Improved ERT2 mutants, inducible cell death systems, and uses thereof

Abstract

Provided herein are mutants of the estrogen receptor alpha ligand binding domain (ER-LBD), and inducible cell death systems comprising mutants of the estrogen receptor alpha ligand binding domain (ER-LBD). Also provided are methods for using the same, e.g., to induce cell death in cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 482,983, filed February 2, 2023, and U.S. Provisional Application No. 63 / 466,147, filed May 12, 2023, each of which is hereby incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains an electronically submitted Sequence Listing XML, which is hereby incorporated by reference in its entirety. The XML copy was created on XX / 20XX, is named XXXXXXX, and is XXX,XXX bytes in size. [Background technology]

[0003] The estrogen receptor (ER) is a ligand-dependent transcription factor that binds to endogenous hormonal ligands, such as estrogen and estradiol. Synthetic ligands that bind to the ER have been developed to treat ER-positive cancers, such as ER-positive breast cancer. For example, the active metabolite of the drug tamoxifen induces nuclear translocation of the ER, antagonizing it in a tissue-selective manner. Tamoxifen and its active metabolites have also been utilized as tools to control nuclear localization in research settings. For example, an ER ligand-binding domain variant known as ERT2 has been used as a fusion protein with Cre recombinase to regulate Cre recombinase-based gene editing in animal model systems. The ability to engineer suicide switch-mediated cell killing using synthetic ligands would also be useful in therapeutic applications, such as in the fields of cell and gene therapy. For example, to address potential toxicity concerns, it would be desirable to design cell and gene therapy products with a "safety" switch, such as an inducible cell death system. Thus, modified ERT2-based systems with improved sensitivity to and / or selectivity for synthetic ligands would be useful for suicide switch-mediated regulated cell killing in clinical settings. Summary of the Invention

[0004] Provided herein, in various embodiments, is an inducible cell death system comprising a polypeptide, wherein the polypeptide comprises a ligand-binding domain and a cell death-inducing domain, wherein the polypeptide is configured, upon contact of the ligand-binding domain with a ligand, to generate a cell death-inducing signal in a cell in which the polypeptide is expressed, and wherein the ligand-binding domain comprises a modified estrogen receptor ligand-binding domain (ER-LBD) corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, wherein one or The multiple additional amino acid substitutions involve reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, positions 517-540, and position 547 of SEQ ID NO:1, optionally in which the modified ER-LBD has greater sensitivity to a non-endogenous ligand compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2, optionally in which the modified ER-LBD has greater sensitivity to a non-endogenous ligand compared to an endogenous ligand as a result of the one or more additional amino acid substitutions, optionally in which the modified ER-LBD has greater selectivity for a non-endogenous ligand compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2, optionally in which the ligand-binding domains of the first polypeptide monomer and the second polypeptide monomer comprise the same additional amino acid substitutions.

[0005] In some embodiments, (a) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391 substitution, optionally wherein the L391 substitution is L391V; (b) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a N413D mutation; (c) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution and a N41 (d) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a H524 substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; (e) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a M421L substitution; (f) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a S46 (g) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a M421L substitution and a S463P substitution; (h) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L384M substitution; (i) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L354I substitution; (j) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L354I substitution. (k) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a Q414E substitution; (l) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L354I substitution and a Q414E substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution;(m) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a M421L substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; (n) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a S463P substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution. (o) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L391V substitution, an N413D mutation, an H524 substitution, and a Q414E substitution, optionally wherein the H524 substitution is an H524L substitution or an H524F substitution; (p) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L391V substitution, an N413D mutation, an H524 substitution, and an L354I substitution, optionally wherein the H524 substitution is an H524L substitution or an H524F substitution;

