Methods and compositions for regulated armouring of cells

Abstract

Provided herein are compositions and methods for regulating the expression of effector molecules using regulatory transcription factors and / or activation-inducible promoters. In one aspect, provided herein are engineered nucleic acids comprising a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polynucleotide (ACP); and a second expression cassette comprising an ACP-responsive promoter and a second exogenous polynucleotide sequence, wherein the ACP is capable of inducing expression of the second expression cassette by binding to the ACP-responsive promoter.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application Nos. 62 / 947,427, filed December 12, 2019, and 63 / 116,103, filed November 19, 2020, each of which is incorporated by reference herein in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on XX / XX / 20XX, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0003] background Tumors employ various direct and indirect suppression strategies to avoid recognition and clearance by the immune system. These evasive strategies can effectively shut down cell therapies. Because non-armored therapies are less effective in solid tumors, combinatorial armoring, which involves the expression of combined effectors, can influence the entire cancer-immune cycle and enhance the activity of cell therapies. However, current cell therapy and gene therapy products cannot be controlled. Unregulated armoring can cause toxicity to the subject. Therefore, additional methods are needed to control and regulate the expression of combinations of effector molecules. Summary of the Invention

[0004] overview In one aspect, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polypeptide (ACP), wherein the first promoter is operably linked to the first exogenous polynucleotide; and an ACP responsive promoter and a polypeptide having the formula: (LE) X wherein E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20; an ACP responsive promoter is operably linked to the second exogenous polynucleotide; and in the first repeat of the (LE) unit, L is absent, and ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter.

[0005] In another aspect, provided herein is a method for producing an expression cassette comprising: (a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activated conditionally regulatory polypeptide (ACP), wherein the first promoter is operably linked to the first exogenous polynucleotide; and (b) an ACP-responsive promoter and a first exogenous polynucleotide sequence encoding an activated conditionally regulatory polypeptide (ACP), the first promoter being operably linked to the first exogenous polynucleotide; and Xand a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X=1 to 20; an ACP responsive promoter is operably linked to the second exogenous polynucleotide; in a first repeat of the (LE) unit, L is absent, and ACP can induce expression of the second expression cassette upon binding to the ACP responsive promoter. In some embodiments, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences. In some embodiments, the first expression cassette and the second expression cassette are encoded by the same polynucleotide sequence. In some embodiments, the first expression cassette and / or the second expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor. In some embodiments, the first expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor. In some embodiments, the second expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor. In some embodiments, the engineered expression system further comprises an additional expression cassette comprising an additional promoter and an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the additional promoter is operably linked to the additional exogenous polynucleotide. In some embodiments, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette or the second expression cassette. In some embodiments, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette. In some embodiments, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the second expression cassette. In some embodiments, the first vector comprises the first expression cassette and, if present, the additional expression cassette, and the second vector comprises the second expression cassette. In some embodiments, the first vector comprises the first expression cassette and the second vector comprises the second expression cassette and, if present, the additional expression cassette.In some embodiments, the first vector comprises the first expression cassette and the second expression cassette, and the second vector comprises additional expression cassettes, if present. In some embodiments, the engineered expression system comprises any of the aspects, features, or embodiments of the engineered nucleic acids described herein, including but not limited to any of the aspects, features, or embodiments described in enumerated embodiments 1-359.

[0006] In some embodiments, the second expression cassette is (LE) X When the molecule comprises two or more units of each linker polynucleotide sequence, each linker polynucleotide sequence is operably linked to the translation of each molecule as a separate polypeptide.

[0007] In some embodiments, the linker polynucleotide sequence encodes a 2A ribosomal skipping tag.

[0008] In some embodiments, the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0009] In some embodiments, the linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0010] In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide.

[0011] In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

[0012] In some embodiments, (LE) X The second expression cassette comprising one or more units of the above further comprises a polynucleotide sequence encoding a secretory signal peptide.

[0013] In some embodiments, for each X, the corresponding secretory signal peptide is operably associated with the effector molecule.

[0014] In some embodiments, each secretory signal peptide comprises the native secretory signal peptide native to the corresponding effector molecule.

[0015] In some embodiments, each secretory signal peptide comprises a non-native secretory signal peptide that is non-native to the corresponding effector molecule.

[0016] In some embodiments, the non-native secretory signal peptide is selected from the group consisting of IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12.

[0017] In some embodiments, the ACP responsive promoter comprises an ACP binding domain and a promoter sequence.

[0018] In some embodiments, the promoter sequence is derived from a promoter selected from the group consisting of minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoter, and tandem repeats thereof.

[0019] In some embodiments, the ACP responsive promoter is a synthetic promoter.

[0020] In some embodiments, the ACP responsive promoter comprises a minimal promoter.

[0021] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites.

[0022] In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter.

[0023] In some embodiments, the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0024] In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0025] In some embodiments, the cytokine is selected from the group consisting of IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α.

[0026] In some embodiments, the chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0027] In some embodiments, the homing molecule is selected from the group consisting of anti-integrin α4β7, anti-MAdCAM, CCR9, CXCR4, SDF1, MMP-2, CXCR1, CXCR7, CCR2;CCR4, and GPR15.

[0028] In some embodiments, the growth factor is selected from the group consisting of FLT3L and GM-CSF.

[0029] In some embodiments, the co-activator molecule is selected from the group consisting of c-Jun, 4-1BBL, and CD40L.

[0030] In some embodiments, the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, a TGFβ inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, and HPGE2.

[0031] In some embodiments, the TGFβ inhibitor is selected from the group consisting of an anti-TGFβ peptide, an anti-TGFβ antibody, a TGFb-TRAP, and combinations thereof.

[0032] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0033] In some embodiments, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0034] In some embodiments, each effector molecule is a human-derived effector molecule.

[0035] In some embodiments, the first foreign polynucleotide sequence further encodes an antigen-recognizing receptor.

[0036] In certain aspects, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polypeptide (ACP) and an antigen-recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an ACP-responsive promoter and a polypeptide having the formula: (LE) X wherein E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20; an ACP responsive promoter is operably linked to the second exogenous polynucleotide; and in the first repeat of the (LE) unit, L is absent, and ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter.

[0037] In certain aspects, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an activated conditional regulatory polypeptide-responsive (ACP-responsive) promoter and a polypeptide having the formula: (LE) X wherein E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20; and the ACP responsive promoter is operably linked to the second exogenous polynucleotide; and wherein in the first repeat of the (LE) unit, L is absent.

[0038] In some embodiments, ACP can induce expression of the second expression cassette by binding to an ACP responsive promoter.

[0039] In some embodiments, the ACP is an antigen-recognizing receptor, and the ACP can induce expression of the second expression cassette following binding of the ACP to a cognate antigen. In some embodiments, the ACP-responsive promoter is an inducible promoter that can be induced by binding of the ACP to a cognate antigen. In some embodiments, the ACP-responsive promoter is derived from the promoter region of a gene that is upregulated following binding of the ACP to a cognate antigen.

[0040] In some embodiments, the ACP responsive promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a synthetic promoter.

[0041] In some embodiments, the ACP responsive promoter comprises a minimal promoter.

[0042] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites.

[0043] In some embodiments, the nucleic acid sequence further comprises a linker polynucleotide sequence located between the first expression cassette and the second expression cassette.

[0044] In some embodiments, the linker polynucleotide sequence operably links the translation of the ACP and each effector molecule as separate polypeptides.

[0045] In some embodiments, the first exogenous polynucleotide sequence further comprises a linker polynucleotide sequence located between the region of the first exogenous polynucleotide sequence encoding the ACP and the region of the first exogenous polynucleotide sequence encoding the antigen-recognizing receptor, hi some embodiments, the linker polynucleotide sequence operably associates with translation of the ACP and the antigen-recognizing receptor as separate polypeptides.

[0046] In some embodiments, the engineered nucleic acid further comprises a linker polynucleotide sequence located between the first expression cassette and the second expression cassette, hi some embodiments, the linker polynucleotide sequence operably associates with translation of the antigen receptor and each effector molecule as separate polypeptides.

[0047] In some embodiments, the first promoter is operably linked to an ACP, a linker polynucleotide sequence, and a first exogenous polynucleotide sequence encoding an antigen-recognizing receptor.

[0048] In some embodiments, the linker polynucleotide sequence encodes a 2A ribosomal skipping tag. In some embodiments, the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A. In some embodiments, the linker polynucleotide sequence encodes an internal ribosome entry site (IRES). In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide. In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

[0049] In some embodiments, the antigen recognition receptor is 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, cadherin 3, cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, claudin 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR , ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FRα, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL- 8, IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY The antigen-recognizing receptor recognizes an antigen selected from the group consisting of ESO1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, survivin, TDGF1, TIM1, TROP2, and WT1. In some embodiments, the antigen-recognizing receptor recognizes GPC3. In some embodiments, the antigen-recognizing receptor recognizes mesothelin (MSLN).

[0050] In some embodiments, the antigen-recognizing receptor comprises an antigen-binding domain.

[0051] In some embodiments, the antigen-binding domain that binds to GPC3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 120), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121), and the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 122), a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123), and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 124).

[0052] In some embodiments, the VH region comprises: EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFA YWGQGTLVTVSA (SEQ ID NO: 125) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 126) The amino acid sequence of the present invention includes an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of

[0053] In some embodiments, the VL region is DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 127), or DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK (SEQ ID NO: 128) The amino acid sequence of the present invention includes an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of

[0054] In some embodiments, the antigen binding domain that binds to MSLN is QVQLVESGGGTVQAGGSLKLACAASGLPRTYNVMGWFRQAPGKEREGVAIIYTTTGATYYRDSVKGRATISQDNAKKSVSLQMNSLRPEDTAIYYCVARQPNSGPWEYWGQGTQVTVSS (SEQ ID NO: 129), or QVKLEESGGGSVQAGGSLRLSCTTSGYTNSYKWMGWFRQAPGQEREGVAVIYTGNDRTYYSDSVKGRFTISRDNAKNMIYLDMTRLRPEDSAVYECAIGHDGAWRYWGQGTQVTVSS (SEQ ID NO: 130) It comprises three complementarity determining regions (CDRs) of a single domain monoclonal antibody having an amino acid sequence of:

[0055] In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb).

[0056] In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0057] In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0058] In some embodiments, the VH and VL are separated by a peptide linker.

[0059] In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain.

[0060] In some embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0061] In some embodiments, the antigen recognition receptor is a CAR.

[0062] In some embodiments, the CAR comprises one or more intracellular signaling domains, each of which is selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0063] In some embodiments, the CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an OX40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and NKG2D.

[0064] In some embodiments, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain.

[0065] In some embodiments, the ACP is a transcriptional modulator.

[0066] In some embodiments, the ACP is a transcriptional repressor.

[0067] In some embodiments, the ACP is a transcriptional activator.

[0068] In some embodiments, the ACP further comprises an inhibitory protease and one or more cognate cleavage sites for the inhibitory protease.

[0069] In some embodiments, the ACP further comprises the hormone binding domain of the estrogen receptor (ERT2 domain).

[0070] In some embodiments, the ACP is a transcription factor.

[0071] In some embodiments, the transcription factor is a zinc finger-containing transcription factor.

[0072] In some embodiments, the ACP comprises a DNA-binding zinc finger protein domain (ZF protein domain) and a transcriptional effector domain.

[0073] In some embodiments, the ZF protein domain is modular in design and consists of a zinc finger array (ZFA).

[0074] In some embodiments, a ZF protein domain comprises between 1 and 10 ZFAs.

[0075] In some embodiments, the effector domain is a herpes simplex virus protein 16 (VP16) activation domain; a VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains (the tripartite activator is known as a VPR activation domain); a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (known as the p300 HAT core activation domain); a Kruppel-associated box (KRAB) repression domain; a truncated Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain; or a WRPW motif of Hairy-related basic helix-loop-helix repressor protein. (SEQ ID NO: 162) (This motif is known as the WRPW repression domain. ("WRPW" disclosed as SEQ ID NO: 162) ); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1α chromoshadow repression domain.

[0076] In some embodiments, one or more cognate cleavage sites for an inhibitory protease are located between the ZF protein domain and the effector domain.

[0077] In some embodiments, the inhibitory protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).

[0078] In some embodiments, the cognate cleavage site comprises an NS3 protease cleavage site.

[0079] In some embodiments, the NS3 protease cleavage site comprises an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site.

[0080] In some embodiments, the NS3 protease can be inhibited by a protease inhibitor.

[0081] In some embodiments, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, the protease inhibitor is grazoprevir. In some embodiments, the protease inhibitor is grazoprevir and elbasvir. In some embodiments, grazoprevir and elbasvir are co-formulated in the pharmaceutical composition. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, grazoprevir and elbasvir are in a 2:1 weight ratio. In some embodiments, grazoprevir is 100 mg per unit dose and elbasvir is 50 mg per unit dose.

[0082] In some embodiments, the ACP can undergo nuclear localization upon binding of the ERT2 domain to tamoxifen or its metabolites.

[0083] In some embodiments, the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0084] In some embodiments, the ACP further comprises a degron, wherein the degron is operably linked to the ACP.

[0085] In some embodiments, the degron is an HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB of influenza A or influenza B (SP2-NB-SP2), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (influenza A virus M2 protein), or any combination thereof. The SP1 and SP2 tandem repeats of the mutant (SP2-SP1-SP2-SP1-SP2), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), APC / C degron, and COP1 E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, actinfilin-binding degron, KEAP1-binding degron, KLHL2- and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphodegron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phospho-dependent degron (SEQ ID NO: 163) , a Siah-binding motif, an SPOP SBC docking motif, and a PCNA-binding PIP box.

[0086] In some embodiments, the degron comprises a cereblon (CRBN) polypeptide substrate domain that can bind to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting the degradation of ACP via the ubiquitin pathway.

[0087] In some embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or fragments thereof, that are capable of drug-inducible binding of CRBN.

[0088] In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a naturally occurring CRBN polypeptide sequence.

[0089] In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 131).

[0090] In some embodiments, the IMiD is an FDA-approved drug.

[0091] In some embodiments, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0092] In some embodiments, the degron is located 5' of the inhibitory protease, 3' of the inhibitory protease, 5' of the ZF protein domain, 3' of the ZF protein domain, 5' of the effector domain, or 3' of the effector domain.

[0093] In some embodiments, the engineered nucleic acid further comprises an insulator.

[0094] In some embodiments, the insulator is located between the first expression cassette and the second expression cassette.

[0095] In some embodiments, the first expression cassette is located in the same orientation relative to the second expression cassette.

[0096] In some embodiments, the first expression cassette is located in the opposite orientation relative to the second expression cassette.

[0097] In some embodiments, the engineered nucleic acid is selected from the group consisting of DNA, cDNA, RNA, mRNA, and naked plasmids.

[0098] In another aspect, provided herein is an engineered nucleic acid, expression system, or expression vector comprising a first expression cassette, a second expression cassette, and / or an additional expression cassette disclosed herein.

[0099] In another aspect, provided herein is a composition comprising an engineered nucleic acid, expression system, or first expression cassette, second expression cassette, and / or additional expression cassettes described herein, and a pharmaceutically acceptable carrier.

[0100] In another aspect, provided herein is an isolated cell comprising an engineered nucleic acid, expression system, or first expression cassette, a second expression cassette, and / or an additional expression cassette described herein or a vector described herein.

[0101] In some embodiments, the engineered nucleic acid is recombinantly expressed.

[0102] In some embodiments, the engineered nucleic acid is expressed from a vector or a selected locus from the genome of the cell.

[0103] In some embodiments, the cell is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the cell is a natural killer (NK) cell.

[0104] In some embodiments, the cells are autologous.

[0105] In some embodiments, the cells are allogeneic.

[0106] In some embodiments, the cell is a tumor cell selected from the group consisting of an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell, a breast tumor cell, a cervical tumor cell, a colon tumor cell, an esophageal tumor cell, a glioma cell, a kidney tumor cell, a liver tumor cell, a lung tumor cell, a melanoma cell, a mesothelioma cell, an ovarian tumor cell, a pancreatic tumor cell, a gastric tumor cell, a testicular yolk sac tumor cell, a prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell.

[0107] In some embodiments, the cells are engineered via transduction with an oncolytic virus.

[0108] In some embodiments, the oncolytic virus is selected from the group consisting of an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic Sindbis virus, and any variant or derivative thereof.

[0109] In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising a first expression cassette and a second expression cassette.

[0110] In some embodiments, the cell is a bacterial cell selected from the group consisting of Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

[0111] In another aspect, provided herein is a composition comprising an isolated cell as described herein and a pharmaceutically acceptable carrier.

[0112] In another aspect, provided herein is a method of treating a subject in need thereof, the method comprising administering a therapeutically effective amount of any of the isolated cells or compositions described herein.

[0113] In another aspect, provided herein is a method of stimulating a cellular immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective amount of any of the isolated cells or compositions described herein.

[0114] In another aspect, provided herein is a method of providing anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the isolated cells or compositions described herein.

[0115] In another aspect, provided herein are methods of treating a subject having cancer, the methods comprising administering a therapeutically effective amount of any of the isolated cells or compositions described herein.

[0116] In another aspect, provided herein is a method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the isolated cells or compositions described herein.

[0117] In some embodiments, the administration comprises systemic administration.

[0118] In some embodiments, the administration comprises intratumoral administration.

[0119] In some embodiments, the isolated cells are from a subject.

[0120] In some embodiments, the isolated cells are allogeneic with respect to the subject.

[0121] In some embodiments, the method further comprises administering a checkpoint inhibitor.

[0122] In some embodiments, the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0123] In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0124] In some embodiments, the tumor is selected from the group consisting of an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0125] In another aspect, provided herein are lipid-based structures, engineered nucleic acids, expression systems, or first expression cassettes, second expression cassettes, and / or additional expression cassettes described herein.

[0126] In some embodiments, the lipid-based structures comprise extracellular vesicles.

[0127] In some embodiments, the extracellular vesicles are selected from the group consisting of nanovesicles and exosomes.

[0128] In some embodiments, the lipid-based structures comprise lipid nanoparticles or micelles.

[0129] In some embodiments, the lipid-based structure comprises a liposome.

[0130] In another aspect, provided herein are compositions comprising the lipid-based structures described herein and a pharmaceutically acceptable carrier.

[0131] In another aspect, provided herein are methods of treating a subject in need thereof, the methods comprising administering a therapeutically effective amount of any of the lipid-based structures or compositions described herein.

[0132] In another aspect, provided herein is a method of stimulating a cellular immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective amount of any of the lipid-based structures or compositions described herein.

[0133] In another aspect, provided herein is a method of providing anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the lipid-based structures or compositions described herein.

[0134] In another aspect, provided herein are methods of treating a subject having cancer, the methods comprising administering a therapeutically effective amount of any of the lipid-based structures or compositions described herein.

[0135] In another aspect, provided herein is a method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the lipid-based structures or compositions described herein.

[0136] In some embodiments, the administration comprises systemic administration.

[0137] In some embodiments, the administration comprises intratumoral administration.

[0138] In some embodiments, the lipid-based structures are capable of engineering cells of a subject.

[0139] In some embodiments, the method further comprises administering a checkpoint inhibitor.

[0140] In some embodiments, the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0141] In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0142] In some embodiments, the tumor is selected from the group consisting of an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0143] In another aspect, provided herein is a nanoparticle, an engineered nucleic acid, an expression system, or a first expression cassette, a second expression cassette, and / or an additional expression cassette as described herein.

[0144] In some embodiments, the nanoparticles comprise an inorganic material.

[0145] In another aspect, provided herein are compositions comprising the nanoparticles described herein.

[0146] In another aspect, provided herein are methods of treating a subject in need thereof, the methods comprising administering a therapeutically effective amount of any of the nanoparticles or compositions described herein.

[0147] In another aspect, provided herein is a method of stimulating a cellular immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective amount of any of the nanoparticles or compositions described herein.

[0148] In another aspect, provided herein is a method of providing anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of any of the nanoparticles or compositions described herein.

[0149] In another aspect, provided herein are methods of treating a subject having cancer, the methods comprising administering a therapeutically effective amount of any of the nanoparticles or compositions described herein.

[0150] In another aspect, provided herein is a method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising any of the nanoparticles or compositions described herein.

[0151] In some embodiments, the administration comprises systemic administration.

[0152] In some embodiments, the administration comprises intratumoral administration.

[0153] In some embodiments, the nanoparticles are capable of manipulating cells of a subject.

[0154] In some embodiments, the method further comprises administering a checkpoint inhibitor.

[0155] In some embodiments, the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0156] In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0157] In some embodiments, the tumor is selected from the group consisting of an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0158] In another aspect, provided herein are viruses engineered to contain the engineered nucleic acids described herein.

[0159] In some embodiments, the virus is selected from the group consisting of a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

[0160] In some embodiments, the virus is an oncolytic virus.

[0161] In some embodiments, the first expression cassette and the second expression cassette are capable of being expressed in a tumor cell.

[0162] In some embodiments, the tumor is selected from the group consisting of an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0163] In some embodiments, the oncolytic virus is selected from the group consisting of an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic Sindbis virus, and any variant or derivative thereof.

[0164] In another aspect, provided herein is a composition comprising an engineered virus or composition.

[0165] In another aspect, provided herein is a method of stimulating a cellular immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective amount of either an engineered virus or composition.

[0166] In another aspect, provided herein is a method of providing anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of either the engineered virus or composition.

[0167] In another aspect, provided herein is a method of treating a subject having cancer, the method comprising administering a therapeutically effective amount of either the engineered virus or composition.

[0168] In another aspect, provided herein is a method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising either an engineered virus or composition.

[0169] In some embodiments, the administration comprises systemic administration.

[0170] In some embodiments, the administration comprises intratumoral administration.

[0171] In some embodiments, the engineered virus infects cells of a subject and expresses the first expression cassette and the second expression cassette.

[0172] In some embodiments, the method further comprises administering a checkpoint inhibitor.

[0173] In some embodiments, the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0174] In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0175] In some embodiments, the tumor is selected from the group consisting of an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0176] In another aspect, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activated conditionally regulatory polypeptide (ACP), wherein the first promoter is operably linked to the first exogenous polynucleotide; and an ACP-responsive promoter and a polypeptide having the formula: (LE) X and a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X=1 to 20; an ACP responsive promoter is operably linked to the second exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent, and ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter.

[0177] In some embodiments, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences.

[0178] In some embodiments, the first expression cassette and the second expression cassette are encoded by a single polynucleotide sequence.

[0179] The second expression cassette is (L1-E) X 156. The engineered cell of any one of Claims 153-155, wherein when said engineered cell comprises two or more units of said L1 linker polynucleotide sequence, each L1 linker polynucleotide sequence is operably associated with translation of each effector molecule as a separate polypeptide.

[0180] In some embodiments, the engineered cell further comprises a second linker polynucleotide sequence, wherein the second linker polynucleotide links the first expression cassette to the second expression cassette.

[0181] In some embodiments, the second linker polynucleotide sequence operably links the translation of each effector molecule and ACP as separate polypeptides.

[0182] In some embodiments, each linker polynucleotide sequence encodes a 2A ribosomal skipping tag.

[0183] In some embodiments, the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0184] In some embodiments, each linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0185] In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide.

[0186] In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

[0187] In some embodiments, (L1-E) X The second expression cassette comprising one or more units of the above further comprises a polynucleotide sequence encoding a secretory signal peptide.

[0188] In some embodiments, for each X, the corresponding secretory signal peptide is operably associated with the effector molecule.

[0189] In some embodiments, each secretory signal peptide comprises the native secretory signal peptide native to the corresponding effector molecule.

[0190] In some embodiments, each secretory signal peptide comprises a non-native secretory signal peptide that is non-native to the corresponding effector molecule.

[0191] In some embodiments, the non-native secretory signal peptide is selected from the group consisting of IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12.

