle (RNP) nucleofection is performed to obtain SUV39H1-KO cells as a control. On day 7, GFP-positive cells are sorted and subsequently analyzed for the expression of SUV39H1 (Western blot) and memory marker CD27, as well as H3K9 trimethylation (FACS). The remaining cells are restimulated with transact on day 14 for a new analysis.

[0186] In another approach, gene-activating CRISPR is used to upregulate AF196970.3 in cells as described in Gene-activating CRISPR (Rankin et al., Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR. J. Vis. Exp.(145), e59233(2019)).

[0187] Using these approaches, AF196970.3 or a portion thereof is overexpressed in human T cells. The level of AF196970.3 is quantified, for example, by RT-PCR. The effect of AF196970.3 overexpression on 1) SUV39H1 protein level (by Western blot), 2) H3K9me3 level (by FACS), 3) T cell phenotype (CD27, CCR7, CD62L expression by FACS) is observed and compared to SUV39H1 inhibition by CRISPR knockout.

[0188] AF196970.3 overexpression inhibits SUV39H1 expression as measured by the level of SUV39H1 protein, the level of SUV39H1 activity, the level of H3K9 trimethylation, or T cell phenotype (an increased memory phenotype as described in International Application No. PCT / EP2020 / 070845. Briefly, CD8+ To observe the expression of central memory T cell surface markers that are important for the memory phenotype of T cells, AF196970.3 overexpressing T cells can be stimulated with aCD3+aCD28 beads for one week, followed by analysis by flow cytometry.Central memory T cell markers CCR7, CD27 and CD62L typically show increased expression levels when Suv39h1 is silenced in T cells.In addition, the fraction of CCR7+CD45RO+CD27+CD62L+ cells that constitute the central memory cell subset also typically increases when Suv39h1 is silenced in T cells.Typically, Suv39h1 silencing increases the fraction of central memory cells.

[0189] The effect of AF196970.3 overexpression on human CAR T cell efficacy in xenogeneic mouse models can also be compared to SUV39H1 inhibition by CRISPR KO.

[0190] Example 4 Effect of lncRNA AF196970.3 inhibition on human T cells Expression of AF196970.3 is inhibited to study its effect on human T cells. The gene is inactivated by CRISPR-Cas9 using gRNAs designed to target the first exon (SEQ ID NO:2) or promoter of AF196970.3.

[0191] Quantify AF196970.3 RNA (by RT-PCR) and SUV39H1 protein (by Western blot) levels. Observe the effect of AF196970.3 inhibition on 1) H3K9me3 levels (by FACS), and 2) T cell phenotype (CD27, CCR7, CD62L expression by FACS).

[0192] Inhibition of AF196970.3 is expected to increase the levels of hSUV39H1.

[0193] Example 5 Design of shRNA silencing Suv39h1 gene and overexpression in human cells Disruption of SUV39H1 histone methyltransferase results in enhanced T cell memory phenotype and in vivo persistence due to lack of suppression of stemness and memory-related genes. Knockout can be used in the context of adoptive T cell therapy and can result in long-term enhancement of CAR T cell activity, which leads to increased survival in preclinical models. Based on this knowledge and the results obtained using lncRNA as shown in the previous examples, short hairpin RNA (shRNA) has been designed to silence Suv39h1 gene expression and inhibit or reduce SUV39H1 protein level and / or activity.

[0194] Figure 7a shows a representation of the shRNA target sequence on the SUV39H1 gene. Figure 7b shows the target and loop sequences of different shRNAs (1-5) targeting SUV39H1. Figure 7c shows the plasmid map of the lentiviral construct used for shRNA expression in cells using the U6 promoter and EGFP reporter. The experimental progress is illustrated in Figure 8a. Briefly, HEK293 cells were cultured at day 0 with 2.10 5Cells were plated with 100% PBS. On day 1, cells were transduced with lentiviral particles at MOI=5 with polybrene (5pg / mL) and removed 24 hours later. On day 4, cells were split. On day 8, cells were harvested and analyzed for GFP and SUV39H1 expression. Flow cytometric comparison of GFP expression levels in untransduced cells with cells transduced with either scrambled shRNA or one of five shRNAs against SUV39H1 is illustrated in FIG. 8b. As illustrated in FIG. 8c, Western blots performed on day 8 revealed a significant reduction in shRNA-transduced cells compared to HEK293 control and SUV39H1 staining compared to actin staining for scrambled shRNA-transduced cells, thus underlying the significant Suv39H1 protein levels when overexpressing one shRNA (1-5) as disclosed herein.