[0006] In some embodiments, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer are 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537 , 538, 539, 540, and 547, optionally in which (i) the one or more positions include position 343 of SEQ ID NO:1, optionally in which the amino acid substitution at position 343 of SEQ ID NO:1 is selected from the group consisting of M343F, M343I, M343L, and M343V; (ii) the one or more positions include position 344 of SEQ ID NO:1, optionally in which the amino acid substitution at position 344 of SEQ ID NO:1 is G344M; (iii) one or more (iv) one or more positions include position 346 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 346 of SEQ ID NO:1 is selected from the group consisting of L346I, L346M, L346F, and L346V; (v) one or more positions include position 347 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 347 of SEQ ID NO:1 is T347D, T347E, T347F. , T347I, T347K, T347L, T347M, T347N, T347Q, T347R, T347S, and T347V; (vi) the one or more positions include position 348 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 348 of SEQ ID NO:1 is N348K; (vii) the one or more positions include position 349 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 349 of SEQ ID NO:1 is selected from the group consisting of L349I, L349M, L349F, and L349V;(viii) the one or more positions include position 350 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 350 of SEQ ID NO:1 is selected from the group consisting of A350F, A350I, A350L, A350M, and A350V; (ix) the one or more positions include position 351 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 351 of SEQ ID NO:1 is selected from the group consisting of D351E, D351F, D351I, D351L, D351M, D351N, D351Q, and D351V. (x) one or more positions include position 352 of SEQ ID NO:1, optionally in which the amino acid substitution at position 352 of SEQ ID NO:1 is R352K; (xi) one or more positions include position 354 of SEQ ID NO:1, optionally in which the amino acid substitution at position 354 of SEQ ID NO:1 is selected from the group consisting of L354I, L354M, L354F, and L354V; (xii) one or more positions include position 380 of SEQ ID NO:1, optionally in which the amino acid substitution at position 380 of SEQ ID NO:1 is R352K. the amino acid substitution is E380Q; (xiii) the one or more positions include position 384 of SEQ ID NO:1, optionally in which the amino acid substitution at position 384 of SEQ ID NO:1 is selected from the group consisting of L384I, L384M, L384F, and L384V; (xiv) the one or more positions include position 386 of SEQ ID NO:1, optionally in which the amino acid substitution at position 386 of SEQ ID NO:1 is I386V; (xv) the one or more positions include position 387 of SEQ ID NO:1, optionally in which (xvi) the one or more positions include position 388 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 388 of SEQ ID NO:1 is selected from the group consisting of M388I, M388L, and M388F; (xvii) the one or more positions include position 389 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 389 of SEQ ID NO:1 is I389M;(xviii) the one or more positions include position 391 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 391 of SEQ ID NO:1 is selected from the group consisting of L391I, L391M, L391F, and L391V, optionally wherein the amino acid substitution at position 391 of SEQ ID NO:1 is L391V; (xix) the one or more positions include position 392 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 392 of SEQ ID NO:1 is V392M; (xx) the one or more positions include position 404 of SEQ ID NO:1. (xxii) one or more positions include position 409 of SEQ ID NO:1, optionally in which the amino acid substitution at position 409 of SEQ ID NO:1 is L409V; (xxiii) one or more positions include position 413 of SEQ ID NO:1, optionally in which the amino acid substitution at position 409 of SEQ ID NO:1 is L409V; (xxiv) one or more positions include position 414 of SEQ ID NO:1, optionally in which the amino acid substitution at position 404 of SEQ ID NO:1 is N407D; (xxv) one or more positions include position 415 of SEQ ID NO:1, optionally in which the amino acid substitution at position 404 of SEQ ID NO:1 is N407D; (xxvi) one or more positions include position 416 of SEQ ID NO:1, optionally in which the amino acid substitution at position 416 of SEQ ID NO:1 is N407D; (xxvi) one or more positions include position 417 of SEQ ID NO:1, optionally in which the amino acid substitution at position 417 of SEQ ID NO:1 is N407D; (xxvi) one or more positions include position 