[0192] In some embodiments, the ACP responsive promoter comprises an ACP binding domain and a promoter sequence.

[0193] In some embodiments, the promoter sequence is derived from a promoter selected from the group consisting of minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoter, and tandem repeats thereof.

[0194] In some embodiments, the ACP responsive promoter is a synthetic promoter.

[0195] In some embodiments, the ACP responsive promoter comprises a minimal promoter.

[0196] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites.

[0197] In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter.

[0198] In some embodiments, the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0199] In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0200] In some embodiments, the cytokine is selected from the group consisting of IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α.

[0201] In some embodiments, the chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0202] In some embodiments, the homing molecule is selected from the group consisting of anti-integrin α4β7, anti-MAdCAM, CCR9, CXCR4, SDF1, MMP-2, CXCR1, CXCR7, CCR2, and GPR15.

[0203] In some embodiments, the growth factor is selected from the group consisting of FLT3L and GM-CSF.

[0204] In some embodiments, the co-activator molecule is selected from the group consisting of c-Jun, 4-1BBL, and CD40L.

[0205] In some embodiments, the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, a TGFβ inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, and HPGE2.

[0206] In some embodiments, the TGFβ inhibitor is selected from the group consisting of an anti-TGFβ peptide, an anti-TGFβ antibody, a TGFb-TRAP, and combinations thereof.

[0207] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0208] In some embodiments, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0209] In some embodiments, each effector molecule is a human-derived effector molecule.

[0210] In some embodiments, the cell further comprises a third expression cassette comprising a third promoter and a third exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the third promoter is operably linked to the third exogenous polynucleotide.

[0211] In some embodiments, the first foreign polynucleotide sequence further encodes an antigen-recognizing receptor.

[0212] In another aspect, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polypeptide (ACP) and an antigen recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an ACP responsive promoter and a polypeptide having the formula: (LE) X and a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X=1 to 20; an ACP responsive promoter is operably linked to the second exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent, and ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter.

[0213] In another aspect, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an activated conditional regulatory polypeptide-responsive (ACP-responsive) promoter and a polypeptide having the formula: (LE) X wherein E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20; and the ACP responsive promoter is operably linked to the second exogenous polynucleotide; and wherein in the first repeat of the (LE) unit, L is absent.

[0214] In some embodiments, the cell further comprises a third expression cassette comprising a third promoter and a third exogenous polynucleotide sequence encoding an activating conditionally controlled polypeptide (ACP), wherein the third promoter is operably linked to the third exogenous polynucleotide.

[0215] In some embodiments, ACP can induce expression of the second expression cassette by binding to an ACP responsive promoter.

[0216] In some embodiments, the ACP is an antigen-recognizing receptor, and the ACP can induce expression of the second expression cassette following binding of the ACP to its cognate antigen. In some embodiments, the ACP-responsive promoter is an inducible promoter that can be induced by binding of the ACP to its cognate antigen. In some embodiments, the ACP-responsive promoter is derived from the promoter region of a gene that is upregulated following binding of the ACP to its cognate antigen.

[0217] In some embodiments, the ACP is an antigen-recognizing receptor, and the ACP can induce expression of the second expression cassette upon binding to its cognate antigen.

[0218] In some embodiments, the ACP responsive promoter is an inducible promoter that can be induced by binding of ACP to its cognate antigen.

[0219] In some embodiments, the ACP responsive promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a synthetic promoter.

[0220] In some embodiments, the ACP responsive promoter comprises a minimal promoter.

[0221] In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites.

[0222] In some embodiments, the first exogenous polynucleotide sequence further comprises a third linker polynucleotide sequence located between the region of the first exogenous polynucleotide sequence encoding the ACP and the region of the first exogenous polynucleotide sequence encoding the antigen-recognizing receptor. In some embodiments, the third linker polynucleotide sequence operably associates with translation of the ACP and the antigen-recognizing receptor as separate polypeptides. In some embodiments, a first promoter is operably linked to the ACP, the third linker polynucleotide sequence, and the first exogenous polynucleotide sequence encoding the antigen-recognizing receptor.

[0223] In some embodiments, the cell further comprises a third linker polynucleotide sequence located between the first expression cassette and the second expression cassette.

[0224] In some embodiments, the third linker polynucleotide sequence operably links the translation of the antigen receptor and each effector molecule as separate polypeptides.

[0225] In some embodiments, the third linker polynucleotide sequence encodes a 2A ribosomal skipping tag. In some embodiments, the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A. In some embodiments, the third linker polynucleotide sequence encodes an internal ribosome entry site (IRES). In some embodiments, the third linker polynucleotide sequence encodes a cleavable polypeptide. In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

[0226] In some embodiments, the third linker polynucleotide sequence operably links the translation of the ACP and the antigen recognition receptor as separate polypeptides.

[0227] In some embodiments, the antigen recognition receptor is 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, cadherin 3, cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, claudin 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR , ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FRα, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL- 8, IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY The antigen-recognizing receptor recognizes an antigen selected from the group consisting of ESO1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, survivin, TDGF1, TIM1, TROP2, and WT1. In some embodiments, the antigen-recognizing receptor recognizes GPC3. In some embodiments, the antigen-recognizing receptor recognizes mesothelin (MSLN).

[0228] In some embodiments, the antigen-recognizing receptor comprises an antigen-binding domain.

[0229] In some embodiments, the antigen-binding domain that binds to GPC3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 120), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121), and the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 122), a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123), and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 124).

[0230] In some embodiments, the VH region comprises: EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFA YWGQGTLVTVSA (SEQ ID NO: 125), or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 126) The amino acid sequence of the present invention includes an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of

[0231] In some embodiments, the VL region is DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 127), or DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK (SEQ ID NO: 128) The amino acid sequence of the present invention includes an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of

[0232] In some embodiments, the antigen binding domain that binds to MSLN is QVQLVESGGGTVQAGGSLKLACAASGLPRTYNVMGWFRQAPGKEREGVAIIYTTTGATYYRDSVKGRATISQDNAKKSVSLQMNSLRPEDTAIYYCVARQPNSGPWEYWGQGTQVTVSS (SEQ ID NO: 129), or QVKLEESGGGSVQAGGSLRLSCTTSGYTNSYKWMGWFRQAPGQEREGVAVIYTGNDRTYYSDSVKGRFTISRDNAKNMIYLDMTRLRPEDSAVYECAIGHDGAWRYWGQGTQVTVSS (SEQ ID NO: 130) It comprises three complementarity determining regions (CDRs) of a single domain monoclonal antibody having an amino acid sequence of:

[0233] In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb).

[0234] In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv).

[0235] In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL).

[0236] In some embodiments, the VH and VL are separated by a peptide linker.

[0237] In some embodiments, the scFv comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain.

[0238] In some embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or a T-cell receptor (TCR).

[0239] In some embodiments, the antigen recognition receptor is a CAR.

[0240] In some embodiments, the CAR comprises one or more intracellular signaling domains selected from the group consisting of a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.

[0241] In some embodiments, the CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an OX40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D transmembrane domain.

[0242] In some embodiments, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain.

[0243] In some embodiments, the ACP is a transcriptional modulator.

[0244] In some embodiments, the ACP is a transcriptional repressor.

[0245] In some embodiments, the ACP is a transcriptional activator.

[0246] In some embodiments, the ACP further comprises an inhibitory protease and one or more cognate cleavage sites for the inhibitory protease.

[0247] In some embodiments, the ACP further comprises the hormone binding domain of the estrogen receptor (ERT2 domain).

[0248] In some embodiments, the ACP is a transcription factor.

[0249] In some embodiments, the ACP is a zinc finger-containing transcription factor.

[0250] In some embodiments, the transcription factor comprises a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain.

[0251] In some embodiments, the ZF protein domain is modular in design and consists of a zinc finger array (ZFA).

[0252] In some embodiments, a ZF protein domain comprises between 1 and 10 ZFAs.

[0253] In some embodiments, the effector domain is a herpes simplex virus protein 16 (VP16) activation domain; a VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator consisting of VP64, p65, and Rta activation domains (the tripartite activator is known as a VPR activation domain); a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (known as the p300 HAT core activation domain); a Kruppel-associated box (KRAB) repression domain; a truncated Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain; or a WRPW motif of Hairy-related basic helix-loop-helix repressor protein. (SEQ ID NO: 162) (This motif is known as the WRPW repression domain. ("WRPW" disclosed as SEQ ID NO: 162) ); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1α chromoshadow repression domain.

[0254] In some embodiments, one or more cognate cleavage sites for an inhibitory protease are located between the ZF protein domain and the effector domain.

[0255] In some embodiments, the inhibitory protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3).

[0256] In some embodiments, the cognate cleavage site comprises an NS3 protease cleavage site.

[0257] In some embodiments, the NS3 protease cleavage site comprises an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site.

[0258] In some embodiments, the NS3 protease can be inhibited by a protease inhibitor.

[0259] In some embodiments, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, the protease inhibitor is grazoprevir. In some embodiments, the protease inhibitor is grazoprevir and elbasvir. In some embodiments, grazoprevir and elbasvir are co-formulated in the pharmaceutical composition. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, grazoprevir and elbasvir are in a 2:1 weight ratio. In some embodiments, grazoprevir is 100 mg per unit dose and elbasvir is 50 mg per unit dose.

[0260] In some embodiments, the ACP can undergo nuclear localization upon binding of the ERT2 domain to tamoxifen or its metabolites.

[0261] In some embodiments, the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0262] In some embodiments, the ACP further comprises a degron, wherein the degron is operably linked to the ACP.

[0263] In some embodiments, the degron is an HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB of influenza A or influenza B (SP2-NB-SP2), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (influenza A virus M2 protein), or any combination thereof. The SP1 and SP2 tandem repeats of the mutant (SP2-SP1-SP2-SP1-SP2), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), APC / C degron, and COP1 E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, actinfilin-binding degron, KEAP1-binding degron, KLHL2- and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphodegron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phospho-dependent degron (SEQ ID NO: 163) , a Siah-binding motif, an SPOP SBC docking motif, and a PCNA-binding PIP box.

[0264] In some embodiments, the degron comprises a cereblon (CRBN) polypeptide substrate domain that can bind to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting the degradation of ACP via the ubiquitin pathway.

[0265] In some embodiments, the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or fragments thereof, that are capable of drug-inducible binding of CRBN.

[0266] In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a naturally occurring CRBN polypeptide sequence.

[0267] In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 131).

[0268] In some embodiments, the IMiD is an FDA-approved drug.

[0269] In some embodiments, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide.

[0270] In some embodiments, the degron is located 5' of the inhibitory protease, 3' of the inhibitory protease, 5' of the ZF protein domain, 3' of the ZF protein domain, 5' of the effector domain, or 3' of the effector domain.

[0271] In some embodiments, the engineered nucleic acid further comprises an insulator.

[0272] In some embodiments, the insulator is located between the first expression cassette and the second expression cassette.

[0273] In some embodiments, the first expression cassette is located in the same orientation relative to the second expression cassette.

[0274] In some embodiments, the first expression cassette is located in the opposite orientation relative to the second expression cassette.

[0275] In some embodiments, the cells comprise a third promoter and a gene encoding a gene of the formula (LE) X wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, and the third promoter is operably linked to the third exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent.

[0276] In some embodiments, the third expression cassette is (LE) X When the L linker polynucleotide sequence comprises two or more units of each effector molecule, each L linker polynucleotide sequence is operatively linked to the translation of each effector molecule as a separate polypeptide.

[0277] In some embodiments, each linker polynucleotide sequence encodes a 2A ribosomal skipping tag.

[0278] In some embodiments, the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0279] In some embodiments, each linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0280] In some embodiments, the linker polynucleotide sequence encodes a cleavable polypeptide.

[0281] In some embodiments, the cleavable polypeptide comprises a furin polypeptide sequence.

[0282] In some embodiments, (LE)X The third expression cassette comprising one or more units of the above further comprises a polynucleotide sequence encoding a secretory signal peptide.

[0283] In some embodiments, for each X, the corresponding secretory signal peptide is operably associated with the effector molecule.

[0284] In some embodiments, each secretory signal peptide comprises the native secretory signal peptide native to the corresponding effector molecule.

[0285] In some embodiments, each secretory signal peptide comprises a non-native secretory signal peptide that is non-native to the corresponding effector molecule.

[0286] In some embodiments, the non-native secretory signal peptide is selected from the group consisting of IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12.

[0287] In some embodiments, the additional promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter.

[0288] In some embodiments, the additional promoter is a constitutive promoter selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0289] In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0290] In some embodiments, the cytokine is selected from the group consisting of IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α.

[0291] In some embodiments, the chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0292] In some embodiments, the homing molecule is selected from the group consisting of anti-integrin α4β7, anti-MAdCAM, CCR9, CXCR4, SDF1, MMP-2, CXCR1, CXCR7, CCR2, and GPR15.

[0293] In some embodiments, the growth factor is selected from the group consisting of FLT3L and GM-CSF.

[0294] In some embodiments, the co-activator molecule is selected from the group consisting of c-Jun, 4-1BBL, and CD40L.

[0295] In some embodiments, the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, a TGFβ inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, and HPGE2.

[0296] In some embodiments, the TGFβ inhibitor is selected from the group consisting of an anti-TGFβ peptide, an anti-TGFβ antibody, a TGFb-TRAP, and combinations thereof.

[0297] In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0298] In some embodiments, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0299] In some embodiments, each effector molecule is a human-derived effector molecule.

[0300] In some embodiments, the cell is selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the cell is a natural killer (NK) cell.

[0301] In some embodiments, the cells are autologous.

[0302] In some embodiments, the cells are allogeneic.

[0303] In some embodiments, the cell is a tumor cell selected from the group consisting of an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell, a breast tumor cell, a cervical tumor cell, a colon tumor cell, an esophageal tumor cell, a glioma cell, a kidney tumor cell, a liver tumor cell, a lung tumor cell, a melanoma cell, a mesothelioma cell, an ovarian tumor cell, a pancreatic tumor cell, a gastric tumor cell, a testicular yolk sac tumor cell, a prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell.

[0304] In some embodiments, the cells were engineered via transduction with an oncolytic virus.

[0305] In some embodiments, the oncolytic virus is selected from the group consisting of an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic Sindbis virus, and any variant or derivative thereof.

[0306] In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising a first expression cassette and a second expression cassette.

[0307] In some embodiments, the cell is a bacterial cell selected from the group consisting of Clostridium beejerinkii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella cholerae suis.

[0308] In another aspect, provided herein is a composition comprising an engineered cell and a pharmaceutically acceptable carrier.

[0309] In another aspect, provided herein are methods of treating a subject in need thereof, the methods comprising administering a therapeutically effective amount of either the engineered cells or the compositions.

[0310] In another aspect, provided herein is a method of stimulating a cellular immune response against tumor cells in a subject, the method comprising administering to a tumor-bearing subject a therapeutically effective amount of either the engineered cells or the composition.

[0311] In another aspect, provided herein is a method of providing anti-tumor immunity in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of either the engineered cells or the composition.

[0312] In another aspect, provided herein are methods of treating a subject with cancer, the methods comprising administering a therapeutically effective amount of either the engineered cells or compositions.

[0313] In another aspect, provided herein is a method of reducing tumor volume in a subject, the method comprising administering to a subject having a tumor a composition comprising either an engineered cell or composition.

[0314] In some embodiments, the administration comprises systemic administration.

[0315] In some embodiments, the administration comprises intratumoral administration.

[0316] In some embodiments, the engineered cells are derived from a subject.

[0317] In some embodiments, the engineered cells are allogeneic with respect to the subject.

[0318] In some embodiments, the method further comprises administering a checkpoint inhibitor.

[0319] In some embodiments, the checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0320] In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0321] In some embodiments, the tumor is selected from the group consisting of an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0322] In some embodiments, the method further comprises administering a protease inhibitor. In some embodiments, the protease inhibitor is administered in an amount sufficient to inhibit the inhibitory protease. In some embodiments, the protease inhibitor is administered before, simultaneously with, or after administration of the engineered cells or a composition comprising the engineered cells. In some embodiments, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, the protease inhibitor is grazoprevir. In some embodiments, the protease inhibitor is grazoprevir and elbasvir. In some embodiments, grazoprevir and elbasvir are co-formulated in the pharmaceutical composition. In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, grazoprevir and elbasvir are in a 2:1 weight ratio. In some embodiments, the grazoprevir is at 100 mg per unit dose and the elbasvir is at 50 mg per unit dose.

[0323] In some embodiments, the method further comprises administering tamoxifen or a metabolite thereof, in some embodiments, the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0324] [The present invention 1001] (a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditionally controlled polypeptide (ACP), the first promoter is operably linked to the first exogenous polynucleotide; the first expression cassette, and (b) an ACP responsive promoter and the formula: (LE) X and a second exogenous polynucleotide sequence having the sequence: During the ceremony, E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20, the ACP responsive promoter is operably linked to the second exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent; the second expression cassette 1. An engineered expression system comprising: the ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter; Optionally, said second expression cassette is (LE) X wherein each linker polynucleotide sequence is operably associated with the translation of each effector molecule as a separate polypeptide, Optionally, (LE) X wherein the second expression cassette further comprises a polynucleotide sequence encoding a secretory signal peptide for each X; Optionally, for each X, the corresponding secretory signal peptide is operably associated with the effector molecule; Optionally, each secretory signal peptide comprises the native secretory signal peptide native to the corresponding effector molecule; Optionally, each secretory signal peptide comprises a non-native secretory signal peptide that is non-native to the corresponding effector molecule; Optionally, each secretory signal peptide comprises a non-native secretory signal peptide that is non-native to the corresponding effector molecule, and optionally the non-native secretory signal peptide is a secretory signal peptide of a molecule selected from the group consisting of IL12, IL2, optimized IL2, trypsiongen-2, Gaussia luciferase, CD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12; Optionally, the first expression cassette is comprised in a first nucleic acid and the second expression cassette is comprised in a second nucleic acid, or the first expression cassette and the second expression cassette are comprised in a single nucleic acid. The engineered expression system. [The present invention 1002] further comprising a linker polynucleotide sequence located between the first expression cassette and the second expression cassette; Optionally, the linker polynucleotide sequence operably links the ACP and each effector molecule to be translated as separate polypeptides; optionally, the linker polynucleotide sequence encodes a 2A ribosomal skipping tag, optionally the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A; Optionally, the linker polynucleotide sequence encodes an internal ribosome entry site (IRES), and Optionally, the linker polynucleotide sequence encodes a cleavable polypeptide, and optionally, the cleavable polypeptide comprises a furin polypeptide sequence. The engineered expression system of the present invention. [The present invention 1003] (a) the ACP responsive promoter comprises an ACP binding domain sequence and a promoter sequence; Optionally, the promoter sequence is derived from a promoter selected from the group consisting of minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoter, and tandem repeats thereof; Optionally, the ACP responsive promoter comprises a synthetic promoter; Optionally, the ACP responsive promoter comprises a minimal promoter; and Optionally, the ACP binding domain comprises one or more zinc finger binding sites. (b) the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter; Optionally, the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb; and / or (c) each effector molecule is independently selected from a therapeutic class, said therapeutic class being selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes; Optionally, the cytokine is selected from the group consisting of IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α; Optionally, the chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1; Optionally, the homing molecule is selected from the group consisting of anti-integrin α4β7, anti-MAdCAM, CCR9, CXCR4, SDF1, MMP-2, CXCR1, CXCR7, CCR2, CCR4, and GPR15; Optionally, the growth factor is selected from the group consisting of FLT3L and GM-CSF; Optionally, the co-activator molecule is selected from the group consisting of c-Jun, 4-1BBL, and CD40L; optionally, the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, a TGFβ inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, and HPGE2; and optionally, the TGFβ inhibitor is selected from the group consisting of an anti-TGFβ peptide, an anti-TGFβ antibody, TGFb-TRAP, and combinations thereof; Optionally, the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREMI antibody, and an anti-TREM2 antibody; Optionally, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof; and Optionally, each effector molecule is a human-derived effector molecule; The engineered expression system of the present invention 1001 or 1002. [The present invention 1004] (a) the first expression cassette and / or the second expression cassette further comprise an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor; or (b) the engineered expression system further comprises an additional expression cassette comprising an additional promoter and an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor; said additional promoter is operably linked to said additional exogenous polynucleotide, and optionally said additional exogenous polynucleotide sequence is encoded by the same polynucleotide as said first expression cassette or said second expression cassette; Optionally, the antigen recognition receptor recognizes GPC3; Optionally, said antigen recognizing receptor comprises an antigen binding domain; Optionally, the antigen-binding domain that binds to GPC3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region; the VH is heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119); A heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 120), and Heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121) and The VL is a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 122); a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123); and Light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence QQYYNYPLT (SEQ ID NO: 124) Including, Optionally, the VH region comprises: TIFF0007792335000001.tif41147 and an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of Optionally, the VL region is TIFF0007792335000002.tif41146 and an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the amino acid sequence of Optionally, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, a F(ab) fragment, a F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb); optionally, the VH and VL are separated by a peptide linker; Optionally, when the antigen-binding domain comprises an scFv, the scFv comprises the structure VH-L-VL or VL-L-VH, wherein VH is the heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain; Optionally, the antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR); Optionally, when the antigen recognition receptor is a CAR, the CAR comprises one or more intracellular signaling domains, each of the one or more intracellular signaling domains being a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a a signaling domain, a GITR intracellular signaling domain, a HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain; Optionally, the CAR comprises a transmembrane domain, wherein the transmembrane domain is selected from the group consisting of a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an OX40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, a NKp44 transmembrane domain, a NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D transmembrane domain; and Optionally, the CAR comprises a spacer region between the antigen binding domain and the transmembrane domain. The engineered expression system of any one of claims 1001 to 1003. [The present invention 1005] the ACP is a transcriptional modulator; Optionally, said ACP is a transcriptional repressor, or said ACP is a transcriptional activator; Optionally, the ACP further comprises an inhibitory protease and one or more cognate cleavage sites for the inhibitory protease; Optionally, the ACP further comprises a hormone binding domain of the estrogen receptor (ERT2 domain), and optionally, when the ERT2 domain binds to tamoxifen or a metabolite thereof, the ACP is capable of undergoing nuclear localization, and optionally, the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen; optionally, said ACP is a transcription factor, optionally, said transcription factor is a zinc finger-containing transcription factor; optionally, the ACP comprises a DNA-binding zinc finger protein domain (ZF protein domain) and a transcriptional effector domain, optionally the ZF protein domain is modular and consists of a zinc finger array (ZFA), and optionally the ZF protein domain comprises 1 to 10 ZFAs; Optionally, the effector domain is a herpes simplex virus protein 16 (VP16) activation domain; a VP64 activation domain, which is an activation domain comprising four tandem copies of VP16; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator of NFκB (VPR) activation domain comprising VP64, p65, and Rta activation domains; a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (p300HAT a core activation domain; a Kruppel-associated box (KRAB) repression domain; a truncated Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain; a Hairy-related basic helix-loop-helix repressor protein WRPW motif (this motif is known as the WRPW repression domain); a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1α chromoshadow repression domain, Optionally, the one or more cognate cleavage sites for the inhibitory protease are located between the ZF protein domain and the effector domain; Optionally, the inhibitory protease is Hepatitis C virus (HCV) nonstructural protein 3 (NS3); Optionally, the cognate cleavage site comprises an NS3 protease cleavage site; Optionally, the NS3 protease cleavage site comprises an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site; Optionally, the NS3 protease may be inhibited by a protease inhibitor; Optionally, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir, or the protease inhibitor is grazoprevir, or the protease inhibitor comprises grazoprevir and elbasvir; Optionally, said grazoprevir and said elbasvir are co-formulated into a pharmaceutical composition; Optionally, the pharmaceutical composition is a tablet; Optionally, said grazoprevir and said elbasvir are in a 2:1 weight ratio; Optionally, said grazoprevir is at 100 mg per unit dose and said elbasvir is at 50 mg per unit dose; Optionally, the ACP further comprises a degron, wherein the degron is operably linked to the ACP; Optionally, the degron is selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB (SP2-NB-SP2 of influenza A or influenza B)), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (tandem repeats of SP1 and SP2 (influenza A or influenza B)), SPmix (tandem repeats of SP1 and SP2 (influenza ... SP2-SP1-SP2-SP1-SP2 of the influenza A virus M2 protein), NS2 (three copies of residues 79-93 of the influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), APC / C degron, COP1 selected from the group consisting of an E3 ligase-binding degron motif, a CRL4-Cdt2-binding PIP degron, an actinfilin-binding degron, a KEAP1-binding degron, a KLHL2- and KLHL3-binding degron, an MDM2-binding motif, an N-degron, a hydroxyproline modification in hypoxia signaling, a plant hormone-dependent SCF-LRR-binding degron, an SCF ubiquitin ligase-binding phosphodegron, a plant hormone-dependent SCF-LRR-binding degron, a DSGxxS phospho-dependent degron, a Siah-binding motif, an SPOP SBC docking motif, and a PCNA-binding PIP box; Optionally, the degron comprises a cereblon (CRBN) polypeptide substrate domain capable of binding to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting degradation of ACP via the ubiquitin pathway; Optionally, the CRBN polypeptide substrate domain is selected from the group consisting of IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof capable of drug-inducible binding of CRBN; Optionally, the CRBN polypeptide substrate domain is a chimeric fusion product of a native CRBN polypeptide sequence; Optionally, the IMiD is an FDA-approved drug; optionally, the IMiD is selected from the group consisting of thalidomide, lenalidomide, and pomalidomide; and Optionally, the degron is located 5' of the inhibitory protease, 3' of the inhibitory protease, 5' of the ZF protein domain, 3' of the ZF protein domain, 5' of the effector domain, or 3' of the effector domain. The engineered expression system of any one of claims 1001 to 1004. [The present invention 1006] (a) the engineered expression system further comprises an insulator, optionally located between the first expression cassette, the second expression cassette, and / or additional expression cassettes, if present; (b) the first expression cassette is localized in the same orientation relative to the second expression cassette, or the first expression cassette is localized in the opposite orientation relative to the second expression cassette; and / or (c) the engineered expression system is a nucleic acid selected from the group consisting of DNA, cDNA, RNA, mRNA, and naked plasmid; 1006. The engineered expression system of any one of claims 1001 to 1005. [The present invention 1007] comprising the first expression cassette, the second expression cassette, and / or the additional expression cassette, if present, of any one of the engineered expression systems of any of claims 1001 to 1006; and Optionally, (a) a first vector comprises the first expression cassette and, if present, the additional expression cassette, and a second vector comprises the second expression cassette; (b) a first vector comprises the first expression cassette and a second vector comprises the second expression cassette and, if present, the additional expression cassette; (c) a first vector comprises the first expression cassette and the second expression cassette, and a second vector comprises, if present, the additional expression cassette; or (d) a vector comprises the first expression cassette, the second expression cassette, and, if present, the additional expression cassette; One or more expression vectors. [The present invention 1008] An isolated cell comprising the engineered expression system of any one of claims 1001 to 1006, or one or more expression vectors of claim 1007, Optionally, the engineered expression system is recombinantly expressed; Optionally, the engineered expression system is expressed from one or more vectors or one or more selected loci from the genome of the cell; Optionally, the cell is selected from the group consisting of a T cell, a CD8+ T cell, a CD4+ T cell, a gamma delta T cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a virus-specific T cell, a natural killer T (NKT) cell, a natural killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a bone marrow cell, a macrophage, a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a human embryonic stem cell (ESC), an ESC-derived cell, a pluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell; and Optionally, the cells are autologous or the cells are allogeneic. The isolated cells. [The present invention 1009] A pharmaceutical composition comprising any one of the engineered expression systems of the present invention 1001 to 1006, one or more expression vectors of the present invention 1007, or an isolated cell of the present invention 1008, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof. [The present invention 1010] A method of treating a subject in need thereof, comprising administering a therapeutically effective amount of the isolated cell of the present invention 1008 or the composition of the present invention 1009. [The present invention 1011] A method for stimulating a cellular immune response against tumor cells in a subject, comprising administering to a tumor-bearing subject a therapeutically effective amount of the isolated cells of the present invention 1008 or the composition of the present invention 1009. [The present invention 1012] A method for providing anti-tumor immunity in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the isolated cell of the present invention 1008 or the composition of the present invention 1009. [The present invention 1013] A method for reducing tumor volume in a subject, comprising administering a composition comprising the isolated cells of invention 1008 or the composition of invention 1009 to a subject having a tumor. [The present invention 1014] (a) the administering comprises systemic administration or intratumoral administration; (b) the isolated cells are derived from the subject, or the isolated cells are allogeneic with respect to the subject; (c) the method further comprises administering a checkpoint inhibitor. (d) the tumor is selected from the group consisting of adenocarcinoma, bladder tumor, brain tumor, breast tumor, cervical tumor, colon tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, melanoma, mesothelioma, ovarian tumor, pancreatic tumor, gastric tumor, testicular yolk sac tumor, prostate tumor, skin tumor, thyroid tumor, and uterine tumor; (e) the method further comprises administering a protease inhibitor; Optionally, the protease inhibitor is administered in an amount sufficient to inhibit an inhibitory protease; Optionally, the protease inhibitor is administered prior to, simultaneously with, or following administration of the engineered cells or the composition comprising the engineered cells; Optionally, the protease inhibitor is selected from the group consisting of simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir, or the protease inhibitor is grazoprevir, or the protease inhibitor comprises grazoprevir and elbasvir; Optionally, when the protease inhibitors comprise grazoprevir and elbasvir, the grazoprevir and the elbasvir are co-formulated in the pharmaceutical composition; Optionally, the pharmaceutical composition is a tablet; optionally, said grazoprevir and said elbasvir are in a 2 to 1 weight ratio; and Optionally, the grazoprevir is at 100 mg per unit dose and the elbasvir is at 50 mg per unit dose; and / or (f) the method further comprises administering tamoxifen or a metabolite thereof; Optionally, the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. Any of the methods 1010 to 1013 of the present invention. [The present invention 1015] A kit for treating and / or preventing cancer, comprising the isolated cells of the present invention 1008 or the pharmaceutical composition of the present invention 1009, optionally further comprising written instructions for using the cells or composition to treat and / or prevent cancer in a subject. These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings. [Brief explanation of the drawings]