[0195] Example 6 Effect of Suv39h1 shRNA overexpression on human T cells The experimental procedure for shRNA (inhibiting Suv39h1 as described above) overexpression in CD8+ T cells is described in Figure 9a. Briefly, T cell activation was performed on day 0. On day 1, cells were transduced with lentiviral particles at MOI=5 with polybrene (4 μg / mL) and removed 24 hours later and IL-2 was added. Cas9 ribonucleoparticle (RNP) nucleofection was performed on day 3 to obtain SUV39H1-KO cells as a control. On day 7, cells were harvested and analyzed for expression of SUV39H1, memory marker CD27, as well as trimethylation of H3K9.

[0196] Flow cytometric comparison of GFP expression levels in untransduced cells with cells transduced with either scrambled shRNA or one of five shRNAs against SUV39H1 is shown in Figure 9b. As shown in Figure 9c, Western blots performed on day 7 revealed a significant reduction in SUV39H1 staining compared to actin staining for shRNA-transduced cells compared to HEK293 controls and for scrambled shRNA-transduced cells, thus underlying the significant Suv39H1 protein levels when overexpressing one shRNA (1-5) as disclosed herein. Consistently, Figure 9d shows that in cells transduced with any one of the five shRNAs, the level of H3K9 trimethylation was significantly reduced when compared to cells transduced with scrambled shRNA and to a similar extent to the levels obtained in Suv39h1 KO cells. Figures 9E-F further illustrate that cells transduced with shRNA exhibit an enhanced memory phenotype, as indicated by increased expression of CD27 at D7 and D14, when compared to cells transduced with scrambled shRNA.

[0197] All patents, patent applications, and publications are incorporated by reference herein in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes.

[0198] Although the above articles and methods of the present disclosure have been described with respect to preferred embodiments and optional features, it will be apparent to those skilled in the art that modifications or combinations may be applied without departing from the spirit and scope of the present disclosure. Such modifications and combinations are intended to fall within the meaning and scope of the present disclosure as defined by the claims. The breadth and scope of the present disclosure should not be limited by any of the exemplary aspects described above, but should be defined only in accordance with the following claims and their equivalents. The terms and expressions that have been used are used as terms of description and not as terms of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents or portions of the features shown and described.

[0199] References [Table 1]

[0200] array SEQ ID NO:1 lncRNA AF196970.3 RNA-seq [ka]

[0201] SEQ ID NO:2 lncRNA AF196970.3 exon 1 RNA-seq [ka]

[0202] SEQ ID NO:3 lncRNA AF196970.3 exon 2 RNA-seq [ka]

[0203] SEQ ID NO:4 lncRNA AF196970.3 exon 3 RNA-seq [ka]

[0204] SEQ ID NO:5 Genomic DNA sequence [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0205] SEQ ID NO:6 lncRNA AF196970.3 cDNA sequence [ka]

[0206] SEQ ID NO:7 lncRNA AF196970.3 exon 1 cDNA sequence [ka]

[0207] SEQ ID NO:8 lncRNA AF196970.3 exon 2 cDNA sequence [ka]

[0208] SEQ ID NO:9 lncRNA AF196970.3 exon 3 cDNA sequence [ka]

[0209] [Table 2]

[0210] [ka]

Claims

1. A cell comprising a first nucleic acid, said first nucleic acid comprising: a heterologous nucleic acid expressing one or more of the nucleotide sequences of SEQ ID NOs: 13 and 26 or fragments thereof capable of inhibiting the expression of SUV39H1 in a cell; or A nucleic acid expressing one or more of SEQ ID NOs: 13 or 26 or fragments thereof capable of inhibiting expression of SUV39H1 in a cell, said nucleic acid being operably linked to a heterologous expression control sequence. That is, cells.

2. 2. The cell of claim 1, wherein the first nucleic acid expresses RNA of at least about 12-50, 50-100, 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800, 800-850, 850-900, 250-750, 500-750 or more bases in length.