418 of SEQ ID NO:1, optionally in which the amino acid substitution at position 418 of SEQ ID NO:1 is N407D; (xxiv) the one or more positions include position 414 of SEQ ID NO:1, optionally in which the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E; (xxv) the one or more positions include position 417 of SEQ ID NO:1, optionally in which the amino acid substitution at position 417 of SEQ ID NO:1 is C417S; (xxvi) the one or more positions include position 418 of SEQ ID NO:1, optionally in which the amino acid substitution at position 418 of SEQ ID NO:1 is C417S. (xxvii) the one or more positions include position 420 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 420 of SEQ ID NO:1 is selected from the group consisting of G420I, G420M, G420F, and G420V; (xxviii) the one or more positions include position 421 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 421 of SEQ ID NO:1 is selected from the group consisting of M421I, M421L, M421F, and M421V;(xxix) one or more positions include position 422 of SEQ ID NO:1, optionally in which the amino acid substitution at position 422 of SEQ ID NO:1 is V422I; (xxx) one or more positions include position 424 of SEQ ID NO:1, optionally in which the amino acid substitution at position 424 of SEQ ID NO:1 is selected from the group consisting of I424L, I424M, I424F, and I424V; (xxxi) one or more positions include position 428 of SEQ ID NO:1, optionally in which the amino acid substitution at position 428 of SEQ ID NO:1 is I424L, I424M, I424F, and I424V. (xxxii) the one or more positions include position 463 of SEQ ID NO:1, optionally in which the amino acid substitution at position 463 of SEQ ID NO:1 is S463P; (xxxiii) the one or more positions include position 517 of SEQ ID NO:1, optionally in which the amino acid substitution at position 517 of SEQ ID NO:1 is M517A; (xxxiv) the one or more positions include position 521 of SEQ ID NO:1, optionally in which the amino acid substitution at position 521 of SEQ ID NO:1 is M517A; (xxxv) the one or more positions include position 522 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 522 of SEQ ID NO:1 is selected from the group consisting of M522I, M522L, and M522V; (xxxvi) the one or more positions include position 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 524 of SEQ ID NO:1 is selected from the group consisting of H524A, H524F, H524I, H524L, and H524V. (xxxvii) the one or more positions include position 525 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 525 of SEQ ID NO:1 is selected from the group consisting of L525F, L525I, L525M, L525N, L525Q, L525S, L525T, and L525V; (xxxviii) the one or more positions include position 526 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 526 of SEQ ID NO:1 is Y526L;(xxxix) one or more positions include position 527 of SEQ ID NO:1, optionally in which the amino acid substitution at position 527 of SEQ ID NO:1 is S527N; (xl) one or more positions include position 528 of SEQ ID NO:1, optionally in which the amino acid substitution at position 528 of SEQ ID NO:1 is selected from the group consisting of M528F, M528I, and M528V; (xli) one or more positions include position 533 of SEQ ID NO:1, optionally in which the amino acid substitution at position 533 of SEQ ID NO:1 is M528F, M528I, and M528V. (xlii) the one or more positions include position 534 of SEQ ID NO:1, optionally in which the amino acid substitution at position 534 of SEQ ID NO:1 is selected from the group consisting of V534Q and V534R; (xliii) the one or more positions include position 536 of SEQ ID NO:1, optionally in which the amino acid substitution at position 536 of SEQ ID NO:1 is selected from the group consisting of L536F, and L536M, L536R, and L536Y. (xliv) the one or more positions include position 537 of SEQ ID NO:1, optionally in which the amino acid substitution at position 537 of SEQ ID NO:1 is selected from the group consisting of Y537E and Y537S; (xlv) the one or more positions include position 538 of SEQ ID NO:1, optionally in which the amino acid substitution at position 538 of SEQ ID NO:1 is selected from the group consisting of D538G and D538K; (xlvi) the one or more positions include position 539 of SEQ ID NO:1, optionally in which (xlvii) one or more positions include position 540 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 540 of SEQ ID NO:1 is selected from the group consisting of L540A and L540F; and / or (xlviii) one or more positions include position 547 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 547 of SEQ ID NO:1 is H547A;