[0325] [Figure 1A] FIG. 1 shows a diagram of an exemplary regulatory TF protein and a TF-inducible gene with the minCMV promoter and mCherry gene. [Figure 1B] 1 shows the expression of mCherry protein in cells expressing both regulatory TFs and an mCherry vector in the presence or absence of asunaprevir. [Figure 1C] FIG. 1 shows a diagram of an exemplary regulatory TF protein and a regulatory TF-inducible gene with the minYB_TATA promoter and mCherry gene. [Figure 1D] 1 shows the expression of mCherry protein in cells expressing both regulatory TFs and an mCherry vector in the presence or absence of asunaprevir. [Figure 2A] FIG. 1 shows a diagram of an exemplary single vector expressing a regulatory TF and a regulatory TF-inducible gene with the minTK promoter and IL-10 gene. [Figure 2B] IL-10 production in cells expressing regulatory TF and IL-10 vectors in the presence or absence of asunaprevir. [Figure 3A] FIG. 1 shows a diagram of an exemplary single vector expressing a regulatory TF and a regulatory TF-inducible gene with the minTK promoter and IL-12 gene. [Figure 3B] IL-12 production in cells expressing both regulatory TF and IL-12 vector in the presence or absence of asunaprevir is shown. [Figure 4A] FIG. 1 shows a diagram of an exemplary single vector expressing a regulatory TF linked to a myc-tagged CAR gene and a regulatory TF-inducible gene with a minYB_TATA promoter and mCherry gene. [Figure 4B] CAR expression in cells expressing regulatory TFs and mCherry vector in the presence and absence of asunaprevir is shown. [Figure 4C] 1 shows the expression of mCherry in cells expressing regulatory TFs and an mCherry vector in the presence or absence of asunaprevir. [Figure 5]Additional exemplary vectors for expressing regulatory TF and effector genes in a single vector system are shown. [Figure 6A] A schematic diagram of the GPC3 CAR construct ("1106") is shown. [Figure 6B] CAR transduction properties in combination with various constructs as assessed by flow cytometry are shown. [Figure 7] The transduction properties of the various constructs as assessed YFP MFI (upper panel) and percentage (lower panel) are shown. [Figure 8A] IL-12 production of various constructs with (right) or without (left) co-expression of CAR is shown. [Figure 8B] IL-15 production of various constructs with (right) or without (left) co-expression of CAR is shown. [Figure 8C] IL-21 production of various constructs with (right) or without (left) co-expression of CAR is shown. [Figure 9A] IL-12 production of various constructs with (bottom) or without (top) co-culture with target HepG2 cells is shown. [Figure 9B] IL-15 production of various constructs with (bottom) or without (top) co-culture with target HepG2 cells is shown. [Figure 9C] IL-21 production of various constructs with (bottom) or without (top) co-culture with target HepG2 cells is shown. [Figure 10A] TNFα production of various constructs with (bottom) or without (top) co-culture with target HepG2 cells is shown. [Figure 10B] IFNg production of various constructs with (bottom) or without (top) co-culture with target HepG2 cells is shown. [Figure 10C] IL-2 production of various constructs with (bottom) or without (top) co-culture with target HepG2 cells is shown. [Figure 11A] A schematic diagram of the GPC3 CAR construct ("1108") is shown. [Figure 11B]CAR transduction properties in combination with various constructs as assessed by flow cytometry are shown. [Figure 12] Tumor size assessed by BLI measurements on days 11, 14, 21, and 24 for mice treated with T cells transduced with various constructs is shown (Figure 13C - IL-15, Figure 13D - IL-12, Figure 13E - IL-21). [Figure 13A] Individual mice treated with T cells without virus (Figure 13A-left panel) or GPC3-CAR T alone without cytokines (Figure 13A-right panel) are shown. [Figure 13B] Individual mice treated with GPC3-CAR T engineered with IL-12 / IL-21 co-expressing armoring are shown. [Figure 13C] Individual mice treated with IL-15 armored engineered GPC3-CAR T are shown. [Figure 13D] Individual mice treated with IL-12 armored engineered GPC3-CAR T are shown. [Figure 13E] Individual mice treated with IL-21 armored engineered GPC3-CAR T are shown. [Figure 14A] TNFα production assessed in the plasma of mice treated with the various constructs at 3 days (upper panel) and 13 days (lower panel) after treatment is shown. [Figure 14B] IFNγ production assessed in the plasma of mice treated with the various constructs at day 3 (top panel) and day 13 (bottom panel) after treatment is shown. [Figure 14C] IL-2 production assessed in the plasma of mice treated with the various constructs at 3 days (upper panel) and 13 days (lower panel) after treatment is shown. [Figure 15A] IL-12 production assessed in the plasma of mice treated with the various constructs at 3 days (upper panel) and 10 days (lower panel) after treatment is shown. [Figure 15B] IL-15 production assessed in the plasma of mice treated with the various constructs at 3 days (upper panel) and 10 days (lower panel) after treatment is shown. [Figure 15C] IL-21 production assessed in the plasma of mice treated with the various constructs at days 3 (top panel) and 10 (bottom panel) after treatment is shown. [Figure 16A] Tumor size (left panel) and human T cell persistence (right panel) of T cells engineered with various constructs at 14 days after tumor injection (3 days after T cell treatment) are shown. [Figure 16B] Tumor size (left panel) and human T cell persistence (right panel) of T cells engineered with various constructs at 21 days after tumor injection (13 days after T cell treatment) are shown. [Figure 17A] Schematic diagrams of various payload expression systems using a tamoxifen-based regulated TF expression system are shown. [Figure 17B] 1 shows reporter expression using various tamoxifen-based regulated TF expression systems after treatment with different amounts of 4-OHT. [Figure 17C] 1 shows reporter expression using various tamoxifen-based regulated TF expression systems after treatment with different amounts of N-desmethyltamoxifen. [Figure 17D] 1 shows reporter expression using various tamoxifen-based regulated TF expression systems after treatment with different amounts of endoxifen. [Figure 17E] 1 shows a summary of reporter expression using various tamoxifen-based regulated TF expression systems after treatment. [Figure 18] 1 shows a summary of CAR expression using various tamoxifen-based regulated TF expression systems after treatment. [Figure 19] 1 shows flow cytometry plots of CAR expression using various tamoxifen-based regulated TF expression systems after treatment. [Figure 20] Schematic diagrams of various payload expression systems using a drug-inducible ACP-based regulated TF expression system are shown. [Figure 21] Schematic diagrams of various payload expression systems using a drug-inducible ACP-based regulated TF expression system are shown. [Figure 22]CAR expression using various drug-inducible ACP-based regulated TF expression systems. [Figure 23A] 1 shows reporter expression using a specific drug-inducible ACP-based regulated TF expression system. [Figure 23B] CAR expression using a specific drug-inducible ACP-based regulated TF expression system. [Figure 24A] 1 shows reporter expression using a specific drug-inducible ACP-based regulated TF expression system. [Figure 24B] CAR expression using a specific drug-inducible ACP-based regulated TF expression system. [Figure 25] A schematic diagram of a drug-inducible ACP (also called "synTF") using the NS3 / NS4 protease cleavage site and VPR transcriptional effector domain (construct "1845") and an expression cassette using the 4xBS minYB-TATA ACP-responsive promoter driving the hIL-12 effector molecule payload is shown. [Figure 26] Figure 1 shows in vitro production of hIL-12 using an ACP-based regulated expression system. Columns from left to right are no drug, 0.1 μM GRZ, and 0.5 μM GRZ, respectively. [Figure 27] 1 shows the experimental design used to evaluate the ACP-based regulatable expression system in vivo. [Figure 28] Fold expansion of T cells engineered with a constitutive hIL-12 expression system or an ACP-based regulated expression system in vivo is shown. [Figure 29] 1 shows the glucose signature of T cells engineered with a constitutive hIL-12 expression system or an ACP-based regulated expression system in vivo. [Figure 30] The percentage of circulating T cells engineered with a constitutive hIL-12 expression system or an ACP-based regulated expression system in vivo is shown. [Figure 31] 1 shows the production of hIL-12 in plasma produced by T cells engineered with a constitutive hIL-12 expression system or an ACP-based regulated expression system in vivo. [Figure 32] Schematic diagrams of various payload expression systems using a drug-inducible ACP-based regulated TF expression system are shown. [Figure 33] 1 shows in vitro production of hIL-12 by T cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir. [Figure 34] 1 shows in vitro production of hIL-15 by T cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir. [Figure 35] CAR expression in T cells transduced with various drug-inducible ACP-based regulatory TF expression systems. [Figure 36] CAR activity using various drug-inducible ACP-based regulated TF expression systems is shown, as assessed by target cell killing (LDH release). [Figure 37] 1 shows in vitro production of hIL-12 by NK cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir. [Figure 38] Schematic diagrams of various payload expression systems using a drug-inducible ACP-based regulated TF expression system are shown. [Figure 39] 1 shows in vitro production of hIL-12 by T cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir. [Figure 40] 1 shows in vitro production of hIL-15 by T cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir. [Figure 41] 1 shows in vitro production of hIL-15 by T cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir. [Figure 42]Blood T cells (hCD3+:hCD45+ shown as % of viable cells) engineered with an in vivo constitutive hIL-12 expression system or a drug-inducible ACP-based regulatory expression system are shown over time for various grazoprevir dosing regimens (day 4: top panel, day 8: middle panel, day 12: bottom panel). *Samples from two groups were lost and not included in the analysis. [Figure 43] Figure 1 shows plasma hIL-12 production on day 4 produced by T cells engineered with an in vivo constitutive hIL-12 expression system or a drug-inducible ACP-based regulated expression system for various grazoprevir dosing regimens. [Figure 44] Figure 1 shows plasma hIL-12 production on days 8 (left panel) and 12 (right panel) produced by T cells engineered with an in vivo constitutive hIL-12 expression system or a drug-inducible ACP-based regulatory expression system for various grazoprevir dosing regimens. [Figure 45] 1 shows an "on / off / on" grazoprevir (Grz) dosing regimen. [Figure 46] Blood T cells (hCD3+:hCD45+ shown as % of viable cells) engineered with an in vivo constitutive hIL-12 expression system or a drug-inducible ACP-based regulatory expression system are shown over time on the indicated days for an "on / off / on" grazoprevir (Grz) dosing regimen. [Figure 47] Plasma hIL-12 production produced by T cells engineered with an in vivo constitutive hIL-12 expression system or a drug-inducible ACP-based regulated expression system is shown over time on the indicated days for an "on / off / on" grazoprevir (Grz) dosing regimen. [Figure 48] Shown are the body weights over time of mice administered T cells engineered with an in vivo constitutive hIL-12 expression system or a drug-inducible ACP-based regulatory expression system on the indicated days for an "on / off / on" grazoprevir (Grz) dosing regimen. [Figure 49]1 shows the workflow of a screen aimed at evaluating promoters that turn on transcription when CAR cells are activated by target cells. [Figure 50] Shown are the constructs and candidate promoters evaluated in a screen for promoters that turn on transcription when CAR cells are activated by target cells. [Figure 51] After 24 hours of culture in the presence (right column) or absence (left column) of HepG2 target cells, quantified mKate expression by flow cytometry is shown for constructs and candidate promoters evaluated in a screen for promoters that turn on transcription when CAR cells are activated by target cells. [Figure 52] After 48 hours of culture in the presence (right column) or absence (left column) of HepG2 target cells, quantified mKate expression by flow cytometry is shown for constructs and candidate promoters evaluated in a screen for promoters that turn on transcription when CAR cells are activated by target cells. [Figure 53] Histograms of mKate expression by flow cytometry are shown for identified promoters that turn on transcription when CAR cells are activated by target cells 24 hours (top panel) and 48 hours (bottom panel) after culturing with HepG2 target cells ("promoter + target"). Also shown are CAR alone cultured in the absence of HepG2 target ("CAR only"), CAR alone cultured in the presence of HepG2 target ("CAR + target"), and CAR + promoter cultured in the absence of HepG2 target ("promoter only"). [Figure 54] 1 shows in vitro production of hIL-12 by T cells transduced with various ACP-based regulated expression systems after treatment with various concentrations of grazoprevir in the presence or absence of elbasvir. DETAILED DESCRIPTION OF THE INVENTION

[0326] Detailed Description definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0327] The term "amelioration" refers to any therapeutically beneficial result in the treatment of a disease state, for example, a cancer disease state, including prevention, reduction in severity or progression, remission, or cure thereof.

[0328] The term "in situ" refers to processes that occur within living cells grown separately from the organism, for example, grown in tissue culture.

[0329] The term "in vivo" refers to a process that occurs within a living organism.

[0330] As used herein, the term "mammal" includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0331] The term percent "identity," with respect to two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage (%) of nucleotide or amino acid residues that are identical when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those of skill in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or over the entire length of the two sequences being compared.

[0332] For sequence comparison, one sequence usually serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0333] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0334] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0335] The term "sufficient amount" means an amount sufficient to produce a desired effect, for example, an amount sufficient to modulate protein aggregation in a cell.

[0336] The term "therapeutically effective amount" is an amount effective for ameliorating symptoms of disease. Prevention can be considered treatment, and thus a therapeutically effective amount can be a "prophylactically effective amount."

[0337] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0338] Engineered Nucleic Acids and Polypeptides Regulation of drug expression in cell therapy is necessary to explore the window of therapeutic efficacy. Described herein is a method using regulatory transcription factors that can drive the expression of any desired effector molecule or combination of effector molecules. This system is versatile, as it can regulate intracellular or membrane-bound proteins, for example, by using a modular protease system that allows for ON or OFF configuration. FDA-approved protease switch drugs can be used and can be administered orally with favorable pharmacokinetic properties. Furthermore, the methods and compositions described herein may be used, for example, for regulated immune-modulating effector expression in cell or gene therapy. Regulatable transcription factors can be used in combination with, for example, CAR T cells, CAR NK cells, TCR T cells, TIL therapy, virus-specific T cells, or other suitable immune cell therapies.

[0339] In one aspect, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polypeptide (ACP) and / or an antigen recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide, and an activation conditional regulatory polypeptide responsive (ACP responsive) promoter and a polypeptide having the formula: (LE) Xand a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X = 1 to 20; an ACP-responsive promoter is operably linked to the second exogenous polynucleotide; in the first repeat of the (LE) unit, L is absent; and optionally, the ACP is capable of inducing expression of the first expression cassette upon binding to the ACP-responsive promoter. In some embodiments, the ACP comprises a drug-inducible domain, such as a tetracycline-responsive domain (e.g., a TetR domain) or an inhibitory protease domain (e.g., an NS3 protease). In some embodiments, the ACP is an antigen-recognizing receptor, and the receptor can induce expression of the second expression cassette upon binding to its cognate antigen (an "activation-inducible system"), e.g., CAR binding to the cognate antigen, and the ACP-responsive promoter comprises a promoter sequence capable of driving expression of the second expression cassette in response to CAR signaling.

[0340] In one aspect, provided herein is (a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polypeptide (ACP) and an antigen-recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an ACP-responsive promoter and a first exogenous polynucleotide sequence having the formula: (LE) X wherein E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20; an ACP responsive promoter is operably linked to the second exogenous polynucleotide; and in the first repeat of the (LE) unit, L is absent, and ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter.

[0341] In one aspect, provided herein is a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an activated conditional regulatory polypeptide-responsive (ACP-responsive) promoter and a polypeptide having the formula: (LE) X and a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X = 1 to 20; an ACP-responsive promoter is operably linked to the second exogenous polynucleotide; and L is absent in the first repeat of the (LE) unit. Expression of the second expression cassette can be induced by ACP binding to the ACP-responsive promoter. The ACP can be a receptor, such as an antigen-recognition receptor, and upon ACP binding to a cognate ligand (e.g., a cognate antigen), downstream signaling following ligand binding can induce expression of the second expression cassette, e.g., from the ACP-responsive promoter. In a non-limiting illustrative example, the ACP can be a chimeric antigen receptor (CAR), and upon CAR binding to its cognate receptor, downstream signaling (e.g., T cell or NK cell receptor signaling) can induce expression of a cytokine payload from the ACP-responsive promoter specific for CAR binding to the target antigen (e.g., cytokine arming). Examples of ACP-responsive promoters useful in activation-inducible systems are described below (see "Promoters").

[0342] In some embodiments, the second expression cassette is (LE) X When the molecule comprises two or more units of each linker polynucleotide sequence, each linker polynucleotide sequence is operably linked to the translation of each molecule as a separate polypeptide.

[0343] X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0344] In some embodiments, a single engineered nucleic acid comprises at least one, two, three, four, five, or more expression cassettes. Generally, each expression cassette refers to a promoter operably linked to a polynucleotide sequence encoding a protein of interest. For example, an ACP, an effector molecule, and an antigen-recognizing receptor may each be encoded by a separate expression cassette on the same engineered nucleic acid (e.g., vector). The expression cassettes can be oriented in any direction relative to each other (e.g., the cassettes can be in the same or opposite orientations). In an exemplary engineered nucleic acid having three or more expression cassettes, the cassettes can be in the same or mixed orientations (e.g., the first and second cassettes can be in the same orientation, while the third cassette is in the opposite orientation). In some embodiments, a first expression cassette is located in the same orientation relative to a second expression cassette. In some embodiments, a first expression cassette is located in the opposite orientation relative to a second expression cassette.