3. A cell described in claim 1 or 2, which is an immune cell or a precursor thereof.

4. 4. The cell of claim 3, wherein the cell is a T cell, a CD4+ T cell, a CD8+ T cell, a CD4+ and CD8+ T cell, a NK cell, a Treg cell, a Tm cell, a memory stem cell (TSCM), a TCM cell, a TEM cell, a monocyte, a dendritic cell, a macrophage, a T cell precursor, a NK cell precursor, a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a hematopoietic stem cell (HSC), an adipose-derived stem cell (ADSC), or a pluripotent stem cell of the myeloid or lymphoid lineage.

5. 5. The cell of any one of claims 1 to 4, further comprising one or more, two or more, or three or more manipulated receptors.

6. A cell according to any one of claims 1 to 5, wherein the cell further comprises a second heterologous nucleic acid that expresses one or more manipulated receptors.

7. 7. The cell of claim 5 or 6, wherein the engineered receptor is a chimeric antigen receptor (CAR) and / or a T cell receptor (TCR), comprising an extracellular antigen-binding domain that specifically binds to an antigen, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain from CD3 zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CDS, CD22, CD79a, CD79b, or CD66d, 2B4, or a fragment of any thereof.

8. 8. The cell of claim 7, wherein the CAR comprises a CD3 zeta intracellular signaling domain or a fragment thereof in which ITAM2 and ITAM3 are inactivated.

9. The cell described in claim 7, wherein the TCR is a modified TCR.

10. The cell described in claim 9, wherein the TCR is a recombinant HLA-independent TCR.

11. 11. The cell of any one of claims 7 to 10, wherein the antigen has a low density on the cell surface of less than about 10,000 molecules, or less than about 5,000 molecules, or less than about 2,000 molecules per cell.

12. The extracellular antigen-binding domain is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, p95HER2, LI-CAM, CD19, CD20, CD22, mesothelin, CEA, claudin 18.2, hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, CD70, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, FcRH5, fetal acetylcholine e receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, BCMA, Lewis 12. The cell of any one of claims 7 to 11, wherein the extracellular antigen-binding domain binds to Y, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D ligand, NY-ESO-1, MART-1, gp100, carcinoembryonic antigen, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate-specific antigen (PSMA), estrogen receptor, progesterone receptor, ephrin B2, CD123, CS-1, c-Met, GD-2, MAGE A3, CE7, Wilms' tumor 1 (WT-1), or wherein the extracellular antigen-binding domain binds to any of the tumor neoantigenic peptides disclosed in International Publication No. WO2021 / 043804.

13. 13. The cell of any one of claims 1 to 12, wherein SUV39H1 expression is reduced or inhibited by at least about 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95%.

14. A polynucleotide comprising a sequence encoding SEQ ID NO: 13 or 26 or comprising SEQ ID NO: 13, optionally said polynucleotide comprising one or more of a modified backbone linkage, a modified sugar moiety, a modified phosphate moiety, a modified nucleobase, or a chemically conjugated moiety.

15. A polynucleotide operably linked to a heterologous expression control sequence, wherein the nucleic acid (a) encodes or expresses RNA comprising the nucleotide sequence of any one of SEQ ID NO: 13 or 26 or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell; or (b) comprises the nucleotide sequence of SEQ ID NO: 13 or a fragment thereof capable of inhibiting the expression of SUV39H1 in a cell, optionally wherein the cell is an immune cell or a precursor thereof.

16. 16. A vector comprising the polynucleotide of claim 14 or 15 and one or more additional expression control sequences, optionally wherein the vector is a viral vector, optionally an adenovirus, adeno-associated virus (AAV), poxvirus, papillomavirus, lentivirus, retrovirus, herpesvirus, foamyvirus, or Semliki Forest virus vector, and including pseudotyped viruses.

17. 17. The polynucleotide or vector of any one of claims 14 to 16 in a delivery vehicle, optionally a liposome, a lipid-containing complex, a nanoparticle, a gold particle, or a polymer complex.

18. A polynucleotide or vector described in any one of claims 14 to 17 for introduction into a cell in vitro, ex vivo, and / or in vivo, wherein the cell is an immune cell or a precursor thereof.

19. 19. A method for generating a cell according to any one of claims 1 to 13, comprising the steps of: (a) introducing into the cell a polynucleotide or vector according to any one of claims 14 to 17; and optionally (b) introducing into the cell a nucleic acid encoding an antigen-specific receptor.

20. 18. A pharmaceutical composition comprising a cell according to any one of claims 1 to 13, a polynucleotide or a vector according to any one of claims 14 to 17, for use in treating a subject suffering from cancer.

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