[0007] In some embodiments, the one or more additional amino acid substitutions in the (a) polypeptide, first polypeptide monomer, and / or second polypeptide monomer are two amino acid substitutions, optionally wherein each of the two amino acid substitutions is at a position in SEQ ID NO: 1 selected from 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525, optionally wherein: (i) the two amino acid substitutions are at positions 345 and 526 of SEQ ID NO: 1; and 348 of SEQ ID NO:1, optionally in which the amino acid substitution at position 345 of SEQ ID NO:1 is L345S and the amino acid substitution at position 348 of SEQ ID NO:1 is N348K; (ii) the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO:1, optionally in which the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 389 of SEQ ID NO:1 is I389M; (iii) the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO:1, optionally in which (iv) the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO:1, optionally in which the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; (v) the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO:1, optionally in which the amino acid substitution at position 354 of SEQ ID NO:1 is L354I; and the amino acid substitution at position 384 of SEQ ID NO:1 is L384M; (vi) the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 387 of SEQ ID NO:1 is L387M; (vii) the two amino acid substitutions are at positions 387 and 391, optionally wherein the amino acid substitution at position 387 of SEQ ID NO:1 is L387M and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F;(viii) the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 387 of SEQ ID NO:1 is L387M; or (ix) the two amino acid substitutions are at positions 384 and 391 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; (b) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer are three amino acid substitutions, optionally wherein each of the three amino acid substitutions is 343, 347, 351, 354, 388, 391, 4 1 is selected from 04, 414, 418, 463, 521, 524, and 525, optionally in which: (i) the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO:1, optionally in which the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; (ii) the three amino acid substitutions are at positions 414, 463, and 524 of SEQ ID NO:1, optionally in which the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L;(c) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer are four amino acid substitutions, optionally wherein each of the four amino acid substitutions is at a position of SEQ ID NO: 1 selected from 343, 347, 351, 354, 384, 388, 391, 404, 413, 418, 463, 521, 524, and 525, optionally wherein: (i) the four amino acid substitutions are at positions 354, 384, 391, 404, 413, 418, 463, 521, 524, and 525 of SEQ ID NO: 1; and 418, optionally in which the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391F, and the amino acid substitution at position 418 of SEQ ID NO:1 is V418I; (ii) four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO:1, optionally in which the amino acid substitution at position 343 of SEQ ID NO:1 is M34 3I, wherein the amino acid substitution at position 388 of SEQ ID NO:1 is M388I, the amino acid substitution at position 521 of SEQ ID NO:1 is G521I, and the amino acid substitution at position 404 of SEQ ID NO:1 is F404L; (iii) four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 524 of SEQ ID NO:1 is H524V, the amino acid substitution at position 347 of SEQ ID NO:1 is T347R, and the amino acid substitution at position 351 of SEQ ID NO:1 is F404L; (iv) four amino acid substitutions are at positions 354, 384, 391, and 463 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, and the amino acid substitution at position 463 of SEQ ID NO:1 is S463P;or (v) the four amino acid substitutions are at positions 384, 391, 413, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; (d) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are at five amino acid substitutions. substitutions, optionally wherein each of the five amino acid substitutions is at a position of SEQ ID NO:1 selected from 354, 384, 391, 409, 413, 414, 421, 463, and 524, optionally wherein (i) the five amino acid substitutions are at positions 384, 409, 413, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, and the amino acid substitution at position 413 of SEQ ID NO:1 is N4 13D, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; (ii) five amino acid substitutions are at positions 391, 413, 414, 463, and 524 of SEQ ID NO:1, optionally in which the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, and the amino acid substitution at position 463 of SEQ ID NO:1 is S463P. and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; (iii) five amino acid substitutions are at positions 391, 414, 421, 463, and 524 of SEQ ID NO:1, optionally in which the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F;(iv) five amino acid substitutions at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; or (v) five amino acid substitutions at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; the amino acid substitutions are at positions 354, 409, 421, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; (e) a polypeptide, a first polypeptide monomer and / or the one or more additional amino acid substitutions of the second polypeptide monomer are six amino acid substitutions, optionally wherein each of the six amino acid substitutions is at a position of SEQ ID NO:1 selected from 354, 384, 391, 409, 413, 414, 421, 463, and 524, optionally wherein: (i) the six amino acid substitutions are at positions 384, 391, 413, 421, 463, and 524 of SEQ ID NO:1, optionally wherein: the amino acid substitution at position 384 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; (ii) six amino acid substitutions at positions 409, 413, 414, 421, 463, and 524 of SEQ ID NO:1;optionally, in which the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; or (iii) six amino acid substitutions are at positions 354, 391, 409, 413, 414, and 524 of SEQ ID NO:1, optionally, in which the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, and the amino acid substitution at position 413 of SEQ ID NO:1 is N421L, M421L, S463P, and H524L. 13D, wherein the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; (f) the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer are seven amino acid substitutions, optionally wherein each of the seven amino acid substitutions is at a position of SEQ ID NO:1 selected from 354, 384, 391, 409, 413, 414, 421, 463, 517, and 524, optionally wherein: (i) the seven amino acid substitutions are at positions 354, 384, 409, 413, 421, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 384 of SEQ ID NO:1 is L384M; (ii) seven amino acid substitutions at positions 354, 391, 413, 421, 463, 517, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; or (iii) seven amino acid substitutions are at positions 354, 391, 413, 414, 421, 517, and 524 of SEQ ID NO:1, optionally in which the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F;or (g) the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are eight amino acid substitutions, optionally in which the eight amino acid substitutions are at positions 384, 391, 409, 413, 421, 463, 517, and 524 of SEQ ID NO:1, optionally in which (i) the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F;