[0345] In some embodiments, one or more engineered nucleic acids may comprise at least one, two, three, four, five, or more expression cassettes. A strategy for regulated armoring comprising two or more engineered nucleic acids may be referred to as an "engineered expression system." In one aspect, the present invention provides a system comprising: (a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activated conditional regulatory polypeptide (ACP), the first promoter being operably linked to the first exogenous polynucleotide; and (2) an ACP-responsive promoter and a polypeptide of the formula: (LE) Xand a second expression cassette comprising a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X=1 to 20; an ACP-responsive promoter is operably linked to the second exogenous polynucleotide; in a first repeat of the (LE) unit, L is absent, and ACP can induce expression of the second expression cassette upon binding to the ACP-responsive promoter. In some embodiments of the expression system, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences. In some embodiments of the expression system, the first expression cassette and the second expression cassette are encoded by the same polynucleotide sequence. In some embodiments of the expression system, the first expression cassette and / or the second expression cassette further comprise an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor. In some embodiments of the expression system, the first expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor. In some embodiments of the expression system, the second expression cassette further comprises an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor. In some embodiments of the expression system, the engineered expression system further comprises an additional expression cassette comprising an additional promoter and an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the additional promoter is operably linked to the additional exogenous polynucleotide. In some embodiments of the expression system, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette or the second expression cassette. In some embodiments of the expression system, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette. In some embodiments of the expression system, the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the second expression cassette. In some embodiments of the expression system, the first vector comprises the first expression cassette and, if present, the additional expression cassette, and the second vector comprises the second expression cassette.In some embodiments of the expression system, the first vector comprises the first expression cassette and the second vector comprises the second expression cassette and, if present, additional expression cassettes. In some embodiments of the expression system, the first vector comprises the first expression cassette and the second expression cassette and, if present, additional expression cassettes.

[0346] Illustrative, non-limiting examples of expression systems include: (1) the antigen-recognition receptor expression cassette and the effector molecule expression cassette can be encoded by a first engineered nucleic acid, and the ACP expression cassette can be encoded by a second engineered nucleic acid; (2) the ACP expression cassette and the effector molecule expression cassette can be encoded by a first engineered nucleic acid, and the antigen-recognition receptor expression cassette can be encoded by a second engineered nucleic acid; and (3) the ACP expression cassette and the antigen-recognition receptor expression cassette can be encoded by a first engineered nucleic acid, and the effector molecule expression cassette can be encoded by a second engineered nucleic acid. In additional illustrative, non-limiting examples, the effector molecule expression cassette can be encoded by a first engineered nucleic acid, and the ACP expression cassette can be encoded by a second engineered nucleic acid.

[0347] In some embodiments, an expression cassette can be multicistronic, i.e., multiple distinct polypeptides (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single mRNA transcript. For example, a multicistronic expression cassette can encode both an ACP and an antigen-recognizing receptor, e.g., both expressed from a single expression cassette driven by a constitutive promoter. In another example, a multicistronic expression cassette can encode both an effector molecule and an antigen-recognizing receptor, e.g., both expressed from a single expression cassette driven by an ACP-responsive promoter. Expression cassettes can be made multicistronic by using various linkers, e.g., a polynucleotide sequence encoding a first protein of interest can be linked to a nucleotide sequence encoding a second protein of interest (e.g., in a 5' to 3' direction: first gene: linker: second gene). Multicistronic features and options are described in the section "Multicistronic and Multiple Promoter Systems."

[0348] In some embodiments, the engineered nucleic acid is selected from DNA, cDNA, RNA, mRNA, and naked plasmids. Also provided herein are expression vectors comprising the engineered nucleic acid.

[0349] In some embodiments, the engineered nucleic acid further comprises an insulator. The insulator can be located between the first expression cassette and the second expression cassette. The insulator can be located between the first expression cassette and the second expression cassette, where both cassettes are in the same orientation relative to each other. The insulator can be located between the first expression cassette and the second expression cassette, where both cassettes are in opposite orientation relative to each other. The insulator is a cis-regulatory element with enhancer-blocking or barrier function. Enhancer-blocker insulators block enhancers from acting on the promoters of adjacent genes. Barrier insulators prevent euchromatin silencing. An example of a suitable insulator of the present disclosure is the A2 insulator described in Liu M, et al., Nat Biotechnol. 2015 Feb;33(2):198-203. Additional insulators are described in West et al., Genes & Dev, 002.16:271-288, all of which are incorporated by reference in their entirety. Other examples of suitable insulators include, but are not limited to, β-globin locus insulators such as A1 insulator, CTCF insulator, gypsy insulator, HS5 insulator, and cHS4 insulator. In some embodiments, the insulator is an A2 insulator, A1 insulator, CTCF insulator, HS5 insulator, gypsy insulator, β-globin locus insulator, or cHS4 insulator. The insulator may be an A2 insulator.

[0350] Activated Conditional Regulatory Polypeptides (ACPs) In some embodiments, the ACP is a transcriptional modulator. In some embodiments, the ACP is a transcriptional repressor. In some embodiments, the ACP is a transcriptional activator. In some embodiments, the ACP is a transcription factor. In some embodiments, the ACP comprises a DNA-binding domain and a transcriptional effector domain. In some embodiments, the transcription factor is a zinc finger-containing transcription factor. In some embodiments, the zinc finger-containing transcription factor can be a synthetic transcription factor. In some embodiments, the DNA-binding domain of the ACP comprises a DNA-binding zinc finger protein domain (ZF protein domain) and an effector domain. In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative) repressor (TetR) domain. In some embodiments, the ACP is an antigen-recognition receptor of the present disclosure.

[0351] Zinc finger protein domains In some embodiments, the ZF protein domain is modular in design and consists of a zinc finger array (ZFA). A zinc finger array contains multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA specific for any desired nucleic acid sequence. The ZF motifs can be directly adjacent to each other or separated by a flexible linker sequence. In some embodiments, the ZFA is an array, string, or chain of tandemly arranged ZF motifs. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1, 3, 14, or 15 zinc finger motifs. A ZFA can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 zinc finger motifs.

[0352] A ZF protein domain can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs. A ZF domain can have 1-10, 1-15, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, 4-5, 4-6, 4-7, 4-8, 4-9, 4-10, 5-6, 5-7, 5-8, 5-9, 5-10, or 5-15 ZFAs. In some embodiments, a ZF protein domain comprises 1-10 ZFA(s). In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least 10 ZFAs.

[0353] An exemplary ZF protein domain is shown in the sequence SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGSQKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS (SEQ ID NO: 88).

[0354] ACP effector domain ACPs can also include an effector domain, such as a transcription effector domain. For example, the transcription effector domain can be the effector domain or activation domain of a transcription factor. The transcription factor activation domain, also known as a transactivation domain, functions as a scaffolding domain of proteins such as transcription coregulators that act to activate or repress gene transcription. Any suitable transcription effector domain may be a herpes simplex virus protein 16 (VP16) activation domain; a VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator containing VP64, p65, and Rta activation domains (the tripartite activator is known as a VPR activation domain); a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (known as the p300 HAT core activation domain); a Kruppel-associated box (KRAB) repression domain; a truncated Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain; or a WRPW motif of Hairy-related basic helix-loop-helix repressor protein. (SEQ ID NO: 162) (This motif is known as the WRPW repression domain. ("WRPW" disclosed as SEQ ID NO: 162) ); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1α chromoshadow repression domain, or any combination thereof.

[0355] In some embodiments, the effector domain is a herpes simplex virus protein 16 (VP16) activation domain; a VP64 activation domain, which is an activation domain consisting of four tandem copies of VP16; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains (the tripartite activator is known as a VPR activation domain); a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (known as a p300 HAT core activation domain); a Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain; or a WRPW motif of Hairy-related basic helix-loop-helix repressor protein. (SEQ ID NO: 162) (This motif is known as the WRPW repression domain. ("WRPW" disclosed as SEQ ID NO: 162) a transcription effector domain selected from: a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1α chromoshadow repression domain.

[0356] Exemplary transcription effector domain protein sequences are shown in Table 8. Exemplary transcription effector domain nucleotide sequences are shown in Table 9. [Table 8]

[0357] [Table 9]

[0358] Drug-inducible domain In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide. For example, in some embodiments, the ACP can be induced by tetracycline (or a derivative thereof) and comprises a TetR domain and a VP16 effector domain. In some embodiments, the ACP can be induced by tamoxifen or a metabolite thereof, e.g., 4-hydroxytamoxifen (4-OHT), and comprises an estrogen receptor variant such as ERT2. In some embodiments, the ACP is a small molecule (e.g., drug)-inducible polypeptide that comprises an inhibitory protease and one or more cognate cleavage sites for the inhibitory protease.

[0359] As used herein, the term "inhibitory protease" refers to a protease that can be inactivated by the presence or absence of a specific agent (e.g., that binds to the protease). In some embodiments, the inhibitory protease is active (cleaves the cognate cleavage site) in the absence of the specific agent and is inactive (does not cleave the cognate cleavage site) in the presence of the specific agent. In some embodiments, the specific agent is a protease inhibitor. In some embodiments, the protease inhibitor specifically inhibits a given inhibitory protease of the present disclosure.

[0360] Non-limiting examples of inhibitory proteases include hepatitis C virus proteases (e.g., NS3 and NS2-3); signal peptidases; proprotein convertases of the subtilisin / kexin family (furin, PCI, PC2, PC4, PACE4, PC5, PC); proprotein convertases that cleave at hydrophobic residues (e.g., Leu, Phe, Val, or Met); proprotein convertases that cleave at small amino acid residues such as Ala or Thr; proopiomelanocortin convertase (PCE); chromaffin; These include insulin granule aspartic protease (CGAP); prohormone thiol proteases; carboxypeptidases (e.g., carboxypeptidase E / H, carboxypeptidase D, and carboxypeptidase Z); aminopeptidases (e.g., arginine aminopeptidase, lysine aminopeptidase, aminopeptidase B); prolyl endopeptidase; aminopeptidase N; insulin-degrading enzyme; calpain; high molecular weight proteases; and caspases 1, 2, 3, 4, 5, 6, 7, 8, and 9.Other proteases include aminopeptidase N, puromycin-sensitive aminopeptidase, angiotensin-converting enzyme, pyroglutamyl peptidase II, dipeptidyl peptidase IV, N-arginine dibasic convertase, endopeptidase 24.15, endopeptidase 24.16, amyloid precursor protein secretases α, β, and γ, angiotensin-converting enzyme secretase, TGFα secretase, TFα secretase, FAS ligand secretase, TNF receptor-I and -II secretase, CD30 secretase, KL1 and KL2 secretase, IL6 receptor secretase, CD43 and CD44 secretase, CD16-1 and CD16-11 secretase, L-selectin secretase, folate receptor secretase, and MMPs. 1, 2, 3, 7, 8, 9, 10, 11, 12, 13, 14, and 15; urokinase plasminogen activator; tissue plasminogen activator; plasmin; thrombin; BMP-I (procollagen C-peptidase); ADAM 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, and 11; and granzymes A, B, C, D, E, F, G, and H.For the investigation of proteases, see, for example, VYH Hook, Proteolytic and cellular mechanisms in prohormone and proprotein processing, RG Landes Company, Austin, Texas, USA (1998), NM Hooper et al., Biochem. J. 321:265-279 (1997), Z. Werb, Cell 91:439-442 (1997); TG Wolfsberg et al., J. Cell Biol. 131:275-278 (1995), K. Murakami and JD Etlinger, Biochem. Biophys. Res. Comm. 146:1249-1259 (1987), T. Berg et al., Biochem. J. 307:313-326 (1995), MJ Myth and JA Trapani, Immunology Today 16:202-206 (1995); RV Talanian et al., J. Biol. Chem. 272:9677-9682 (1997), and NA Thomberry et al., J. Biol. Chem. 272:17907-17911 (1997), the disclosures of which are incorporated herein by reference.

[0361] As used herein, the term "cognate cleavage site" refers to the specific sequence or sequence motif recognized and cleaved by an inhibitory protease. A protease cleavage site comprises the specific amino acid sequence or motif recognized by the protease during proteolytic cleavage, typically comprising 1 to 6 amino acids surrounding either side of the scissile bond that bind to the active site of the protease and are used to recognize it as a substrate.

[0362] Other proteases can be used, including those listed above and in Table 1. When a protease is selected, its cognate cleavage site and a protease inhibitor known in the art to bind to and inhibit that protease can be used in combination. Exemplary combinations for use are shown in Table 1 below. Representative sequences of proteases are available from public databases, including UniProt, through the uniprot.org website. The UniProt accession numbers of the proteases are also shown in Table 1 below.

[0363] [Table 1] TIFF0007792335000006.tif185165TIFF0007792335000007.tif231165TIFF0007792335000008.tif221165TIFF000 7792335000009.tif255165TIFF0007792335000010.tif208165TIFF0007792335000011.tif195165TIFF00077923350 00012.tif205165TIFF0007792335000013.tif212165TIFF0007792335000014.tif179165TIFF0007792335000015.t if232165TIFF0007792335000016.tif203165TIFF0007792335000017.tif205165TIFF0007792335000018.tif169165

[0364] In some embodiments, one or more cognate cleavage sites of the inhibitory protease are located between the DNA-binding domain and the effector domain of the ACP. In some embodiments, the inhibitory protease is hepatitis C virus (HCV) nonstructural protein 3 (NS3). In some embodiments, the cognate cleavage site comprises an NS3 protease cleavage site. In some embodiments, the NS3 protease cleavage site comprises an NS3 / NS4A, NS4A / NS4B, NS4B / NS5A, or NS5A / NS5B junction cleavage site.

[0365] In some embodiments, the NS3 protease can be inhibited by a protease inhibitor. Any suitable protease inhibitor can be used, including, but not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir, or any combination thereof. In some embodiments, the protease inhibitor is selected from simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir. In some embodiments, the protease inhibitor is grazoprevir. In some embodiments, the protease inhibitor is a combination of grazoprevir and elbasvir (an NS5A inhibitor of the hepatitis C virus NS5A replication complex). Grazoprevir and elbasvir can be co-formulated as a pharmaceutical composition, such as in tablet form (e.g., tablets available under the trade name Zepatier®). Grazoprevir and elbasvir can be co-formulated in a 2:1 weight ratio, e.g., in a unit dose of 100 mg grazoprevir and 50 mg elbasvir, respectively (e.g., tablets available under the trade name Zepatier®). For example, any protease inhibitor structurally similar to grazoprevir having the following general formula (I) can be used: TIFF0007792335000019.tif72128In formula, TIFF0007792335000020.tif18128 is one or more rings selected from the group consisting of: TIFF0007792335000021.tif139128R 1 -CO2R 10 and -CONR 10 SO2R 6 R 2 is -CH=CH2; R 3 is C1-C6 alkyl; R 6 is a C3 cycloalkyl; Y is selected from the group consisting of -OC(O)-; Z is a direct bond; M is C1-C 12 Alkylene and C2-C 12 alkenylene, wherein M is substituted with 1 to 2 substituents F independently selected from the group consisting of C1-C8 alkyl and =CH2; X is -(CH2) 0-3 O-, and when present, TIFF0007792335000022.tif4128 is -(CH2) 0-3 are connected to each R 10 is independently H. Grazoprevir, elbasvir, and combinations thereof are described in U.S. Patent Nos. 9,738,661; 7,973,040; and 8,871,759 and U.S. Patent Publication No. US20160243128, each of which is incorporated by reference herein for all purposes.

[0366] In some embodiments, the ACP of the present disclosure comprises a small molecule (e.g., drug)-inducible hormone-binding domain (ERT2 domain) of the estrogen receptor. In some embodiments, the ERT2 domain is an estrogen receptor variant that binds to tamoxifen and its metabolites but not estradiol. Non-limiting examples of tamoxifen metabolites may include 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. In some embodiments, when expressed in a cell and in the absence of a small molecule (e.g., tamoxifen or its metabolite), the ACP comprising the ERT2 domain binds to HSP90 and is retained in the cytoplasm of the cell. In some embodiments, upon introduction of a small molecule (e.g., tamoxifen or its metabolite), the small molecule displaces HSP90 bound to the ERT2 domain, thereby allowing the ACP comprising the ERT2 domain to translocate to the nucleus of the cell.

[0367] Thus, in some embodiments, an ACP of the present disclosure that includes an ERT2 domain can undergo nuclear localization upon binding of the ERT2 domain to tamoxifen or a metabolite thereof, in some embodiments, the tamoxifen metabolite is selected from 4-hydroxy-tamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0368] Degradation sequences and degrons In some embodiments, the ACP further comprises a degron, wherein the degron is operably linked to the ACP, hi some embodiments, the degron is located 5' of the inhibitory protease, 3' of the inhibitory protease, 5' of the DNA-binding domain, 3' of the DNA-binding domain, 5' of the effector domain, or 3' of the effector domain.

[0369] As used herein, the terms "degron" and "degron domain" refer to proteins or portions thereof that are important in regulating the rate of protein degradation. Various degrons known in the art can be used in various embodiments of the present disclosure, including, but not limited to, short amino acid sequences, structural motifs, and exposed amino acids. Degrons identified from various organisms can be used. Degrons and degron pathways are generally known; see, for example, Varshazsky A., PNAS 2019 Jan 8;116(2):358-366 (incorporated herein by reference).

[0370] As used herein, the term "degradation sequence" refers to a sequence that promotes degradation of an associated protein through either the proteasome or the autophagy-lysosomal pathway. Degradation sequences known in the art can be used in various embodiments of the present disclosure. In some embodiments, the degradation sequence comprises a degron identified from an organism, or a variant thereof. In some embodiments, the degradation sequence is a polypeptide that, when fused to a protein, destabilizes the protein such that the half-life of the protein is reduced by at least two-fold. Many different degradation sequences / signals (e.g., of the ubiquitin-proteasome system) are known in the art, and any of these may be used as provided herein. The degradation sequence may be operably linked to a cellular receptor, but need not be adjacent to it, as long as the degradation sequence still functions to induce degradation of the cellular receptor. In some embodiments, the degradation sequence induces rapid degradation of the cellular receptor. For a discussion of degradation sequences and their functions in protein degradation, see, for example, Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425, Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221, Schrader et al. (2009) Nat. Chem. Biol. 5(11):815-822, Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690, Tasaki et al. (2007) Trends Biochem Sci. 32(11):520-528, Meinnel et al. (2006) Biol. Chem. 387(7):839-851, and Kim et al. (2013) Autophagy 9(7):1100-1103, Varshavsky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3):1268-1278; (incorporated herein by reference).

[0371] In some embodiments, the degron or degradation sequence is selected from the group consisting of HCV NS4 degron, PEST (two copies of residues 277-307 of human IκBα), GRR (residues 352-408 of human p105), DRR (residues 210-295 of yeast Cdc34), SNS (tandem repeats of SP2 and NB of influenza A or influenza B (SP2-NB-SP2), RPB (four copies of residues 1688-1702 of yeast RPB), SPmix (influenza A virus M2 protein), and the like. The SP1 and SP2 tandem repeats of the mutant (SP2-SP1-SP2-SP1-SP2), NS2 (three copies of residues 79-93 of influenza A virus NS protein), ODC (residues 106-142 of ornithine decarboxylase), Nek2A, mouse ODC (residues 422-461), mouse ODC_DA (residues 422-461 of mODC containing D433A and D434A point mutations), APC / C degron, and COP1 E3 ligase-binding degron motif, CRL4-Cdt2-binding PIP degron, actinfilin-binding degron, KEAP1-binding degron, KLHL2- and KLHL3-binding degron, MDM2-binding motif, N-degron, hydroxyproline modification in hypoxia signaling, plant hormone-dependent SCF-LRR-binding degron, SCF ubiquitin ligase-binding phosphodegron, plant hormone-dependent SCF-LRR-binding degron, DSGxxS phospho-dependent degron (SEQ ID NO: 163) , Siah-binding motif, SPOP SBC docking motif, as well as PCNA-binding PIP box.

[0372] In some embodiments, the degron comprises a cereblon (CRBN) polypeptide substrate domain that can bind to CRBN in response to an immunomodulatory drug (IMiD), thereby promoting degradation of ACP via the ubiquitin pathway. In some embodiments, the CRBN polypeptide substrate domain is selected from IKZF1, IKZF3, CKla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or fragments thereof, that are capable of drug-inducible binding of CRBN. In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a native CRBN polypeptide sequence. In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 131).

[0373] In some embodiments, the immunomodulatory drug (IMiD) is an FDA-approved drug. In some embodiments, the IMiD is selected from thalidomide, lenalidomide, and pomalidomide.

[0374] promoter In some embodiments, an engineered nucleic acid of the present disclosure comprises a first expression cassette comprising a first promoter operably linked to an exogenous polynucleotide sequence encoding an activating conditional regulatory polypeptide (ACP). In some embodiments, an engineered nucleic acid of the present disclosure comprises a second expression cassette comprising an ACP-responsive promoter operably linked to a second exogenous polynucleotide sequence encoding one or more effector molecules. In some embodiments, the first expression cassette and the second expression cassette are each encoded by separate engineered nucleic acids of the present disclosure. In other embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid of the present disclosure.

[0375] In some embodiments, the ACP responsive promoter of the present disclosure comprises an ACP binding domain and a promoter sequence, hi some embodiments, the ACP responsive promoter is operably linked to a nucleotide sequence encoding an effector molecule.

[0376] In some embodiments, the engineered nucleic acid comprises an ACP-responsive promoter operably linked to a nucleotide sequence encoding an effector molecule. In some embodiments, the engineered nucleic acid comprises an ACP-responsive promoter operably linked to a nucleotide sequence encoding at least two effector molecules. For example, the engineered nucleic acid may comprise an ACP-responsive promoter operably linked to a nucleotide sequence encoding at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 effector molecules. In some embodiments, the engineered nucleic acid comprises a promoter operably linked to a nucleotide sequence encoding one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more effector molecules.

[0377] A "promoter" refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of transcription of the remainder of the nucleic acid sequence. A promoter may also contain subregions to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of a nucleic acid sequence that it controls. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation with respect to the nucleic acid sequence that it regulates to control ("drive") transcription initiation and / or expression of that sequence.

[0378] A promoter can be one naturally associated with a gene or sequence, as can be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter can be referred to as "endogenous." In some embodiments, a coding nucleic acid sequence can be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coding sequence in its natural environment. Such promoters can include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not "naturally occurring," such as those containing different elements and / or mutations in different transcriptional regulatory regions that manipulate expression through genetic engineering methods known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences can be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0379] The promoter of an engineered nucleic acid of the present disclosure may be an "inducible promoter," which refers to a promoter that is characterized by modulating transcriptional activity (e.g., initiating or activating) when in the presence of, affected by, or contacted with a signal. The signal may be an endogenous or usually exogenous condition (e.g., light), compound (e.g., chemical or non-chemical), or protein (e.g., cytokine) that contacts the inducible promoter in such a way that it is active in modulating transcriptional activity from the inducible promoter. Activating transcription may involve acting directly on the promoter to drive transcription, or acting indirectly on the promoter by inactivating a repressor that prevents the promoter from driving transcription. Conversely, deactivating transcription may involve acting directly on the promoter to prevent transcription, or acting indirectly on the promoter by activating a repressor that in turn acts on the promoter.

[0380] A promoter is "responsive" or "regulated" to a local tumor condition (e.g., inflammation or hypoxia) or signal if transcription from the promoter is activated, inactivated, increased, or decreased in the presence of that condition or signal. In some embodiments, a promoter contains a response element. A "response element" is a short sequence of DNA within the promoter region that binds to specific molecules (e.g., transcription factors) that regulate (control) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, but are not limited to, phloretin regulatable control element (PEACE), zinc finger DNA binding domain (DBD), interferon gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar;17(3):121-34, incorporated herein by reference), interferon stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 Apr9;279(15):15652-61, incorporated herein by reference), NF-κB response element (Wang, V. et al. Cell Reports. 2012;2(4):824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al. J of Biol Chem. 1996;271:9503-9509, incorporated herein by reference). Other response elements are encompassed herein. Response elements can also contain tandem repeats (e.g., consecutive repetitions of the same nucleotide sequence encoding the response element) to generally enhance the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be designated 2x, 3x, 4x, 5x, etc. to indicate the number of repeats present.