[0008] Also provided herein, in various embodiments, is an inducible cell death system comprising a first polypeptide and a second polypeptide monomer, wherein the first and second polypeptide monomers each comprise a ligand-binding domain and a cell death-inducing domain, wherein the first and second polypeptide monomers are configured to oligomerize upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in a cell in which the first and second polypeptide monomers are expressed, and wherein the ligand-binding domain comprises a modified estrogen receptor ligand-binding domain (ER-LBD) comprising an amino acid sequence corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER- The LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) additional amino acid substitutions, wherein the additional amino acid substitutions, with reference to SEQ ID NO: 1, are: (i) a L384M substitution, a L391V substitution, a N413D substitution, a M421L substitution, a S463P substitution, and a H52 4L substitution, (ii) an L391V substitution, an N413D substitution, a Q414E substitution, an S463P substitution, and an H524F substitution, (iii) an L354I substitution, an L391V substitution, an N413D substitution, a Q414E substitution, an M421L substitution, an M517A substitution, and an H524F substitution, or (iv) an L354I substitution, an L391V substitution, an L409V substitution, an N413D substitution, a Q414E substitution, and an H524L substitution.

[0009] In some embodiments, the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an N413D mutation, an H524 substitution, and an S463P substitution, optionally in which: (a) the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L391V substitution, an L409V substitution, a Q414E substitution, an N413D substitution, an S463P substitution, an M517A substitution, and an H524L substitution, optionally in which: the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 90 or 103; (b) the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L391V substitution, an L409V substitution, a Q414E substitution, an N413D substitution, an S463P substitution, an M517A substitution, and an H524L substitution, optionally in which: the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 90 or 103; the amino acid substitutions include a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L384M substitution, and a H524L substitution, optionally wherein the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 91 or 104; (c) the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 91 or 104; The one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95%, 97%, 100%, 105%, 110%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186%, 187%, 188(d) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95% identical to SEQ ID NO: 93 or 106. , 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 94 or 107; (e) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an M421L substitution, and an H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 94 or 107. (f) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a L354I substitution, and a H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 95 or 108. (g) comprising an amino acid sequence that is 8%, 99%, or 100% identical to SEQ ID NO: 96 or 109; and (g) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, an L384M substitution, and an H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95%, 97%, 100%, 109% identical to SEQ ID NO: 96 or 109.(h) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 88%, 99%, or 100% identical to SEQ ID NO: 97 or 110. (i) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include an L391V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an L384M substitution, and an H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 98 or 111; , 90%, 95%, 97.5%, 98%, 99%, or 100% identical amino acid sequence to SEQ ID NO: 99 or 112; (j) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 99 or 112; (k) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include an N413D substitution, an S463P substitution, an L354I substitution, an L384M substitution, and an H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 100 or 113;or (l) one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer include an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, and an H524L substitution, optionally wherein the polypeptide, first polypeptide monomer, and / or second polypeptide monomer is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 101 or 114. 5%, 97.5%, 98%, 99%, or 100% identical amino acid sequence, optionally in which the ligand is a non-endogenous ligand, optionally in which the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen, optionally in which the non-endogenous ligand comprises a tamoxifen metabolite, optionally in which the non-endogenous ligand is endoxifen.