[0381] Non-limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF-β responsive promoters) are shown in Table 2, which shows the promoter and transcription factor design and the effect of the inducer molecule on transcription factor (TF) and transgene transcription (T) (B: ​​binding, D: dissociation, nd: undetermined) (A: activation, DA: deactivation, DR: derepression) (Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m and references cited therein). Other non-limiting examples of inducible promoters include those shown in Table 3.

[0382] [Table 2] TIFF0007792335000024.tif228165TIFF0007792335000025.tif99165

[0383] [Table 3] TIFF0007792335000027.tif104165

[0384] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1 alpha (EF1a) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter comprising the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter. In some embodiments, the promoter is a constitutive promoter. Exemplary constitutive promoters are listed in Table 4.

[0385] [Table 4] TIFF0007792335000029.tif223165TIFF0007792335000030.tif185165TIFF0007792335000031.tif139165TIFF0007792335000032.tif221165 TIFF0007792335000033.tif229165TIFF0007792335000034.tif192165TIFF0007792335000035.tif202165TIFF0007792335000036.tif179165

[0386] In some embodiments, the promoter sequence is derived from a promoter selected from minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoter, and tandem repeats thereof.

[0387] In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, the constitutive promoter is selected from CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0388] In some embodiments, the ACP responsive promoter is a synthetic promoter. In some embodiments, the ACP responsive promoter comprises a minimal promoter. In some embodiments, the ACP binding domain comprises one or more zinc finger binding sites. The ACP binding domain can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc finger binding sites. In some embodiments, the ACP binding domain comprises one zinc finger binding site. In some embodiments, the ACP binding domain comprises two zinc finger binding sites. In some embodiments, the ACP binding domain comprises three zinc finger binding sites. In some embodiments, the ACP binding domain comprises four zinc finger binding sites. An exemplary ACP binding domain containing a zinc finger binding site is set forth in the sequence cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 92).

[0389] In some embodiments, the ACP-responsive promoter comprises an enhancer that promotes transcription when an antigen-recognizing receptor binds to a cognate antigen, for example, an antigen expressed on a target cell. Enhancers include, but are not limited to, enhancers enriched in ATAC-seq of activated T cells (Gate et al. Nat Genet. Author manuscript; available in PMC 2019 Jan 9, incorporated herein by reference for all purposes) or enhancers associated with up-regulated genes in single-cell RNA-seq data (Xhangolli et al. Genomics Proteomics Bioinformatics. 2019 Apr;17(2):129-139. doi: 10.1016 / j.gpb.2019.03.002; incorporated herein by reference for all purposes). Enhancers can be synthetic enhancers, for example, pairs of transcription factors known or predicted to be up-regulated in activated T cells or NK cells. Synthetic enhancers may contain multiple repeats of transcription factor binding sites, for example, four repeats of two different transcription factor binding sites in the aaaabbbb or abababab composition. Illustrative, non-limiting examples of genes in which enhancers can be induced include, but are not limited to, ATF2, ATF7, BACH1, BATF, Bcl-6, Blimp-1, BMI1, CBFB, CREB1, CREM, CTCF, E2F1, EBF1, EGR1, ETV6, FOS, FOXA1, FOXA2, GATA3, HIF1A, IKZF1, IKZF2, IRF4, JUN, JUNB, JUND, Lef1, NFAT, NFIA, NFIB, NFKB, NR2F1, Nur77, PU.1, RELA, RUNX3, SCRT1, SCRT2, SP1, STAT4, STAT5A, T-Bet, Tcf7, ZBED1, ZNF143, or ZNF217.

[0390] In some embodiments, the ACP-responsive promoter includes a promoter that stimulates transcription when a receptor binds its cognate ligand, such as in an activation-induced system. In some embodiments, the ACP-responsive promoter includes a promoter that stimulates transcription when an antigen-recognizing receptor binds its cognate antigen, e.g., an antigen expressed on a target cell. For example, if the ACP is an antigen receptor (e.g., a CAR), the ACP-responsive promoter can include a promoter that is induced by signaling following the antigen receptor binding to its cognate antigen. The ACP-responsive promoter includes a promoter that has increased transcriptional activity in activated T cells and / or NK cells. The ACP-responsive promoter can include a promoter derived from a gene that is upregulated in activated cells such as T cells and / or NK cells. The ACP-responsive promoter can include a promoter derived from a gene that exhibits increased transcription factor binding in activated cells such as T cells and / or NK cells. The induced promoter can include a genomic region 2 kb upstream of the gene. The induced promoter can include a genomic region −100 bp downstream of the transcription start site of the gene. The induced promoter may include a genomic region 2 kbp upstream of the gene to -100 bp downstream of the transcription start site of the gene. The induced promoter may include a genomic region upstream of the translation start site of the gene. The induced promoter may include a genomic region 2 kb upstream of the translation start site of the gene. The induced promoter may include one or more enhancers identified in the promoter region. The ACP-responsive promoter may include, but is not limited to, promoters from the CCL3, CCL4, or MTA2 genes. The ACP-responsive promoter may include, but is not limited to, the CCL3 promoter region (e.g., SEQ ID NO: 156), the CCL4 promoter region (e.g., SEQ ID NO: 157), and / or the MTA2 promoter region (e.g., SEQ ID NO: 158).The ACP-responsive promoter may include an enhancer present in the CCL3 promoter region (e.g., SEQ ID NO: 156), the CCL4 promoter region (e.g., SEQ ID NO: 157), and / or the MTA2 promoter region (e.g., SEQ ID NO: 158). The ACP-responsive promoter may include a synthetic promoter. For example, the ACP-responsive promoter may include an antigen-inducible enhancer or promoter sequence combined with another promoter, such as a minimal promoter (e.g., minimal AdeP or YB-TATA). The ACP-responsive promoter may include a synthetic enhancer, such as a promoter containing multiple repeats of a transcription factor binding site. In an illustrative, non-limiting example, an ACP-responsive promoter, including a synthetic promoter, may include five repeats of the NFAT transcription factor binding site in combination with a minimal Ade promoter (5×NFAT_inAdeP).

[0391] Multicistronic and multi-promoter systems In some embodiments, the engineered nucleic acid is configured to produce multiple effector molecules, for example, the nucleic acid may be configured to produce between 2 and 20 different effector molecules.In some embodiments, the nucleic acid is selected from the group consisting of 2 to 20, 2 to 19, 2 to 18, 2 to 17, 2 to 16, 2 to 15, 2 to 14, 2 to 13, 2 to 12, 2 to 11, 2 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 20, 3 to 19, 3 to 18, 3 to 17, 3 to 16, 3 to 15, 3 to 14, 3 to 13, 3 to 12, 3 to 11, 3 to 10, 3 to 9, 3 to 8, 3 to 7, 3 to 6, 3 to 5, 3 to 4, 4 to 20, 4 to 19, 4 to 18, 4 to 17, 4 to 16, 4 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 1 10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8- 19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-1 Configured to produce 9, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 effector molecules.In some embodiments, the nucleic acid is configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 effector molecules.

[0392] In some embodiments, the engineered nucleic acid can be multicistronic, i.e., multiple distinct polypeptides (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single mRNA transcript. The engineered nucleic acid can be multicistronic by using various linkers, for example, a polynucleotide sequence encoding a first exogenous polynucleotide or effector molecule can be linked to a nucleotide sequence encoding a second exogenous polynucleotide or effector molecule (e.g., in the 5' to 3' direction: first gene: linker: second gene). The linker polynucleotide sequence can encode a 2A ribosomal skipping element, such as T2A. Other 2A ribosomal skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosomal skipping element allows for the production of distinct polypeptides encoded by the first and second genes during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, and following expression, the cleavable linker polypeptide is cleaved to produce the separate polypeptides encoded by the first and second genes. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., a Gly-Ser-Gly sequence), that further facilitates cleavage.

[0393] In some embodiments, the second expression cassette is (L1-E) X When the L1 linker polynucleotide sequence comprises two or more units of each effector molecule, each L1 linker polynucleotide sequence is operably linked to the translation of each effector molecule as a separate polypeptide.

[0394] The linker can encode an internal ribosome entry site (IRES) that, during translation, produces distinct polypeptides encoded by the first and second genes. The linker can encode a splice acceptor site, such as a viral splice acceptor site.

[0395] The linker can be a combination of linkers, such as a furin-2A linker, which can produce a separate polypeptide through further cleavage of the furin site to allow 2A ribosomal skipping followed by complete removal of the 2A residue. In some embodiments, the linker combination can include a furin sequence, a flexible linker, and a 2A linker. Thus, in some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0396] In general, a multicistronic system can express any number of genes or portions thereof using any number or combination of linkers (e.g., an engineered nucleic acid can encode first, second, and third effector molecules, each separated by a linker, such that distinct polypeptides encoded by the first, second, and third effector molecules are produced).

[0397] As used herein, a "linker" may refer to a polypeptide that connects a first polypeptide sequence and a second polypeptide sequence, or to a multicistronic linker as described above.

[0398] Effector molecules Any suitable effector molecule known in the art can be encoded by the engineered nucleic acid or expressed by the engineered cell. Suitable effector molecules can be grouped into therapeutic classes based on structural similarity, sequence similarity, or function. Therapeutic classes of effector molecules include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0399] In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0400] In some embodiments, the effector molecule is a chemokine. Chemokines are small cytokines or signaling proteins secreted by cells that can induce directional chemotaxis within the cell. Chemokines can be classified into four major subfamilies: CXC, CC, CX3C, and XC, all of which exert their biological effects by selectively binding to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that can be encoded by the engineered nucleic acids of the present disclosure include CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1, or any combination thereof. In some embodiments, the chemokine is selected from CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0401] In some embodiments, the effector molecule is a cytokine. Non-limiting examples of cytokines that can be encoded by the engineered nucleic acids of the present disclosure include IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, TNF-α, or any combination thereof. In some embodiments, the cytokine is selected from IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α.

[0402] In some embodiments, the engineered nucleic acid is configured to produce at least one homing molecule. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., a tumor), or organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, the homing molecule functions to recognize and / or initiate interaction of the engineered cell with the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, PDGFR, anti-integrin α4β7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15, or any combination thereof. In some embodiments, the homing molecule is selected from anti-integrin α4β7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

[0403] In some embodiments, the engineered nucleic acid is configured to produce at least one growth factor. Growth factors suitable for use as effector molecules include, but are not limited to, FLT3L and GM-CSF, or any combination thereof. In some embodiments, the growth factor is selected from FLT3L and GM-CSF.

[0404] In some embodiments, the engineered nucleic acid is configured to produce at least one co-activator molecule. Co-activator molecules suitable for use as effector molecules include, but are not limited to, c-Jun, 4-1BBL, and CD40L, or any combination thereof. In some embodiments, the co-activator molecule is selected from c-Jun, 4-1BBL, and CD40L.

[0405] The "tumor microenvironment" is the cellular milieu in which a tumor resides, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. & Weinberg, R.Nature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F.R. et al. J. Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J. Cell Biochem 101(4), 805-15, 2007). Tumor microenvironment modifiers suitable for use as effector molecules include, but are not limited to, adenosine deaminase, TGFβ inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof. In some embodiments, the tumor microenvironment modifier is selected from adenosine deaminase, TGFβ inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

[0406] In some embodiments, the engineered nucleic acid is configured to produce at least one TGFβ inhibitor. TGFβ inhibitors suitable for use as effector molecules include, but are not limited to, anti-TGFβ peptides, anti-TGFβ antibodies, TGFβ-TRAP, or combinations thereof. In some embodiments, the TGFβ inhibitor is selected from anti-TGFβ peptides, anti-TGFβ antibodies, TGFβ-TRAP, and combinations thereof.

[0407] In some embodiments, the engineered nucleic acid is configured to produce at least one immune checkpoint inhibitor. Immune checkpoint inhibitors suitable for use as effector molecules include, but are not limited to, an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREMI antibody, and an anti-TREM2 antibody, or any combination thereof. In some embodiments, the immune checkpoint inhibitor is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREMI antibody, and an anti-TREM2 antibody.

[0408] Exemplary immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda® - Merck), nivolumab (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio® - Pfizer), durvalumab (anti-PD-L1; MEDI4736 / Im finzi® - Mediimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq® - Roche / Genentech), BMS-936559 (anti-PD-L1-BMS), tremelimumab (anti-CTLA-4; Mediimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy® - BMS), lirilumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0409] In some embodiments, the engineered nucleic acid is configured to produce at least one VEGF inhibitor. VEGF inhibitors suitable for use as effector molecules include, but are not limited to, anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof. In some embodiments, the VEGF inhibitor comprises anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

[0410] In some embodiments, each effector molecule is a human-derived effector molecule.

[0411] secretion signal Generally, one or more effector molecules contain a secretory signal peptide (also referred to as a signal peptide or signal sequence) at the N-terminus of the effector molecule to direct the newly synthesized protein into the appropriate protein processing pathway for secretion or membrane insertion. In embodiments having two or more effector molecules, each effector molecule can contain a secretory signal (S). In embodiments having two or more effector molecules, each effector molecule can contain a secretory signal (LE) so that each effector molecule is secreted from the engineered cell. In embodiments, X The second expression cassette, comprising one or more units of the formula: (LSE), further comprises a polynucleotide sequence encoding a secretory signal peptide (S). In embodiments, for each X, the corresponding secretory signal peptide is operably associated with an effector molecule. In embodiments, the second expression cassette comprises an ACP-responsive promoter and a polynucleotide sequence encoding a signal peptide of the formula: (LSE) X and a second exogenous polynucleotide sequence having:

[0412] The secretory signal peptide operably associated with the effector molecule can be a native secretory signal peptide, a naturally occurring secretory signal peptide (e.g., a secretory signal peptide typically endogenously associated with a given effector molecule). The secretory signal peptide operably associated with the effector molecule can be a non-native secretory signal peptide, a naturally occurring secretory signal peptide. A non-native secretory signal peptide can facilitate improved expression and function, such as maintained secretion, in certain environments, such as the tumor microenvironment. Non-limiting examples of non-native secretory signal peptides are shown in Table 5.

[0413] [Table 5] TIFF0007792335000038.tif231165TIFF0007792335000039.tif202165

[0414] antigen-recognition receptor Certain aspects of the present disclosure relate to engineered nucleic acids comprising an antigen-recognizing receptor. In some embodiments, the engineered nucleic acids of the present disclosure comprise a first expression cassette further comprising an antigen-recognizing receptor. In some embodiments, the first expression cassette comprises a first exogenous polynucleotide sequence encoding an ACP and a polynucleotide sequence encoding the antigen-recognizing receptor operably linked to a first promoter. Antigen recognition receptors suitable for use as effector molecules include 5T4, ADAM9, ADGRE2, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, cadherin 3, cadherin 6, CCR1, CCR4, CD117, CD123, CD131, CD133, CD138, CD142, CD166, CD25, CD244, CD30, CD300LF, CD33, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CD93, CEA, CEACAM5, claudin 18.2, CLEC12A, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR, EM B, ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FR alpha, FRb, FLT3, GAPT, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-1RAP, IL-13R, IL-13Ra, IL-13Ra2, IL-8, IL-15, IL 1RAP, integrin aV, KIT, L1CAM, LAMP1, LAT2, Lewis Y, LeY, LILRA2, LILRB2, LIV-1, LRRC, LY6E, MCSP, mesothelin (MSLN), MLC1, MS4A3, MUC1, MUC16, MUC1C, MYADM, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY The antibody recognizes antigens including, but not limited to, ESO1, Ovarin, P-cadherin, pan-Erb2, PIEZO1, PRAM1, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLC22A16, SLC17A9, SLITRK6, SPNS3, SSTR2, STEAP1, survivin, TDGF1, TIM1, TROP2, VSTM1, and WT1, or any combination thereof.

[0415] In some embodiments, the antigen recognition receptor is 5T4, ADAM9, AFP, AXL, B7-H3, B7-H4, B7-H6, C4.4, CA6, cadherin 3, cadherin 6, CCR4, CD123, CD133, CD138, CD142, CD166, CD25, CD30, CD352, CD37, CD38, CD44, CD56, CD66e, CD70, CD71, CD74, CD79b, CD80, CEA, CEACAM5, claudin 18.2, cMet, CSPG4, CTLA, DLK1, DLL3, DR5, EGFR , ENPP3, EpCAM, EphA2, EphrinA4, ETBR, FGFR2, FGFR3, FRα, FRb, GCC, GD2, GFRa4, gpA33, GPC3, gpNBM, GPRC5, HER2, IL-13R, IL-13Ra, IL-13Ra2, IL- 8, IL-15, IL1RAP, Integrin aV, KIT, L1CAM, LAMP1, Lewis Y, LeY, LIV-1, LRRC, LY6E, MCSP, Mesothelin, MUC1, MUC16, MUC1C, NaPi2B, Nectin 4, NKG2D, NOTCH3, NY It recognizes an antigen selected from ESO1, Ovarin, P-cadherin, pan-Erb2, PSCA, PSMA, PTK7, ROR1, S Aures, SCT, SLAMF7, SLITRK6, SSTR2, STEAP1, survivin, TDGF1, TIM1, TROP2, and WT1.

[0416] In some embodiments, the antigen-recognizing receptor recognizes GPC3. The antigen-recognizing receptor that recognizes GPC3 may comprise an anti-binding domain that binds to GPC3. In some embodiments, the antigen-binding domain that binds to GPC3 comprises a heavy chain variable (VH) region and a light chain variable (VL) region, wherein the VH comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119), a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 120), and a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121), and the VL comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 122), a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123), and a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 124). In some embodiments, an antigen-binding domain that binds GPC3 comprises a heavy chain complementarity-determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119). In some embodiments, an antigen-binding domain that binds GPC3 comprises a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence of RIRNKTNNYATYYADSVKA (SEQ ID NO: 120). In some embodiments, an antigen-binding domain that binds GPC3 comprises a heavy chain complementarity-determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121). In some embodiments, an antigen-binding domain that binds GPC3 comprises a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence of KSSQSLLYSSNQKNYLA (SEQ ID NO: 122). In some embodiments, an antigen-binding domain that binds GPC3 comprises a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123). In some embodiments, the antigen-binding domain that binds to GPC3 comprises a light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence of QQYYNYPLT (SEQ ID NO: 124).

[0417] In some embodiments, an antigen-binding domain that binds to GPC3 comprises a VH region having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of EVQLVETGGGMVQPEGSLKLSCAASGFTFNKNAMNWVRQAPGKGLEWVARIRNKTNNYATYYADSVKARFTISRDDSQSMLYLQMNNLKIEDTAMYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 125) or EVQLVESGGGLVQPGGSLRLSCAASGFTFNKNAMNWVRQAPGKGLEWVGRIRNKTNNYATYYADSVKARFTISRDDSKNSLYLQMNSLKTEDTAVYYCVAGNSFAYWGQGTLVTVSA (SEQ ID NO: 126).

[0418] In some embodiments, the antigen-binding domain that binds to GPC3 is DIVMSQSPSSLVVSIGEKVTMTCKSSQSLLYSSNQKNYLAWYQQKPGQSPKLLIYWASSRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQYYNYPLTFGAGTKLELK (SEQ ID NO: 127), or DIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASSRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYNYPLTFGQGTKLEIK (SEQ ID NO: 128) The VL region has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of

[0419] In some embodiments, the antigen-recognizing receptor recognizes MSLN. The antigen-recognizing receptor that recognizes MSLN may comprise an anti-binding domain that binds to MSLN. In some embodiments, the antigen-binding domain that binds to MSLN is QVQLVESGGGTVQAGGSLKLACAASGLPRTYNVMGWFRQAPGKEREGVAIIYTTTGATYYRDSVKGRATISQDNAKKSVSLQMNSLRPEDTAIYYCVARQPNSGPWEYWGQGTQVTVSS (SEQ ID NO: 129), or QVKLEESGGGSVQAGGSLRLSCTTSGYTNSYKWMGWFRQAPGQEREGVAVIYTGNDRTYYSDSVKGRFTISRDNAKNMIYLDMTRLRPEDSAVYECAIGHDGAWRYWGQGTQVTVSS (SEQ ID NO: 130) In some embodiments, the antigen-binding domain that binds to MSLN comprises a single domain binding domain having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence of QVQLVESGGGTVQAGGSLKLACAASGLPRTYNVMGWFRQAPGKEREGVAIIYTTTGATYYRDSVKGRATISQDNAKKSVSLQMNSLRPEDTAIYYCVARQPNSGPWEYWGQGTQVTVSS (SEQ ID NO: 129), or QVKLEESGGGSVQAGGSLRLSCTTSGYTNSYKWMGWFRQAPGQEREGVAVIYTGNDRTYYSDSVKGRFTISRDNAKNMIYLDMTRLRPEDSAVYECAIGHDGAWRYWGQGTQVTVSS (SEQ ID NO: 130) In some embodiments, the antigen-binding domain that binds to MSLN comprises each of the CDR sequences from a single-domain binding domain having the amino acid sequence QVQLVESGGGTVQAGGSLKLACAASGLPRTYNVMGWFRQAPGKEREGVAIIYTTTGATYYRDSVKGRATISQDNAKKSVSLQMNSLRPEDTAIYYCVARQPNSGPWEYWGQGTQVTVSS (SEQ ID NO: 129), or QVKLEESGGGSVQAGGSLRLSCTTSGYTNSYKWMGWFRQAPGQEREGVAVIYTGNDRTYYSDSVKGRFTISRDNAKNMIYLDMTRLRPEDSAVYECAIGHDGAWRYWGQGTQVTVSS (SEQ ID NO: 130) The single domain comprises one or more CDR sequences derived from a binding domain having the following structure:

[0420] In some embodiments, the first expression cassette further comprises a linker polynucleotide sequence located between the ACP and the antigen-recognizing receptor.

[0421] In some embodiments, the antigen-recognizing receptor comprises an antigen-binding domain. In some embodiments, the antigen-binding domain comprises an antibody, an antigen-binding fragment of an antibody, an F(ab) fragment, an F(ab') fragment, a single-chain variable fragment (scFv), or a single-domain antibody (sdAb). In some embodiments, the antigen-binding domain comprises a single-chain variable fragment (scFv). In some embodiments, the scFv comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). In some embodiments, the VH and VL are separated by a peptide linker.

[0422] An scFv has a variable domain of a light chain (VL) connected from its C-terminus to the N-terminus of the variable domain of a heavy chain (VH) by a polypeptide chain. Alternatively, an scFv consists of polypeptide chains, with the C-terminus of the VH connected to the N-terminus of the VL by a polypeptide chain. In some embodiments, an scFv comprises the structure VH-L-VL or VL-L-VH, where VH is a heavy chain variable domain, L is a peptide linker, and VL is a light chain variable domain.

[0423] An sdAb is a molecule in which one variable domain of an antibody specifically binds to an antigen in the absence of other variable domains.

[0424] The F(ab) fragment contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), together with the variable domains of the light and heavy chains, VL and VH, respectively. The F(ab)' fragment differs from the Fab fragment in that it has a few additional residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. The F(ab')2 fragment contains two Fab' fragments linked by disulfide bonds near the hinge region.

[0425] In some embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the antigen-recognizing receptor is a CAR. In some embodiments, the CAR comprises one or more intracellular signaling domains, wherein the one or more intracellular signaling domains are selected from a CD3 zeta chain intracellular signaling domain, a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, and a MyD88 intracellular signaling domain.In some embodiments, the CAR comprises a CD3 zeta chain intracellular signaling domain, and one or more additional intracellular signaling domains (e.g., costimulatory domains) selected from a CD97 intracellular signaling domain, a CD11a-CD18 intracellular signaling domain, a CD2 intracellular signaling domain, an ICOS intracellular signaling domain, a CD27 intracellular signaling domain, a CD154 intracellular signaling domain, a CD8 intracellular signaling domain, an OX40 intracellular signaling domain, a 4-1BB intracellular signaling domain, a CD28 intracellular signaling domain, a ZAP40 intracellular signaling domain, a CD30 intracellular signaling domain, a GITR intracellular signaling domain, an HVEM intracellular signaling domain, a DAP10 intracellular signaling domain, a DAP12 intracellular signaling domain, a MyD88 intracellular signaling domain, a 2B4 intracellular signaling domain, a CD16a intracellular signaling domain, a DNAM-1 intracellular signaling domain, a KIR2DS1 intracellular signaling domain, a KIR3DS1 intracellular signaling domain, an NKp44 intracellular signaling domain, an NKp46 intracellular signaling domain, an FceRlg intracellular signaling domain, an NKG2D intracellular signaling domain, and an EAT-2 intracellular signaling domain.

[0426] In some embodiments, the CAR further comprises a transmembrane domain, wherein the transmembrane domain is selected from a CD8 transmembrane domain, a CD28 transmembrane domain, a CD3 zeta chain transmembrane domain, a CD4 transmembrane domain, a 4-1BB transmembrane domain, an OX40 transmembrane domain, an ICOS transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, a LAG-3 transmembrane domain, a 2B4 transmembrane domain, a BTLA transmembrane domain, an OX40 transmembrane domain, a DAP10 transmembrane domain, a DAP12 transmembrane domain, a CD16a transmembrane domain, a DNAM-1 transmembrane domain, a KIR2DS1 transmembrane domain, a KIR3DS1 transmembrane domain, an NKp44 transmembrane domain, an NKp46 transmembrane domain, an FceRlg transmembrane domain, and an NKG2D transmembrane domain.

[0427] In some embodiments, the CAR further comprises a spacer region (e.g., a hinge domain) between the antigen-binding domain and the transmembrane domain. A spacer or hinge domain is any oligopeptide or polypeptide that functions to link a transmembrane domain to an extracellular domain and / or an intracellular signaling domain within a polypeptide chain. The spacer or hinge domain provides flexibility to the inhibitory chimeric receptor or tumor-targeting chimeric receptor, or domains thereof, or prevents steric hindrance of the inhibitory chimeric receptor or tumor-targeting chimeric receptor, or domains thereof. In some embodiments, the spacer or hinge domain can comprise up to 300 amino acids (e.g., 10-100 amino acids, or 5-20 amino acids). In some embodiments, one or more spacer domain(s) can be included in other regions of the inhibitory chimeric receptor or tumor-targeting chimeric receptor.

[0428] Exemplary spacer or hinge domains may include, but are not limited to, an IgG domain (such as an IgG1 hinge, an IgG2 hinge, an IgG3 hinge, or an IgG4 hinge), an IgD hinge domain, a CD8α hinge domain, and a CD28 hinge domain. In some embodiments, the spacer or hinge domain is an IgG domain, an IgD domain, a CD8α hinge domain, or a CD28 hinge domain.

[0429] Exemplary spacer or hinge domain protein sequences are shown in Table 6. Exemplary spacer or hinge domain nucleotide sequences are shown in Table 7.

[0430] [Table 6]

[0431] [Table 7] TIFF0007792335000042.tif138165

[0432] Transmembrane domains, spacer or hinge domains, and intracellular domains suitable for use in CARs are generally described in Stoiber et al, Cells 2019, 8(5), 472, Guedan et al, Mol Therapy: Met & Clinic Dev, 2019 12:145-156, and Sadelain et al, Cancer Discov; 2013, 3(4); 388-98, each of which is incorporated herein by reference in its entirety.

[0433] In some embodiments, the CAR further comprises a secretory signal peptide. Any suitable secretory signal peptide of the present disclosure may be used.

[0434] Post-transcriptional regulatory elements In some embodiments, an engineered nucleic acid of the present disclosure comprises a post-transcriptional regulatory element (PRE). A PRE can enhance gene expression by enabling tertiary RNA structure stability and 3' end formation. Non-limiting examples of PREs include Hepatitis B virus PRE (HPRE) and Woodchuck Hepatitis virus PRE (WPRE). In some embodiments, the post-transcriptional regulatory element is a Woodchuck Hepatitis virus Post-transcriptional Regulatory Element (WPRE). In some embodiments, a WPRE comprises the α, β, and γ components of a WPRE element. In some embodiments, a WPRE comprises the α component of a WPRE element.

[0435] Engineered cells Also provided herein are cells comprising one or more engineered nucleic acids of the present disclosure, and methods for producing the cells. These cells are referred to herein as "engineered cells." Generally, these cells comprising one or more engineered nucleic acids do not occur in nature. In some embodiments, the cells are isolated cells that recombinantly express one or more engineered nucleic acids. In some embodiments, the engineered one or more nucleic acids are expressed from one or more vectors or from a selected locus from the genome of the cell. In some embodiments, the cells are engineered to contain a first nucleic acid comprising a promoter operably linked to a nucleotide sequence encoding an activating conditional control polypeptide (ACP), such as a transcription factor, and / or an antigen-recognition receptor. In some embodiments, the transcription factor comprises an inhibitory protease and a cognate cleavage site. In some embodiments, the transcription factor comprises a degron. In some embodiments, the ACP is an antigen-recognition receptor.

[0436] In some embodiments, the engineered cells comprise a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activating conditional regulatory polypeptide (ACP) and / or an antigen-recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide; and an ACP-responsive promoter and a first exogenous polynucleotide sequence having the formula: (LE) X and a second exogenous polynucleotide sequence having the formula: wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X=1 to 20; an ACP-responsive promoter is operably linked to the second exogenous polynucleotide; in the first repeat of the (LE) unit, L is absent; and ACP is capable of inducing expression of the second expression cassette upon binding to the ACP-responsive promoter. The ACP is an antigen-recognizing receptor, which is capable of inducing expression of the second expression cassette upon binding to its cognate antigen.

[0437] In some embodiments, X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0438] In some embodiments, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences within the engineered cell. For example, in some embodiments, the engineered cell comprises two engineered nucleic acids: a first engineered nucleic acid comprising a polynucleotide sequence encoding the first expression cassette, and a second engineered nucleic acid comprising a polynucleotide sequence encoding the second expression cassette. In an illustrative example, the effector molecule expression cassette can be encoded by a first engineered nucleic acid within the engineered cell, and the ACP expression cassette can be encoded by a second engineered nucleic acid within the engineered cell. In another illustrative example, the effector molecule expression cassette can be encoded by a first engineered nucleic acid, the ACP expression cassette can be encoded by a second engineered nucleic acid, and the antigen recognition receptor expression cassette can be encoded by a third engineered nucleic acid within the engineered cell.

[0439] In some embodiments, the first expression cassette and the second expression cassette are encoded by a single polynucleotide sequence within the engineered cell. For example, in some embodiments, the engineered cell comprises a single engineered nucleic acid comprising polynucleotide sequences encoding both the first expression cassette and the second expression cassette. Other illustrative examples include, but are not limited to, (1) the antigen-recognition receptor expression cassette and the effector molecule expression cassette can be encoded by a first engineered nucleic acid, and the ACP expression cassette can be encoded by a second engineered nucleic acid; (2) the ACP expression cassette and the effector molecule expression cassette can be encoded by a first engineered nucleic acid, and the antigen-recognition receptor expression cassette can be encoded by a second engineered nucleic acid; or (3) the ACP expression cassette and the antigen-recognition receptor expression cassette can be encoded by a first engineered nucleic acid, and the effector molecule expression cassette can be encoded by a second engineered nucleic acid.

[0440] In some embodiments, the expression cassette of a polynucleotide sequence in an engineered cell can be multicistronic, i.e., multiple distinct polypeptides (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single mRNA transcript. For example, a multicistronic expression cassette can encode both an ACP and an antigen-recognizing receptor, e.g., both expressed from a single expression cassette driven by a constitutive promoter. In another example, a multicistronic expression cassette can encode both an effector molecule and an antigen-recognizing receptor, e.g., both expressed from a single expression cassette driven by an ACP-responsive promoter. Expression cassettes can be made multicistronic by using various linkers, e.g., a polynucleotide sequence encoding a first protein of interest can be linked to a nucleotide sequence encoding a second protein of interest (e.g., in a 5' to 3' direction: first gene: linker: second gene). Multicistronic features and options are described in the section "Multicistronic and Multiple Promoter Systems."

[0441] In some embodiments, the second expression cassette is (LE) X In some embodiments, the linker polynucleotide sequence comprises two or more units of (LE), each L linker polynucleotide sequence being operatively associated with the translation of each effector molecule as a separate polypeptide. X The second expression cassette comprising one or more units of further comprises a polynucleotide sequence encoding a secretory signal peptide. In some embodiments, for each X, the corresponding secretory signal peptide is operably associated with an effector molecule. In some embodiments, each secretory signal peptide comprises a native secretory signal peptide native to the corresponding effector molecule. In some embodiments, each secretory signal peptide comprises a non-native secretory signal peptide non-native to the corresponding effector molecule. In some embodiments, the non-native secretory signal peptide is selected from IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12.

[0442] In some embodiments, the cells are engineered to contain an additional expression cassette comprising an additional promoter operably linked to an additional exogenous nucleotide sequence encoding an additional effector molecule, e.g., an effector molecule that stimulates an immune response. In some embodiments, the engineered cells comprise an additional promoter and a gene encoding a gene of the formula (LE) Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, and the additional promoter is operably linked to the additional exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent.

[0443] X can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more.

[0444] In some embodiments, the additional expression cassette is (LE) X In some embodiments, the linker polynucleotide sequence comprises two or more units of (LE), each L linker polynucleotide sequence being operatively associated with the translation of each effector molecule as a separate polypeptide. X The additional expression cassette comprising one or more units of further comprises a polynucleotide sequence encoding a secretory signal peptide. In some embodiments, for each X, the corresponding secretory signal peptide is operably associated with an effector molecule. In some embodiments, each secretory signal peptide comprises a native secretory signal peptide native to the corresponding effector molecule. In some embodiments, each secretory signal peptide comprises a non-native secretory signal peptide non-native to the corresponding effector molecule. In some embodiments, the non-native secretory signal peptide is selected from the group consisting of IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, CD8, human IgKVIICD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12.

[0445] In some embodiments, the first promoter and / or additional promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, the first promoter and / or additional promoter is a constitutive promoter selected from CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0446] The engineered cells of the present disclosure can contain engineered nucleic acids integrated into the genome of the cell. The engineered cells can contain engineered nucleic acids that are expressible without being integrated into the genome of the cell, for example, engineered in a transient expression system such as a plasmid or mRNA.

[0447] The present disclosure also encompasses additivity and synergy between the effector molecule(s) and the engineered cells that produce them. In some embodiments, the cells are engineered to produce one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) effector molecules, each of which may modulate a different tumor-mediated immune suppression mechanism. In other embodiments, the cells are engineered to produce at least one effector molecule that is not naturally produced by the cells. Such an effector molecule may, for example, complement the function of an effector molecule naturally produced by the cells.

[0448] In some embodiments, cells (e.g., tumor cells, red blood cells, platelet cells, or bacterial cells) are engineered to produce one or more effector molecules. For example, cells may be engineered to produce 1 to 20 different effector molecules. In some embodiments, the cells are selected from the group consisting of 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3 ~13, 3~12, 3~11, 3~10, 3~9, 3~8, 3~7, 3~6, ​​3~5, 3~4, 4~20, 4~19, 4~18, 4~17, 4~16, 4~15, 4~14, 4~13, 4~12, 4~11, 4~10, 4~9, 4~8, 4~7, 4~6, 4~5, 5~20, 5~19, 5~18, 5~17, 5~16, 5~15, 5~14, 5~13, 5~12, 5~11, 5~10, 5~9, 5~8, 5~7, 5~6, 6~20, 6~19, 6~18, 6~17, 6~16, 6~15, 6~1 4, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19 , 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16,In some embodiments, the cells are engineered to produce 14-15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18-20, 18-19, or 19-20 effector molecules. In some embodiments, the cells are engineered to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 effector molecules.

[0449] In some embodiments, the engineered cells comprise a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activated conditionally regulatory polypeptide (ACP), wherein the first promoter is operably linked to the first exogenous polynucleotide, and an ACP-responsive promoter and a polypeptide having the formula: (LE) X and a second expression cassette comprising a second exogenous polynucleotide sequence having the formula: (LE) wherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1 to 20, an ACP responsive promoter is operably linked to the second exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent, and the ACP is capable of inducing expression of the second expression cassette by binding to the ACP responsive promoter. In some embodiments, cells are engineered to contain multiple engineered nucleic acids, e.g., at least two engineered nucleic acids, each comprising a first promoter and a first exogenous polynucleotide sequence encoding an ACP, and a second expression cassette comprising a second exogenous polynucleotide sequence having the formula: (LE) Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, and X=1 to 20. In some embodiments, the second exogenous polynucleotide sequence encodes at least one (e.g., 1, 2, or 3) effector molecule. The second exogenous polynucleotide sequence may encode at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or more effector molecules. For example, cells may be engineered to contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or more engineered nucleic acids, each encoding a first expression cassette comprising a promoter operably linked to an ACP polynucleotide sequence and a second expression cassette comprising an ACP-responsive promoter and an exogenous nucleotide sequence encoding at least one (e.g., one, two, three, or more) effector molecule. In some embodiments, cells are engineered to contain two, three, four, five, six, seven, eight, nine, ten, or more engineered nucleic acids, each encoding a first expression cassette comprising a promoter operably linked to an ACP polynucleotide sequence and a second expression cassette comprising an ACP-responsive promoter and an exogenous nucleotide sequence encoding at least one (e.g., one, two, three, or more) effector molecule.

[0450] In some embodiments, the engineered cell further comprises a third expression cassette comprising a third promoter and a third exogenous polynucleotide sequence encoding an antigen-recognizing receptor, wherein the third promoter is operably linked to the third exogenous polynucleotide. In some embodiments, the first exogenous polynucleotide sequence further encodes an antigen-recognizing receptor.

[0451] In some embodiments, the engineered cells comprise a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditional regulatory polypeptide (ACP) and / or an antigen recognizing receptor, wherein the first promoter is operably linked to the first exogenous polynucleotide, and an activation conditional regulatory polypeptide-responsive (ACP-responsive) promoter and a polypeptide having the formula: (LE) Xwherein E comprises a polynucleotide sequence encoding an effector molecule, L comprises a linker polynucleotide sequence, X=1-20, an ACP-responsive promoter is operably linked to the second exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent, and the ACP can induce expression of the second expression cassette by binding to the ACP-responsive promoter. In embodiments in which the first exogenous polynucleotide sequence encodes an antigen-recognizing receptor, the engineered cell may further comprise a third expression cassette comprising a third promoter and a third exogenous polynucleotide sequence encoding an activating conditional regulatory polypeptide (ACP), wherein the third promoter is operably linked to the third exogenous polynucleotide. Exemplary antigen-recognizing receptors include chimeric antigen receptors (CARs) or T cell receptors (TCRs). In some embodiments, the ACP can induce expression of the second expression cassette by binding to the ACP-responsive promoter. In some embodiments, the ACP is an antigen-recognizing receptor, and the ACP can induce expression of the second expression cassette upon binding to its cognate antigen. In some embodiments, the ACP-responsive promoter is an inducible promoter that can be induced by binding of the ACP to its cognate antigen.

[0452] The engineered cell may comprise an engineered nucleic acid encoding at least one of the above-described linkers, e.g., a polypeptide linking a first polypeptide sequence and a second polypeptide sequence, one or more of the above-described multicistronic linkers, one or more additional promoters operably linked to additional ORFs, or a combination thereof. In some embodiments, the first expression cassette and the second expression cassette are encoded by separate polynucleotide sequences. In some embodiments, the first expression cassette and the second expression cassette are encoded by a single polynucleotide sequence. In some embodiments, the second expression cassette is encoded by (L1-E) XWhen the engineered cell comprises two or more units of L1, each L1 linker polynucleotide sequence is operably associated with translation of each effector molecule as a separate polypeptide. In some embodiments, the engineered cell further comprises a second linker polynucleotide sequence, wherein the second linker polynucleotide links the first expression cassette to the second expression cassette. In some embodiments, the second linker polynucleotide sequence is operably associated with translation of each effector molecule and ACP as separate polypeptides.

[0453] In some embodiments, cells (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) are engineered to produce effector molecules independently selected from a therapeutic class, where the therapeutic class is selected from cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0454] In some embodiments, cells of the disclosure (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) are engineered to produce a chemokine. In some embodiments, the chemokine is selected from CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0455] In some embodiments, cells of the disclosure (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) are engineered to produce a cytokine, in some embodiments, selected from IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α.

[0456] In some embodiments, cells of the present disclosure (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) are engineered to produce at least one homing molecule. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., a tumor), or organ). A "homing molecule" refers to a molecule that directs a cell to a target site. In some embodiments, the homing molecule functions to recognize and / or initiate interaction of the engineered cell to the target site. In some embodiments, the homing molecule is selected from anti-integrin α4β7, anti-MAdCAM, CCR9, CXCR4, SDF1, MMP-2, CXCR1, CXCR7, CCR2, CCR4, and GPR15.

[0457] In some embodiments, cells of the present disclosure (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) are engineered to produce at least one growth factor, which in some embodiments is selected from FLT3L and GM-CSF.

[0458] In some embodiments, cells of the present disclosure (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) are engineered to produce at least one co-activator molecule, in some embodiments, the co-activator molecule is selected from c-Jun, 4-1BBL, and CD40L.

[0459] In some embodiments, cells (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) of the present disclosure are engineered to produce at least one TGFβ inhibitor, hi some embodiments, the TGFβ inhibitor is selected from an anti-TGFβ peptide, an anti-TGFβ antibody, a TGFβ-TRAP, and combinations thereof.

[0460] In some embodiments, cells (e.g., T cells, immune cells, stem cells, tumor cells, red blood cells, or platelet cells) of the disclosure are engineered to produce at least one immune checkpoint inhibitor, which in some embodiments is selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody.

[0461] Exemplary immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda® - Merck), nivolumab (anti-PD-1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio® - Pfizer), durvalumab (anti-PD-L1; MEDI4736 / Im finzi® - Mediimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq® - Roche / Genentech), BMS-936559 (anti-PD-L1-BMS), tremelimumab (anti-CTLA-4; Mediimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy® - BMS), lirilumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0462] In some embodiments, the cells of the present disclosure (e.g., tumor cells, red blood cells, platelet cells, or bacterial cells) are engineered to produce at least one VEGF inhibitor. In some embodiments, the VEGF inhibitor comprises an anti-VEGF antibody, an anti-VEGF peptide, or a combination thereof.

[0463] In some embodiments, each effector molecule is a human-derived effector molecule.

[0464] Type of engineered cells The engineered or isolated cells of the present disclosure can be human cells. The engineered or isolated cells can be human primary cells. The engineered primary cells can be tumor-infiltrating primary cells. The engineered primary cells can be primary T cells. The engineered primary cells can be hematopoietic stem cells (HSCs). The engineered primary cells can be natural killer cells. The engineered primary cells can be any somatic cell. The engineered primary cells can be MSCs. In some embodiments, the engineered cells are derived from a subject. In some embodiments, the engineered cells are allogeneic with respect to the subject.

[0465] The engineered cells of the present disclosure can be isolated from a subject, for example, a subject known or suspected to have cancer. Cell isolation methods are known to those skilled in the art and include, but are not limited to, sorting techniques based on cell surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. The engineered cells can be allogeneic with respect to the subject undergoing treatment. Allogeneic modified cells can be HLA-matched to the subject undergoing treatment. The engineered cells can be cultured cells, for example, ex vivo cultured cells. The engineered cells can be ex vivo cultured cells, such as primary cells isolated from a subject. The cultured cells can be cultured with one or more cytokines.

[0466] In some embodiments, the engineered or isolated cells of the present disclosure are selected from T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells. In some embodiments, the engineered cells are natural killer (NK) cells. In some embodiments, the engineered cells are autologous. In some embodiments, the engineered cells are allogeneic.

[0467] In some embodiments, the engineered cells of the present disclosure are tumor cells selected from adenocarcinoma cells, bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colon tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, mesothelioma cells, ovarian tumor cells, pancreatic tumor cells, gastric tumor cells, testicular yolk sac tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0468] In some embodiments, the engineered cells of the present disclosure are bacterial cells selected from Clostridium beejerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella cholerae suis.

[0469] The present disclosure also provides a method comprising culturing the engineered cells of the present disclosure. The methods for culturing the engineered cells described herein are known. Those skilled in the art will recognize that the culture conditions will depend on the specific engineered cells of interest. Those skilled in the art will recognize that the culture conditions will depend on the specific downstream use of the engineered cells, for example, the specific culture conditions for subsequent administration of the engineered cells to a subject.

[0470] How to manipulate cells Also provided herein are compositions and methods for engineering cells to produce an activating conditional regulatory polypeptide (ACP) and one or more effector molecules encoded by any engineered nucleic acid comprising the first and second expression cassettes described herein.

[0471] Generally, cells are engineered to produce ACP and effector molecules by introducing (i.e., delivering) into the cytosol and / or nucleus of the cells one or more polynucleotides of the present disclosure comprising a first promoter and an exogenous polynucleotide sequence encoding ACP, and a second expression cassette comprising an ACP-responsive promoter and a second exogenous sequence encoding one or more effector molecules. For example, the polynucleotide expression cassette encoding the ACP polypeptide and one or more effector molecules can be any of the engineered nucleic acids described herein. Delivery methods include, but are not limited to, viral-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. Those skilled in the art will understand that the choice of delivery method may depend on the particular cell type to be engineered.

[0472] In some embodiments, the engineered cells are transduced with an oncolytic virus, including, but not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesiculovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variants or derivatives thereof. In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising a first expression cassette and a second expression cassette, hi some embodiments, the oncolytic virus further comprises a third expression cassette.

[0473] Viruses, including any of the oncolytic viruses described herein, can be recombinant viruses encoding one or more transgenes encoding one or more effector molecules, such as any of the engineered nucleic acids described herein. Viruses, including any of the oncolytic viruses described herein, can be recombinant viruses encoding one or more transgenes encoding one or more of two or more effector molecules, such as any of the engineered nucleic acids described herein. In some embodiments, cells are engineered via transduction with an oncolytic virus.

[0474] Viral-mediated delivery Viral vector-based delivery platforms can be used to engineer cells. Generally, viral vector-based delivery platforms engineer cells by introducing (i.e., delivering) them into host cells. For example, viral vector-based delivery platforms can engineer cells by introducing any of the engineered nucleic acids described herein. Viral vector-based delivery platforms can be nucleic acids, and therefore, engineered nucleic acids can also include nucleic acids derived from engineered viruses. Such engineered viral-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0475] Viral vector-based delivery platforms may encode multiple engineered nucleic acids, genes, or transgenes within the same nucleic acid. For example, nucleic acids derived from engineered viruses, such as recombinant or engineered viruses, may encode one or more transgenes, including, but not limited to, any of the engineered nucleic acids described herein encoding one or more effector molecules. One or more transgenes encoding one or more effector molecules can be configured to express one or more effector molecules. In addition to one or more transgenes (e.g., transgenes encoding one or more effector molecules), viral vector-based delivery platforms may also encode one or more genes, referred to as cis-acting elements or cis-acting genes, such as viral genes required for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.).