[0010] In some embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of generating an oligomerization and / or cell death-inducing signal at (a) a concentration of 0.25 nM endoxifen or less, and / or 0.04 nM 4-OHT or less; (b) a concentration of 2.5 nM endoxifen or less, and / or 0.4 nM 4-OHT or less; (c) a concentration of at least 0.001 pM 4-OHT; or (d) a concentration of at least 0.01 pM 4-OHT.

[0011] In some embodiments, the cell death domain is derived from: (a) a protein selected from the following: caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53-upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF related death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, and purine nucleoside phosphorylase, optionally wherein the death-inducing domain comprises the caspase 9-derived amino acid sequence of SEQ ID NO: 48 or 125, and optionally wherein the caspase domain or functional fragment thereof does not comprise a caspase activation and recruitment domain (CARD) domain sequence;or (b) a transcription factor comprising a nucleic acid binding domain and a transcription effector domain, wherein the transcription factor is configured to generate a cell death-inducing signal by inducing expression of a caspase domain or a functional fragment thereof, optionally wherein the caspase is selected from caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or a functional fragment thereof, and wherein the caspase is selected from diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF- R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53 upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, or purine nucleoside phosphorylase;

[0012] Also provided herein, in various embodiments, is an isolated polynucleotide comprising a nucleotide sequence that encodes a polypeptide, a first polypeptide monomer, and / or a second polypeptide monomer provided herein.

[0013] Also provided herein are heterologous constructs comprising a promoter operably linked to a polynucleotide provided herein.

[0014] Also provided herein, in various embodiments, are plasmids or vectors that include the heterologous constructs provided herein.

[0015] Also provided herein, in various embodiments, are cells comprising the heterologous constructs, plasmids, or vectors provided herein.

[0016] Also provided herein, in various embodiments, is a molecular switch for generating a cell death-inducing signal in a cell, comprising: (a) an inducible cell death system, isolated polynucleotide, heterologous construct, plasmid, vector, or cell provided herein, wherein the inducible cell death system is capable of generating a cell death-inducing signal in the cell; and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD generates a cell death-inducing signal in the cell, optionally wherein the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen, optionally wherein the non-endogenous ligand comprises a tamoxifen metabolite, optionally wherein the non-endogenous ligand is endoxifen, optionally wherein: (i) the first polypeptide monomer and the second polypeptide monomer are at a concentration of 0.25 nM endoxifen or less and / or 0.04 nM endoxifen or less. (ii) the first polypeptide monomer and the second polypeptide monomer are capable of generating an oligomerization and / or cell death-inducing signal at a concentration of 2.5 nM endoxifen or less and / or at a concentration of 0.4 nM 4-OHT or less; (iii) the first polypeptide monomer and the second polypeptide monomer are capable of generating an oligomerization and / or cell death-inducing signal at a concentration of at least 0.001 pM 4-OHT; or (iv) the first polypeptide monomer and the second polypeptide monomer are capable of generating an oligomerization and / or cell death-inducing signal at a concentration of at least 0.01 pM 4-OHT.

[0017] Also provided herein, in various embodiments, is a method of inducing oligomerization of a chimeric protein, comprising: (i) transforming a cell with a heterologous construct encoding any one of the inducible cell death systems of any one of claims 1-8, the isolated polynucleotide of claim 9, the heterologous construct of claim 10, or the plasmid or vector of claim 11; and (ii) contacting the transformed cell with a non-endogenous ligand of a modified estrogen receptor ligand binding domain (ER-LBD), optionally wherein: (a) the method comprises culturing the transformed cell under conditions suitable for expression of the inducible cell death system prior to inducing oligomerization and / or inducing cell death. (b) the transformed cell is in a human or an animal, and wherein contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal; and / or (c) the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen, optionally wherein the non-endogenous ligand comprises a tamoxifen metabolite, optionally wherein the non-endogenous ligand is endoxifen, optionally wherein the non-endogenous ligand is administered at a concentration at which the non-endogenous ligand is substantially inactive against wild-type estrogen receptor alpha of SEQ ID NO: 1.

[0018] Also provided herein, in various embodiments, is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein: (i) the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein (ii) the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are a N413D substitution, a S463P substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; (iii) the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L384M substitution, and a H524L substitution. (iv) a modified ER-LBD comprising (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, with reference to SEQ ID NO:1;(v) the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1; (vi) the modified ER-LBD comprises (a) G (vii) a modified ER-LBD comprising (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; (viii) a modified ER-LBD comprising (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, and a H524L substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO:1; (ix) a modified ER-LBD comprising (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:1;and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; (x) a modified ER-LBD comprising (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and if and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1; (xi) a modified ER-LBD comprising (a) a G400V amino acid substitution, (xii) a modified ER-LBD comprising (a) a L391V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a L384M substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, with reference to SEQ ID NO: 1;(xiii) the modified ER-LBD comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to any one of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID NO:114, and optionally wherein the modified ER-LBD has greater sensitivity and / or selectivity to a non-endogenous ligand compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2 or compared to an endogenous ligand, as a result of one or more additional amino acid substitutions, optionally wherein the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen, optionally wherein the endogenous ligand is estradiol, and optionally wherein the modified ER-LBD further comprises a V595A amino acid substitution;

[0019] Also provided herein, in various embodiments, is a chimeric protein comprising a polypeptide of interest fused to the modified ER-LBD of claim 15, optionally wherein the polypeptide of interest comprises a nucleic acid binding domain, optionally wherein the nucleic acid binding domain comprises a zinc finger domain, optionally wherein the zinc finger domain comprises the sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 84, optionally wherein the chimeric protein comprises a chimeric transcription factor, and optionally wherein the polypeptide of interest comprises a nucleic acid binding domain and a transcription regulator domain, optionally wherein the transcription regulator domain The transcriptional activator is a transcriptional activator, optionally wherein the transcriptional activator comprises a herpes simplex virus protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFκB(p65); an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains (VPR activation domain); a tripartite activator comprising VP64, p65, and HSF1 activation domains (VPH activation domain); and a histone acetyltransferase core domain of human E1A-associated protein p300 (p300 activation domain). HAT core activation domain), and the transcriptional activator is selected from the group consisting of a herpes simplex virus protein 16 (VP16) activation domain; an activation domain containing four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFκB(p65); an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator containing VP64, p65, and Rta activation domains (VPR activation domain); a tripartite activator containing VP64, p65, and HSF1 activation domains (VPH activation domain);and the histone acetyltransferase core domain of human E1A-associated protein p300 (p300 HAT core activation domain), optionally wherein the transcriptional activator is a p65 transcriptional activator comprising the amino acid sequence DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64);

[0020] Also provided herein is an isolated polynucleotide molecule comprising a nucleotide sequence encoding a modified ER-LBD or chimeric protein provided herein.

[0021] Also provided herein are heterologous constructs comprising a promoter operably linked to a polynucleotide molecule provided herein.

[0022] Also provided herein, in various embodiments, are cells comprising the heterologous constructs provided herein.

[0023] Also provided herein, in various embodiments, is a molecular switch for regulating transcription of a gene of interest, comprising: (a) the chimeric protein of claim 16 or a heterologous construct encoding the chimeric protein, wherein the chimeric protein binds to a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to the gene of interest; and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD induces the chimeric protein to regulate transcription of the gene of interest, optionally wherein: (i) the non-endogenous ligand is 4-hydroxytamoxifen, N-desmethyltamoxifen, (ii) the gene of interest encodes a polypeptide selected from the group consisting of a cytokine, a chemokine, a homing molecule, a growth factor, a cell death regulator, a co-activation molecule, a tumor microenvironment modifier, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme; (iii) the molecular switch further comprises an additional construct comprising a CTF-responsive promoter operably linked to the gene of interest; (iv) the heterologous construct and the additional construct are comprised in a single vector; and / or (v) the heterologous construct is comprised in a first vector, and the additional construct is comprised in a second vector.