[0476] Viral vector-based delivery platforms may include multiple viral vectors, such as separate viral vectors encoding engineered nucleic acids, genes, or transgenes, referred to herein as trans-acting elements or trans-acting genes. For example, helper-dependent viral vector-based delivery platforms may provide additional genes necessary for viral infectivity and / or virus production on one or more additional, separate vectors in addition to a vector encoding one or more effector molecules. A single viral vector may deliver multiple engineered nucleic acids, such as a single vector delivering an engineered nucleic acid configured to produce two or more effector molecules. Multiple viral vectors may deliver multiple engineered nucleic acids, such as multiple vectors delivering one or more engineered nucleic acids configured to produce one or more effector molecules. The number of viral vectors used may depend on the packaging capacity of the viral vector-based vaccine platform, and those skilled in the art will be able to select the appropriate number of viral vectors.

[0477] In general, any viral vector-based system can be used for the in vitro production of molecules such as effector molecules, or can be used in in vivo and ex vivo gene therapy procedures, for example, to deliver engineered nucleic acids encoding one or more effector molecules in vivo. The selection of an appropriate viral vector-based system depends on various factors, such as the size of the cargo / payload, the immunogenicity of the viral system, the intended target cell, the intensity and timing of gene expression, and other factors recognized by those skilled in the art.

[0478] Viral vector-based delivery platform can be RNA-based virus or DNA-based virus.Exemplary viral vector-based delivery platform includes but is not limited to herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana vesiculovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, Maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, morbillivirus, lentivirus, replicating retrovirus, rhabdovirus, Seneca Valley virus, Sindbis virus, and any variant or derivative thereof.Other exemplary viral vector-based delivery platforms have been described in the art, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including but not limited to second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (see, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1):45-61; Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18; Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors). containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43(1):682-690; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880).

[0479] These sequences may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into a host cell, the infected cell (i.e., the engineered cell) expresses, and in some cases may secrete, one or more effector molecules. Vaccinia vectors and methods useful for immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the disclosure herein.

[0480] Viral vector-based delivery platforms can be viruses that target tumor cells, referred to herein as oncolytic viruses. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indiana vesiculovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic Maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replicating retrovirus, oncolytic rhabdovirus, oncolytic Seneca Valley virus, oncolytic Sindbis virus, and any variant or derivative thereof. Any of the oncolytic viruses described herein can be recombinant oncolytic viruses that include one or more transgenes (e.g., engineered nucleic acids) that encode one or more effector molecules. The transgenes encoding the one or more effector molecules can be configured to express the one or more effector molecules.

[0481] In some embodiments, the virus is selected from a lentivirus, a retrovirus, an oncolytic virus, an adenovirus, an adeno-associated virus (AAV), and a virus-like particle (VLP).

[0482] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats (LTRs) with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, which are then used to integrate one or more engineered nucleic acids (e.g., transgenes encoding one or more effector molecules) into target cells to provide persistent transgene expression. Retrovirus-based delivery systems include, but are not limited to, delivery systems based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); PCT / US94 / 05700). Other retroviral systems include the Phoenix retroviral system.

[0483] Viral vector-based delivery platform can be lentivirus-based.Generally, lentivirus vector is a retroviral vector that can transduce or infect non-dividing cells and usually produces high viral titers.Lentivirus-based delivery platform can be HIV-based, such as ViraPower system (ThermoFisher) or pLenti system (Cell Biolabs).Lentivirus-based delivery platform can be SIV or FIV-based. Other exemplary lentiviral-based delivery platforms are described in further detail in U.S. Patent Nos. 7,311,907, 7,262,049, 7,250,299, 7,226,780, 7,220,578; 7,211,247, 7,160,721, 7,078,031, 7,070,993, 7,056,699, 6,955,919, each of which is incorporated herein by reference for all purposes.

[0484] Viral vector-based delivery platforms can be adenovirus-based. Generally, adenovirus-based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titers and expression levels, and can be produced in large quantities using a relatively simple system. Generally, adenoviruses do not usually integrate into the host genome, so they can be used for transient expression of transgenes in infected cells. Adenovirus-based delivery platforms are described in further detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649; WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655, each of which is incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in further detail in U.S. Pat. Nos. 5,585,362, 6,083,716, 7,371,570, 7,348,178, 7,323,177, 7,319,033, 7,318,919, and 7,306,793 and International Patent Application WO 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0485] The viral vector-based delivery platform may be based on adeno-associated virus (AAV). Adeno-associated virus ("AAV") vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for in vitro production of effector molecules or can be used in in vivo and ex vivo gene therapy procedures, for example, to deliver engineered nucleic acids encoding one or more effector molecules in vivo (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368, 5,436,146, 6,632,670, 6,642,051, 7,078,387, 7,314,912, 6,498,244, 7,906,111; U.S. Patent Publication Nos. US2003-0138772, US2007 / 0036760, and US2009 / 0197338; Gao, et al. See, e.g., et al., J. Virol, 78(12):6381-6388 (June 2004), Gao, et al., Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003), and International Patent Applications WO2010 / 138263 and WO93 / 24641, Kotin, Human Gene Therapy 5:793-801 (1994), Muzyczka, J. Clin. Invest. 94:1351 (1994) (each of which is incorporated herein by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in further detail in U.S. Pat. No. 5,173,414, Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985), Tratschin et al., Mol. Cell. Biol. 4:2072-2081 (1984), Hermonat & Muzyczka, PNAS 81:64666470 (1984), and Samuiski et al., J. Virol. 63:03822-3828 (1989), each of which is incorporated herein by reference for all purposes.Generally, AAV-based vectors comprise a capsid protein having an amino acid sequence corresponding to any one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, and variants thereof.

[0486] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. Generally, VLPs are constructed by producing viral structural proteins and purifying the resulting viral particles. Following purification, a cargo / payload (e.g., any of the engineered nucleic acids described herein) is then encapsulated ex vivo within the purified particles. Thus, VLP production maintains the separation of nucleic acids encoding viral structural proteins from nucleic acids encoding cargo / payload. Viral structural proteins used for VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. Purified viral particles can be denatured and reassembled in the presence of the desired cargo using methods known in the art to produce VLPs. VLP production is described in further detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777), incorporated herein by reference for all purposes.

[0487] Viral vector-based delivery platforms can be engineered to target (i.e., infect) a range of cells, a narrow subset of cells, or specific cells. Generally, the envelope protein selected for a viral vector-based delivery platform determines the viral tropism. Viruses used in viral vector-based delivery platforms can be pseudotyped to target specific cells of interest. Viral vector-based delivery platforms are pantropic and can infect a variety of cells. For example, a pantropic viral vector-based delivery platform may contain a VSV-G envelope. Viral vector-based delivery platforms are amphotropic and can infect mammalian cells. Therefore, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0488] Lipid structure delivery system The engineered nucleic acids of the present disclosure (e.g., any of the engineered nucleic acids described herein) can be introduced into cells using a lipid-mediated delivery system. Generally, lipid-mediated delivery systems use a structure consisting of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. The lipid structure delivery system can deliver cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0489] Lipid-based nanoparticles may include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid preparations. As used herein, "liposome" is a generic term encompassing in vitro preparations of lipid vesicles formed by encapsulating a desired cargo, such as an engineered nucleic acid, within a lipid shell or lipid aggregate, such as any of the engineered nucleic acids described herein. Liposomes may be characterized as having a vesicular structure with a bilayer membrane generally comprising phospholipids and an internal medium generally comprising an aqueous composition. Liposomes include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. Liposomes may be unilamellar. Liposomes may be multilamellar. Liposomes may be multivesicular. Liposomes may be positively, negatively, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and sterols such as cholesterol.The selection of lipids is generally guided by considering the desired purpose, for example, the criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in bloodstream.A variety of methods for preparing liposomes are available, as described in, for example, Szoka et al., Ann.Rev.Biophys.Bioeng.9;467(1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0490] Multilamellar liposomes spontaneously form when lipids, including phospholipids, are suspended in an excess amount of aqueous solution so that multiple lipid layers are separated by aqueous media. Water and dissolved solutes are trapped in a closed structure between the lipid bilayers after the lipid components self-rearrange. Desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids such as any of the engineered nucleic acids described herein, viral vectors, viral-based delivery systems, etc.) can be encapsulated within the aqueous interior of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of the liposome, entrapped in the liposome, complexed with the liposome, or associated with the liposome for delivery to a target entity. Lipophilic molecules or molecules with lipophilic regions can also be dissolved or associated with the lipid bilayer.

[0491] Liposomes for use in accordance with the present embodiments can be prepared by different methods, as known to those skilled in the art. Liposome preparation is described in further detail in WO2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patent Nos. 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706, each of which is incorporated herein by reference for all purposes.

[0492] The liposomes can be cationic liposomes. Examples of cationic liposomes are described in further detail in U.S. Patent Nos. 5,962,016, 5,030,453, 6,680,068, U.S. Patent Application No. 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each of which is incorporated herein by reference in its entirety.

[0493] Lipid-mediated gene delivery methods are described, for example, in WO96 / 18372, WO93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988), U.S. Patent No. 5,279,833, Rose U.S. Patent No. 5,279,833, WO91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987), each of which is incorporated herein by reference for all purposes.

[0494] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. Exosomes range in size from 30 to 100 nm in diameter. Their surface consists of a lipid bilayer derived from the plasma membrane of the donor cell, contains cytosol derived from the cell that produced the exosome, and displays membrane proteins derived from the parent cell on their surface. Exosomes useful for nucleic acid delivery are known to those skilled in the art and are described in further detail, for example, in U.S. Patent No. 9,889,210 (incorporated herein by reference for all purposes).

[0495] As used herein, the term "extracellular vesicle" or "EV" refers to a cell-derived vesicle that contains a membrane enclosing an internal space. Generally, extracellular vesicles include all membrane-bound vesicles with a diameter smaller than the cell from which they originate. Typically, extracellular vesicles range in diameter from 20 nm to 1000 nm and can contain a variety of macromolecular cargoes, either within the internal space, displayed on the outer surface of the extracellular vesicle, and / or spanning the membrane. Cargo can include nucleic acids (e.g., any of the engineered nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. By way of example, and without limitation, extracellular vesicles include apoptotic bodies, cell fragments, cell-derived vesicles derived from direct or indirect manipulation (e.g., by continuous extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). The extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0496] As used herein, the term "exosome" refers to small (20-300 nm in diameter, more preferably 40-200 nm in diameter) cell-derived vesicles that contain a membrane surrounding an internal space and are generated from cells by direct plasma membrane budding or fusion of late endosomes with the plasma membrane. Exosomes contain lipids or butyrate and polysaccharides, and optionally contain a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, e.g., an engineered nucleic acid described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Exosomes are derived from producer cells and can be isolated from producer cells based on their size, density, biochemical parameters, or a combination thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of producer cells. Exosomes and the preparation of exosomes are described in further detail in WO2016 / 201323, which is incorporated herein by reference in its entirety.

[0497] As used herein, "nanovesicles" (also referred to as "microvesicles") refer to small (20-250 nm diameter, more preferably 30-150 nm diameter) cell-derived vesicles that comprise a membrane surrounding an internal space and are generated from cells by direct or indirect manipulation, and that would not be generated by the producer cells without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulation of producer cells includes, but is not limited to, continuous extrusion, treatment with alkaline solution, sonication, or a combination thereof. Nanovesicle production may, in some cases, result in the destruction of the producer cells. Preferably, the nanovesicle population is substantially free of producer cell-derived vesicles that are generated by direct budding from the plasma membrane or by fusion of late endosomes with the plasma membrane. Nanovesicles comprise lipids or butyric acid and polysaccharides, and optionally comprise a payload (e.g., a therapeutic agent), a receptor (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, e.g., an engineered nucleic acid described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. Once derived from producer cells according to such manipulation, nanovesicles can be isolated from the producer cells based on their size, density, biochemical parameters, or a combination thereof.

[0498] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that rely on the amphiphilic properties of lipids to form membrane- or vesicle-like structures (Riley 2017). These vesicles typically deliver cargo / payloads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. The lipids used in LNP formation can be cationic, anionic, or neutral. Lipids can be synthetic or naturally derived, and in some cases, biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates, including but not limited to polyethylene glycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat-soluble vitamins. Lipid compositions generally contain defined mixtures of materials, such as cationic, neutral, anionic, and amphiphilic lipids. In some cases, specific lipids are included to prevent LNP aggregation, prevent lipid oxidation, or provide functional groups that facilitate the attachment of additional moieties. Lipid composition can affect overall LNP size and stability. In one example, the lipid composition contains dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to contain one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. Furthermore, LNPs can be further engineered or functionalized to facilitate targeting to specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0499] Micelles are generally spherical synthetic lipid structures formed using single-chain lipids, with the hydrophilic heads of the single-chain lipids forming the outer layer or membrane, and the hydrophobic tails of the single-chain lipids forming the core of the micelle. Micelle generally refers to a lipid structure that contains only a lipid monolayer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25 (7): 1501-1513), which is incorporated herein by reference for all purposes.

[0500] Nucleic acid vectors, such as expression vectors, directly exposed to serum can result in several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Similarly, viral delivery systems directly exposed to serum can trigger undesirable immune responses and / or neutralization of the viral delivery system. Therefore, encapsulation of engineered nucleic acids and / or viral delivery systems can be used to avoid degradation while also avoiding potential off-target effects. In certain instances, the engineered nucleic acids and / or viral delivery systems are fully encapsulated within the delivery vehicle, e.g., the aqueous interior of LNPs. Encapsulation of engineered nucleic acids and / or viral delivery systems within LNPs can be performed using techniques well known to those skilled in the art, such as microfluidic mixing and droplet generation performed on a microfluidic droplet generation device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In one example, a desired lipid formulation, e.g., an MC3- or MC3-like-containing composition, is provided to a droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agent, thereby fully encapsulating the delivery vector and desired agent within the MC3- or MC3-like-based LNPs. In one example, the droplet generating device can control the size range and size distribution of the generated LNPs. For example, LNPs can have sizes ranging from 1 to 1,000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1,000 nanometers. Following droplet generation, the delivery vehicles (e.g., engineered nucleic acid and / or viral delivery systems) encapsulating the cargo / payload can be further processed or manipulated to prepare them for administration.

[0501] Nanoparticle Delivery Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Importantly, nanomaterial vehicles can be made of non-immunogenic materials, generally avoiding eliciting immunity against the delivery vector itself. These materials include, but are not limited to, lipids (as described above), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery—A Review. Nanomaterials 2017, 7(5), 94), incorporated herein by reference for all purposes.

[0502] Genome editing A genome editing system can be used to engineer a host genome to encode an engineered nucleic acid, for example, an engineered nucleic acid of the present disclosure. Generally, a "genome editing system" refers to any system for integrating an exogenous gene into the genome of a host cell. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and viral vector-based delivery platforms.

[0503] A transposon system can be used to integrate an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure, into a host genome. A transposon generally comprises a terminal inverted repeat (TIR) ​​flanking a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon with a cargo flanked by the TIR in cis or trans. The transposon system can be a retrotransposon system or a DNA transposon system. Generally, the transposon system randomly integrates the cargo / payload (e.g., an engineered nucleic acid) into the host genome. Examples of transposon systems include systems that use transposons from the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, described in further detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380) and U.S. Patent Nos. 6,489,458, 6,613,752, and 7,985,739, each of which is incorporated by reference for all purposes. Another example of a transposon system is the PiggyBac transposon system, described in further detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is incorporated by reference for all purposes.

[0504] Nuclease genome editing systems can be used to engineer a host genome to encode engineered nucleic acids, such as the engineered nucleic acids of the present disclosure. Without wishing to be bound by theory, nuclease-mediated gene editing systems used to introduce exogenous genes generally utilize the cell's natural DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. Briefly, following damage to genomic DNA (usually a double-strand break), the cell can eliminate the damage by using another DNA source with identical or substantially identical sequences at both the 5' and 3' ends as a template during DNA synthesis to repair the damage. Under natural circumstances, HDR can use other chromosomes present in the cell as templates. In gene editing systems, an exogenous polynucleotide is introduced into a cell and used as a homologous recombination template (HRT or HR template). Generally, an additional exogenous sequence not originally present in the chromosome, containing a lesion contained between the 5' and 3' complementary ends within the HRT (e.g., a gene or portion of a gene), can be incorporated (i.e., integrated) into a given genomic locus during templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to a first region of the endogenous genomic target locus, a nucleotide sequence identical to a second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the engineered nucleic acids described herein, e.g., any of the engineered nucleic acids encoding one or more effector molecules).

[0505] In some instances, the HR template may be linear. Examples of linear HR templates include, but are not limited to, linearized plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain instances, the HR template may be circular, such as a plasmid. Circular templates may include supercoiled templates.

[0506] With respect to the exogenous sequence to be introduced, the identical or substantially identical sequences present at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms). HR arms can be identical (i.e., 100% identical) to regions of the endogenous genomic target locus. In some examples, HR arms can be substantially identical to regions of the endogenous genomic target locus. Although substantially identical HR arms can be used, it can be advantageous for the HR arms to be identical, since the efficiency of the HDR pathway may be affected by HR arms with less than 100% identity.

[0507] Each HR arm, i.e., the 5' and 3' HR arms, can be the same size or different sizes. The length of each HR arm can be 50, 100, 200, 300, 400, or 500 bases or more, respectively. While HR arms can generally be any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. HR arms can be identical or substantially identical to a region of the endogenous genomic target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to a region of the endogenous genomic target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to a region of the endogenous genomic target locus within a certain distance from the cleavage site, for example, 1 base pair, 10 base pairs or less, 50 base pairs or less, or 100 base pairs or less of each other.

[0508] Nuclease genome editing systems can use a variety of nucleases to cleave target genomic loci, including, but not limited to, clustered regularly interspaced short palindromic repeats (CRISPR) family nucleases or derivatives thereof, transcription activator-like effector nucleases (TALENs) or derivatives thereof, zinc finger nucleases (ZFNs) or derivatives thereof, and homing endonucleases (HEs) or derivatives thereof.

[0509] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, for example, an engineered nucleic acid encoding one or more of the effector molecules described herein. CRISPR systems are described in further detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), the teachings of which are incorporated herein by reference. Generally, CRISPR-mediated gene editing systems include a CRISPR-associated (Cas) nuclease and RNA(s) that direct cleavage to specific target sequences. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which consists of a Cas9 nuclease and RNA(s) with a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. A crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity to a target sequence (a "defined nucleotide sequence"), e.g., a genomic sequence, via base-pair hybridization; and an RNA domain that hybridizes to the tracrRNA. The tracrRNA can interact with a nuclease (e.g., Cas9) and thereby facilitate its recruitment to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can also be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is shown herein, other CRISPR systems, such as the Cpf1 system, can also be used. Nucleases can include derivatives thereof, such as Cas9 functional variants, e.g., Cas9 "nickase" mutants, which generally mediate only a single-strand cleavage of a defined nucleotide sequence, as opposed to the complete double-strand cleavage typically generated by the Cas9 enzyme.

[0510] Generally, components of a CRISPR system interact with each other to form a ribonucleoprotein (RNP) complex that mediates sequence-specific cleavage. In some CRISPR systems, each component can be generated individually and used to form an RNP complex. In some CRISPR systems, each component can be generated individually in vitro and contacted with each other (i.e., "complexed") in vitro to form an RNP complex. The in vitro-generated RNPs can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, e.g., the cytosol and / or nucleus of a T cell. The in vitro-generated RNP complexes can be delivered to cells by various means, including, but not limited to, electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In a specific example, the in vitro-generated RNP complexes can be delivered to cells using the Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, the MaxCyte electroporation system, the Miltenyi CliniMACS electroporation system, the Neon electroporation system, and the BTX electroporation system. CRISPR nucleases, such as Cas9, can be produced in vitro (i.e., synthesized and purified) using various protein production techniques known in the art. CRISPR RNAs, such as sgRNAs, can be produced in vitro (i.e., synthesized and purified) using various RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0511] The in vitro generated RNP complex can be complexed with various ratios of nuclease to gRNA. The in vitro generated RNP complex can also be used in different amounts in CRISPR-mediated editing systems. For example, depending on the number of cells you want to edit, you can adjust the total amount of RNP added, such as adding less RNP complex if you are editing a large number of cells in the reaction.

[0512] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) is encoded by a separate polynucleotide, and each polynucleotide can be introduced into a cell together or separately. In some CRISPR systems, each component is encoded by a single polynucleotide (i.e., a multi-promoter or multicistronic vector, see the description of an exemplary multicistronic system below), which can be introduced into a cell. After the CRISPR components encoded by each polynucleotide are expressed in the cell (e.g., nucleases are translated and CRISPR RNA is transcribed), an RNP complex can be formed in the cell, which can then induce site-specific cleavage.

[0513] Some RNPs can be engineered to have moieties that facilitate delivery of the RNP to the nucleus. For example, the Cas9 nuclease can have a nuclear localization signal (NLS) domain, whereby either when delivering the Cas9 RNP complex to the cytosol of a cell, or after translation of Cas9 and subsequent RNP formation, the NLS can facilitate further transport of the Cas9 RNP to the nucleus.

[0514] The engineered cells described herein can be engineered using non-viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using non-viral methods. The engineered cells described herein can be engineered using viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using viral methods, for example, adenovirus, retrovirus, lentivirus, or any of the other viral-based delivery methods described herein.

[0515] In some CRISPR systems, multiple CRISPR compositions can be provided, each of which can individually target the same gene or common genomic locus with multiple target nucleotide sequences.For example, two separate CRISPR compositions can be provided, and cleavage can be directed at two different target nucleotide sequences that are within a certain distance from each other.In some CRISPR systems, multiple CRISPR compositions can be provided, and each of which can individually target the opposite strand of the same gene or common genomic locus.For example, two separate CRISPR "nickase" compositions can be provided, and cleavage can be directed at the same gene or common genomic locus on the opposite strand.

[0516] In general, the functionality of the CRISPR-mediated editing system described herein can be applied to other nuclease-based genome editing systems. TALENs are engineered site-specific nucleases consisting of the DNA-binding domain of a TALE (transcription activator-like effector) and the catalytic domain of the restriction endonuclease Fokl. By altering the amino acids present in the highly variable residue region of the DNA-binding domain monomer, various artificial TALENs can be created that target different nucleotide sequences. The DNA-binding domain then directs the nuclease to the target sequence, creating a double-strand break. TALEN-based systems are described in further detail in U.S. Ser. Nos. 12 / 965,590, 8,450,471, 8,440,431, 8,440,432, 10,172,880, and 13 / 738,381, all of which are incorporated herein by reference in their entireties. ZFN-based editing systems are described in further detail in U.S. Patent Nos. 6,453,242, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,030,215, 6,794,136, 7,067,317, 7,262,054, 7,070,934, 7,361,635, 7,253,273, and U.S. Patent Publication Nos. 2005 / 0064474, 2007 / 0218528, and 2005 / 0267061, which are incorporated by reference in their entireties for all purposes.

[0517] Other engineered delivery systems A variety of additional means for introducing an engineered nucleic acid (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity, e.g., any of the lipid structures described herein.

[0518] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of applying an electric field to temporarily permeabilize the outer membrane or shell of the target cell or entity, thereby internalizing the cargo / payload into the internal compartment of the target cell or entity. Generally, this method involves placing a cell or target entity between two electrodes in a solution containing the cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cell is then disrupted, i.e., permeabilized, by applying a transient set voltage, allowing the cargo to enter the interior of the entity, e.g., the cytoplasm of the cell. In the case of cells, at least some, if not most, of the cells remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., cell number, cargo concentration, recovery conditions, voltage, time, volume, pulse type, pulse length, volume, cuvette length, composition of electroporation solution, etc.) vary depending on several factors, including, but not limited to, the type of cell or other recipient entity, the cargo to be delivered, the desired internalization efficiency, and the desired viability. Optimization of such criteria is within the skill of one of ordinary skill in the art. Various devices and protocols can be used for electroporation. Examples include, but are not limited to, the Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ System, and Bio-Rad® Electroporation System.

[0519] Other means for introducing an engineered nucleic acid (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity include, but are not limited to, sonication, gene guns, hydrodynamic injection, and cell membrane deformation by physical means.

[0520] Compositions and methods for in vivo delivery of naked plasmids or engineered mRNA, such as mRNA, are described in detail in Kowalski et al. (Mol Ther. 2019 Apr 10;27(4):710-728) and Kaczmarek et al. (Genome Med. 2017;9:60.), each of which is incorporated herein by reference for all purposes.

[0521] How to use Methods for treating disease are also encompassed by the present disclosure. The methods comprise administering a therapeutically effective amount of an engineered nucleic acid, engineered cell, or isolated cell, as described above. In some aspects, provided herein are methods for treating a subject in need of treatment, the methods comprising administering a therapeutically effective amount of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0522] In some aspects, provided herein are methods of stimulating a cellular immune response against tumor cells in a subject, the methods comprising administering a therapeutically effective amount of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0523] In some aspects, provided herein are methods of providing anti-tumor immunity in a subject, the methods comprising administering to a subject in need thereof a therapeutically effective amount of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0524] In some aspects, provided herein are methods of treating a subject having cancer, the methods comprising administering a therapeutically effective amount of any of the engineered cells, isolated cells, or compositions disclosed herein.

[0525] In some aspects, provided herein are methods of reducing tumor volume in a subject, the methods comprising administering to a subject having a tumor a composition comprising any of the engineered cells, isolated cells, or compositions disclosed herein.

[0526] In some embodiments, the administration comprises systemic administration. In some embodiments, the administration comprises intratumoral administration. In some embodiments, the isolated cells are derived from the subject. In some embodiments, the isolated cells are allogeneic with respect to the subject.

[0527] In some embodiments, the method further comprises administering a checkpoint inhibitor selected from an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, an anti-VISTA antibody, an anti-KIR antibody, an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-HVEM antibody, an anti-BTLA antibody, an anti-GAL9 antibody, an anti-A2AR antibody, an anti-phosphatidylserine antibody, an anti-CD27 antibody, an anti-TNFα antibody, an anti-TREM1 antibody, and an anti-TREM2 antibody. In some embodiments, the method further comprises administering an anti-CD40 antibody.

[0528] In some embodiments, the tumor is selected from an adenocarcinoma, a bladder tumor, a brain tumor, a breast tumor, a cervical tumor, a colon tumor, an esophageal tumor, a glioma, a kidney tumor, a liver tumor, a lung tumor, a melanoma, a mesothelioma, an ovarian tumor, a pancreatic tumor, a gastric tumor, a testicular yolk sac tumor, a prostate tumor, a skin tumor, a thyroid tumor, and a uterine tumor.

[0529] Some methods involve selecting a subject (or patient population) that has a tumor (or cancer) and treating the subject with an engineered cell or delivery vehicle that modulates tumor-mediated immune suppression mechanisms.

[0530] The methods provided herein also include delivering a preparation of engineered cells or a delivery vehicle. In some embodiments, the preparation is a substantially pure preparation, e.g., containing less than 5% (e.g., less than 4%, 3%, 2%, or 1%) of cells other than the engineered cells. The preparation may be 1×10 5 cells / kg~1×10 7 cells / kg of cells.

[0531] The methods provided herein also include administering a drug or pharmaceutical composition in combination with a therapeutically effective amount of any of the engineered cells, isolated cells, or compositions disclosed herein to induce ACP and / or inhibit inhibitory proteases. For example, tamoxifen or its metabolites (e.g., 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, or endoxifen) can be administered to induce ACP. The drug or pharmaceutical agent can be administered before, with, simultaneously with, and / or after administration of any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical agent can be administered sequentially. The drug or pharmaceutical agent can be administered with or simultaneously with administration of any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical agent can be administered at a separate interval (e.g., before or after) administration of any of the engineered cells, isolated cells, or compositions disclosed herein. The drug or pharmaceutical agent can be administered both together / concurrently with any of the engineered cells, isolated cells, or compositions disclosed herein, and at separate intervals. The drug or pharmaceutical composition and the engineered cells, isolated cells, or compositions can be administered via different routes; for example, the drug or pharmaceutical composition can be administered orally, and the engineered cells, isolated cells, or compositions can be administered intraperitoneally, intravenously, subcutaneously, or by any other route suitable for administration, as will be understood by one of skill in the art.

[0532] The specific dose level and frequency of administration for a particular patient may vary widely and will depend upon a variety of factors, including the activity of the particular compound employed, the metabolic stability and length of action of that compound, age, body weight, general health, sex, diet, mode and time of administration, excretion rate, drug combination, the severity of the particular condition, and the host being treated.

[0533] The methods provided herein include administering a protease inhibitor. In some embodiments, NS3 protease can be inhibited by the protease inhibitor. Any suitable protease inhibitor can be used, including, but not limited to, simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir, or any combination thereof. In some embodiments, the protease inhibitor is selected from simeprevir, danoprevir, asunaprevir, cilprevir, boceprevir, sovaprevir, paritaprevir, telaprevir, grazoprevir, glecaprevir, and voxiloprevir.

[0534] In some embodiments, the protease inhibitor is grazoprevir. In some embodiments, the protease inhibitor is a combination of grazoprevir and elbasvir (an NS5A inhibitor of the hepatitis C virus NS5A replication complex). Grazoprevir and elbasvir can be co-formulated as a pharmaceutical composition, such as in tablet form (e.g., tablets available under the trade name Zepatier®). Grazoprevir and elbasvir can be co-formulated in a 2:1 weight ratio, e.g., in a unit dose of 100 mg grazoprevir and 50 mg elbasvir, respectively (e.g., tablets available under the trade name Zepatier®). The protease inhibitor can be administered at a dose capable of inhibiting the inhibitory protease domain of ACP. The protease inhibitor can be administered at a dose approved for another indication. As an illustrative, non-limiting example, Zepatier can be administered at a dose approved for the treatment of HCV.

[0535] Grazoprevir, in combination with elbasvir, can be administered orally in a dosage range of 0.001 to 1,000 mg / kg of mammalian (e.g., human) body weight per day, in single or divided doses. A single dosage range is 0.01 to 500 mg / kg of body weight per day, orally, in single or divided doses. Another dosage range is 0.1 to 100 mg / kg of body weight per day, orally, in single or divided doses. For oral administration, grazoprevir, in combination with elbasvir, can be provided in tablet or capsule form containing 1.0 to 500 mg of the active ingredient, particularly 1, 5, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, and 750 mg, to allow for symptomatic adjustment of the dosage to the patient being treated. Generally, the total daily dose of grazoprevir in combination with elbasvir can range from about 1 to about 2500 mg per day, but will necessarily vary depending on the treatment target, patient, and route of administration. In one embodiment, the dose of grazoprevir in combination with elbasvir is about 10 to about 1000 mg / day, administered in a single dose or in 2 to 4 divided doses. In another embodiment, the dose of grazoprevir in combination with elbasvir is about 1 to about 500 mg / day, administered in a single dose or in 2 to 4 divided doses. In yet another embodiment, the dose of grazoprevir in combination with elbasvir is about 1 to about 100 mg / day, administered in a single dose or in 2 to 4 divided doses. In yet another embodiment, the dose of grazoprevir in combination with elbasvir is about 1 to about 50 mg / day, administered in a single dose or in 2 to 4 divided doses. In another embodiment, the dose of grazoprevir, including in combination with elbasvir, is about 500 to about 1500 mg / day, administered in a single dose or in 2 to 4 divided doses. In yet another embodiment, the dose of grazoprevir, including in combination with elbasvir, is about 500 to about 1000 mg / day, administered in a single dose or in 2 to 4 divided doses. In yet another embodiment, the dose of grazoprevir, including in combination with elbasvir, is about 100 to about 500 mg / day, administered in a single dose or in 2 to 4 divided doses.

[0536] In vivo expression The methods provided herein also include delivering compositions in vivo that can generate engineered cells described herein (e.g., that can deliver any of the engineered nucleic acids described herein to cells in vivo). Such compositions include any of the viral-mediated delivery platforms, any of the lipid structure delivery systems, any of the nanoparticle delivery systems, any of the genome editing systems, or any of the other engineered delivery systems described herein that can engineer cells in vivo.

[0537] The methods provided herein also include in vivo delivery of a composition capable of producing any of the effector molecules described herein. The methods provided herein also include in vivo delivery of a composition capable of producing two or more effector molecules described herein. Compositions capable of in vivo production of effector molecules include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production of effector molecules can be naked mRNA or naked plasmids.

[0538] Pharmaceutical Composition The engineered nucleic acids or engineered cells can be formulated into pharmaceutical compositions. These compositions can contain, in addition to one or more engineered nucleic acids or engineered cells, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The exact nature of the carrier or other material can depend on the route of administration, for example, oral, intravenous, transdermal or subcutaneous, nasal, intramuscular, or intraperitoneal.

[0539] Pharmaceutical compositions for oral administration can be in the form of tablets, capsules, powders, or liquids. Tablets can contain solid carriers such as gelatin or adjuvants. Liquid pharmaceutical compositions generally contain liquid carriers such as water, petroleum, animal or vegetable oils, mineral oil, or synthetic oil. Physiological saline, dextrose, or other sugar solutions, or glycols such as ethylene glycol, propylene glycol, or polyethylene glycol can be included.

[0540] For intravenous, transdermal or subcutaneous injection, or injection at a painful site, the active ingredient is in the form of a pyrogen-free parenterally acceptable aqueous solution having suitable pH, isotonicity and stability.Those skilled in the art can easily prepare suitable solutions using isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection, etc.Preservatives, stabilizers, buffers, antioxidants, and / or other additives can be included as necessary.

[0541] Whether administered to an individual is a polypeptide, nucleic acid, small molecule, or other pharmaceutically useful compound according to the present disclosure, the administration is preferably a "therapeutically effective amount" or a "prophylactically effective amount" (in some cases, prevention can be considered treatment), which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the protein aggregation disorder being treated. Prescribing treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians, and will usually take into account the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0542] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0543] Further embodiments Set forth below are enumerated embodiments that describe particular embodiments of the present invention.

[0544] Embodiment 1: An engineered nucleic acid comprising: a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding an activation conditionally controlled polypeptide (ACP), the first promoter is operably linked to the first exogenous polynucleotide; the first expression cassette, and b) an ACP responsive promoter and the formula: (LE) X and a second exogenous polynucleotide sequence having the sequence: During the ceremony, E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence; X=1 to 20, the ACP responsive promoter is operably linked to the second exogenous polynucleotide, and in the first repeat of the (LE) unit, L is absent; the second expression cassette Including, The ACP can induce expression of the second expression cassette by binding to the ACP responsive promoter. The engineered nucleic acid.

[0545] Embodiment 2: The second expression cassette is (LE) X wherein each linker polynucleotide sequence is operably associated with translation of each effector molecule as a separate polypeptide.

[0546] Embodiment 3: The engineered nucleic acid of embodiment 1 or embodiment 2, wherein the linker polynucleotide sequence encodes a 2A ribosomal skipping tag.

[0547] Embodiment 4: The engineered nucleic acid of embodiment 3, wherein the 2A ribosomal skipping tag is selected from the group consisting of P2A, T2A, E2A, and F2A.

[0548] Embodiment 5: The engineered nucleic acid of any one of embodiments 1-2, wherein the linker polynucleotide sequence encodes an internal ribosome entry site (IRES).

[0549] Embodiment 6: The engineered nucleic acid of any one of embodiments 1 to 5, wherein the linker polynucleotide sequence encodes a cleavable polypeptide.

[0550] Embodiment 7: The engineered nucleic acid of embodiment 6, wherein said cleavable polypeptide comprises a furin polypeptide sequence.

[0551] Embodiment 8: (LE) X 8. The engineered nucleic acid of any one of embodiments 1 to 7, wherein said second expression cassette comprising one or more units of, for each X, further comprises a polynucleotide sequence encoding a secretory signal peptide.

[0552] Embodiment 9: The engineered nucleic acid of embodiment 8, wherein for each X, the corresponding secretory signal peptide is operably associated with the effector molecule.

[0553] Embodiment 10: The engineered nucleic acid of embodiment 8 or embodiment 9, wherein each secretory signal peptide comprises the native secretory signal peptide native to the corresponding effector molecule.

[0554] Embodiment 11: The engineered nucleic acid of any one of embodiments 8 to 10, wherein each secretory signal peptide comprises a non-native secretory signal peptide that is non-native to the corresponding effector molecule.

[0555] Embodiment 12: The engineered nucleic acid of embodiment 11, wherein the non-native secretory signal peptide is a secretory signal peptide of a molecule selected from the group consisting of IL12, IL2, optimized IL2, trypsinogen-2, Gaussia luciferase, CD5, CD8, human IgKVII, mouse IgKVII, VSV-G, prolactin, serum albumin preprotein, azurocidin preprotein, osteonectin, CD33, IL6, IL8, CCL2, TIMP2, VEGFB, osteoprotegerin, serpin E1, GROα, GM-CSFR, GM-CSF, and CXCL12.

[0556] Embodiment 13: The engineered nucleic acid of any one of embodiments 1 to 12, wherein the ACP responsive promoter comprises an ACP binding domain sequence and a promoter sequence.

[0557] Embodiment 14: The engineered nucleic acid of embodiment 13, wherein the promoter sequence is derived from a promoter selected from the group consisting of minP, an NFkB response element, a CREB response element, an NFAT response element, an SRF response element 1, an SRF response element 2, an AP1 response element, a TCF-LEF response element promoter fusion, a hypoxia response element, a SMAD binding element, a STAT3 binding site, minCMV, YB_TATA, minTK, an inducer molecule responsive promoter, and tandem repeats thereof.

[0558] Embodiment 15: The engineered nucleic acid of any one of embodiments 1 to 14, wherein the ACP responsive promoter comprises a synthetic promoter.

[0559] Embodiment 16: The engineered nucleic acid of any one of embodiments 1 to 15, wherein the ACP responsive promoter comprises a minimal promoter.

[0560] Embodiment 17: The engineered nucleic acid of any one of embodiments 12 to 16, wherein the ACP binding domain comprises one or more zinc finger binding sites.

[0561] Embodiment 18: The engineered nucleic acid of any one of embodiments 1 to 17, wherein the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter.

[0562] Embodiment 19: The engineered nucleic acid of embodiment 18, wherein the constitutive promoter is selected from the group consisting of CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0563] Embodiment 20: The engineered nucleic acid of any one of embodiments 1 to 19, wherein each effector molecule is independently selected from a therapeutic class, said therapeutic class being selected from the group consisting of cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0564] Embodiment 21: The engineered nucleic acid of Embodiment 20, wherein the cytokine is selected from the group consisting of IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α.

[0565] Embodiment 22: The engineered nucleic acid of embodiment 20, wherein the chemokine is selected from the group consisting of CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0566] Embodiment 23: The engineered nucleic acid of embodiment 20, wherein the homing molecule is selected from the group consisting of anti-integrin α4β7, anti-MAdCAM, CCR9, CXCR4, SDF1, MMP-2, CXCR1, CXCR7, CCR2, CCR4, and GPR15.

[0567] Embodiment 24: The engineered nucleic acid of embodiment 20, wherein the growth factor is selected from the group consisting of FLT3L and GM-CSF.

[0568] Embodiment 25: The engineered nucleic acid of embodiment 20, wherein the co-activator molecule is selected from the group consisting of c-Jun, 4-1BBL, and CD40L.

[0569] Embodiment 26: The engineered nucleic acid of embodiment 20, wherein the tumor microenvironment modifier is selected from the group consisting of adenosine deaminase, a TGFβ inhibitor, an immune checkpoint inhibitor, a VEGF inhibitor, and HPGE2.

[0570] Embodiment 27: The engineered nucleic acid of embodiment 26, where...

Claims

1. An engineered expression system for use in therapy, said expression system comprising: (a) a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence encoding a transcriptional modulator; the first promoter is operably linked to the first exogenous polynucleotide, and the transcriptional modulator comprises (i) a DNA-binding domain, (ii) a transcriptional effector domain, and (iii) a hormone-binding domain of an estrogen receptor (ERT2 domain); the ERT2 domain is capable of undergoing nuclear localization upon binding to tamoxifen or its metabolites; the first expression cassette, (b) a transcriptional modulator responsive promoter and a promoter of the formula: (L-E) Ｘ a second expression cassette comprising a second exogenous polynucleotide sequence having During the ceremony, E comprises a polynucleotide sequence encoding an effector molecule; L comprises a linker polynucleotide sequence, and X=1 to 20; wherein the effector molecule is a cytokine; the transcriptional modulator-responsive promoter is operably linked to the second exogenous polynucleotide, and L is absent in the first repeat of the (LE) unit; the second expression cassette, and (c) an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor comprising an antigen-binding domain, wherein the first expression cassette and / or the second expression cassette comprises the additional exogenous polynucleotide sequence; or an additional expression cassette comprising an additional promoter and an additional exogenous polynucleotide sequence encoding an antigen-recognizing receptor comprising an antigen-binding domain, wherein the engineered expression system further comprises the additional expression cassette; Including, the transcriptional modulator is capable of inducing expression of the second expression cassette by binding to the transcriptional modulator-responsive promoter; (i) the first expression cassette is comprised within a first nucleic acid and the second expression cassette is comprised within a second nucleic acid, or (ii) the first expression cassette and the second expression cassette are comprised within a single nucleic acid. The engineered expression system.

2. The second expression cassette is (LE) Ｘ 10. The engineered expression system of claim 1, wherein when the engineered expression system comprises two or more units of each effector molecule, each linker polynucleotide sequence is operatively associated with the translation of each effector molecule as a separate polypeptide.

3. the first expression cassette and the second expression cassette are contained within a single nucleic acid, and the expression system further comprises a linker polynucleotide sequence located between the first expression cassette and the second expression cassette; the linker polynucleotide sequence operably links the translation of the transcriptional modulator and each effector molecule as separate polypeptides; and / or the linker polynucleotide sequence encodes a 2A ribosomal skipping tag, an internal ribosome entry site (IRES), or a cleavable polypeptide; 3. The engineered expression system of claim 1 or 2.

4. (a) the transcriptional modulator-responsive promoter comprises a transcriptional modulator binding domain sequence and a promoter sequence; the promoter sequence is derived from a promoter selected from the group consisting of minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia response element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoter, synthetic promoter, minimal promoter, and tandem repeats thereof; and / or the transcriptional modulator binding domain sequence comprises one or more zinc finger binding sites; (b) the first promoter comprises a constitutive promoter, an inducible promoter, or a synthetic promoter; and / or (c) the cytokine is selected from the group consisting of IL1-β, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-γ, and TNF-α; Each cytokine is of human origin. The engineered expression system of any one of claims 1 to 3.

5. the additional promoter is operably linked to the additional exogenous polynucleotide, and the additional exogenous polynucleotide sequence is encoded by the same polynucleotide as the first expression cassette or the second expression cassette; and / or the antigen-recognition receptor recognizes GPC3, and the antigen-binding domain binds to GPC3, the antigen-binding domain binding to GPC3 comprising a heavy chain variable (VH) region and a light chain variable (VL) region; The VH is a heavy chain complementarity determining region 1 (CDR-H1) having the amino acid sequence of KNAMN (SEQ ID NO: 119); a heavy chain complementarity-determining region 2 (CDR-H2) having the amino acid sequence RIRNKTNNYATYYADSVKA (SEQ ID NO: 120); and Heavy chain complementarity determining region 3 (CDR-H3) having the amino acid sequence of GNSFAY (SEQ ID NO: 121) and The VL is a light chain complementarity-determining region 1 (CDR-L1) having the amino acid sequence KSSQSLLYSSNQKNYLA (SEQ ID NO: 122); a light chain complementarity-determining region 2 (CDR-L2) having the amino acid sequence of WASSRES (SEQ ID NO: 123); and Light chain complementarity-determining region 3 (CDR-L3) having the amino acid sequence QQYYNYPLT (SEQ ID NO: 124) Including, The engineered expression system of any one of claims 1 to 4.

6. 6. The engineered expression system of any one of claims 1 to 5, wherein the transcriptional modulator is a transcriptional repressor or a transcriptional activator.

7. The engineered expression system described in any one of claims 1 to 6, wherein the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

8. (a) the engineered expression system further comprises an insulator, wherein the insulator is located between the first expression cassette, the second expression cassette, and / or additional expression cassettes, if present; (b) the first expression cassette is localized in the same orientation relative to the second expression cassette, or the first expression cassette is localized in the opposite orientation relative to the second expression cassette; and / or (c) the engineered expression system is a nucleic acid selected from the group consisting of DNA, cDNA, RNA, mRNA, and a naked plasmid; The engineered expression system of any one of claims 1 to 7.

9. (LE) The second expression cassette comprising one or more units of X further comprises a polynucleotide sequence encoding a secretory signal peptide for each X; for each X, the corresponding secretory signal peptide is operably associated with the effector molecule; and each secretory signal peptide comprises a natural secretory signal peptide native to the corresponding effector molecule, or a non-natural secretory signal peptide non-native to the corresponding effector molecule; 9. The engineered expression system of any one of claims 1 to 8.

10. 10. The engineered expression system of any one of claims 1 to 9, comprising the first expression cassette, the second expression cassette, and / or the further expression cassette, if present; (a) a first vector comprises the first expression cassette and, if present, the additional expression cassette, and a second vector comprises the second expression cassette; (b) a first vector comprises the first expression cassette and a second vector comprises the second expression cassette and, if present, the additional expression cassette; (c) a first vector comprises the first expression cassette and the second expression cassette, and a second vector comprises, if present, the additional expression cassette; or (d) a vector comprises the first expression cassette, the second expression cassette, and, if present, the additional expression cassette. One or more expression vectors for use in therapy.

11. 11. An isolated cell for use in therapy comprising an engineered expression system according to any one of claims 1 to 9 or one or more expression vectors according to claim 10, the engineered expression system is recombinantly expressed; the engineered expression system is expressed from one or more vectors or one or more selected loci from the genome of the cell; the cells are selected from the group consisting of T cells, CD8+ T cells, CD4+ T cells, gamma delta T cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, virus-specific T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages, monocytes, dendritic cells, erythrocytes, platelet cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells; and / or the cells are autologous or the cells are allogeneic; The isolated cells.

12. 12. A pharmaceutical composition comprising the engineered expression system of any one of claims 1 to 9, one or more expression vectors of claim 10, or the isolated cell of claim 11, and a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, or a combination thereof.

13. 13. A medicament for use in (i) a method for stimulating a cellular immune response against tumor cells in a tumor-bearing subject, or (ii) a method for providing anti-tumor immunity in a subject, or (iii) a method for reducing tumor volume in a subject, the medicament comprising the isolated cell of claim 11 or the composition of claim 12.

14. (a) the medicament is administered by systemic administration or intratumoral administration; (b) the isolated cells are derived from the subject, or the isolated cells are allogeneic with respect to the subject; (c) the method further comprises administering a checkpoint inhibitor. (d) the tumor is selected from the group consisting of adenocarcinoma, bladder tumor, brain tumor, breast tumor, cervical tumor, colon tumor, esophageal tumor, glioma, kidney tumor, liver tumor, lung tumor, melanoma, mesothelioma, ovarian tumor, pancreatic tumor, gastric tumor, testicular yolk sac tumor, prostate tumor, skin tumor, thyroid tumor, and uterine tumor; and / or (e) the method further comprises administering tamoxifen or a metabolite thereof; the tamoxifen metabolite is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen; The pharmaceutical composition according to claim 13.

15. 13. A kit for treating and / or preventing cancer, comprising the isolated cells of claim 11 or the pharmaceutical composition of claim 12, the kit further comprising written instructions for using the cells or composition to treat and / or prevent cancer in a subject.

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