[0024] Also provided herein, in various embodiments, are methods for modulating the localization of a chimeric protein, comprising: (a) transforming a cell with a heterologous construct encoding a chimeric protein provided herein; and (b) inducing nuclear localization of the chimeric protein by contacting the transformed cell with a non-endogenous ligand, optionally, wherein the method further comprises culturing the transformed cell under conditions suitable for expression of the chimeric protein prior to contacting the transformed cell with the non-endogenous ligand, and / or optionally, contacting the transformed cell with the non-endogenous ligand. The species construct and the additional construct are contained in a single vector, or the heterologous construct is contained in a first vector and the additional construct is contained in a second vector, and / or optionally, wherein the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen, and / or optionally, wherein the non-endogenous ligand is administered at a concentration at which the non-endogenous ligand is substantially inactive against the wild-type estrogen receptor alpha of SEQ ID NO: 1.

[0025] Also provided herein, in some embodiments, are modified estrogen receptor ligand-binding domains (ER-LBDs) with improved sensitivity and / or selectivity for non-endogenous ligands, such as tamoxifen and its metabolites. Also provided herein, in some embodiments, are chimeric proteins comprising the modified ER-LBDs described herein, molecular switches, polynucleotides encoding the modified ER-LBDs and chimeric proteins described herein, cells encoding the polynucleotides described herein or expressing the modified ER-LBDs and chimeric proteins described herein, and methods of using the modified ER-LBDs, chimeric proteins, polynucleotides, molecular switches, or cells described herein.

[0026] The modified ER-LBD and chimeric proteins described herein (e.g., any of the polypeptides, first polypeptide monomers, and / or second polypeptide monomers of the inducible cell death system herein) have greater sensitivity to and / or selectivity for non-endogenous ligands (e.g., 4-hydroxytamoxifen, also referred to as 4-OHT) compared to ERT2. ERT2 is the ligand-binding domain of ER containing a G400V, M543A, and L544A amino acid substitution (see SEQ ID NO: 2, SEQ ID NO: 3A). ERT2 can also contain a V595A amino acid substitution in addition to G400V / M543A / L544A (see SEQ ID NO: 3, SEQ ID NO: 3B). The mean peak plasma concentration after a typical clinical dose of tamoxifen is in the nanomolar range (e.g., approximately 40 ng / mL). Furthermore, ERT2 can be responsive to endogenous ligands, such as estradiol. Thus, the improved sensitivity to and / or selectivity of the modified ER-LBD and chimeric proteins comprising the modified ER-LBD for non-endogenous ligands enables the use of ER-based systems for suicide switch-mediated killing.

[0027] Provided herein is an inducible cell death system comprising a polypeptide, wherein the polypeptide comprises a ligand-binding domain and a cell death-inducing domain, wherein the polypeptide is configured, upon contact of the ligand-binding domain with a ligand, to generate a cell death-inducing signal in a cell in which the polypeptide is expressed, and wherein the ligand-binding domain comprises a modified estrogen receptor ligand-binding domain (ER-LBD) comprising an amino acid sequence corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises: (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are with reference to one or more regions selected from positions 343-354, 380-392, 404-463, and 517-540, and position 547 of SEQ ID NO: 1.

[0028] In some embodiments, the modified ER-LBD may further comprise other modifications, such as amino acid substitutions, deletions, and / or insertions (with reference to SEQ ID NO: 1). Such other modifications may be within or outside of positions 343-354, 380-392, 404-463, 517-540, and / or 547 with reference to SEQ ID NO: 1. Such other modifications may be within or outside of positions 283-594 with reference to SEQ ID NO: 1.

[0029] In some aspects, the polypeptide is or comprises a first polypeptide monomer, and the inducible cell death system further comprises a second polypeptide monomer, and wherein the first polypeptide monomer and the second polypeptide monomer each comprise a ligand-binding domain and a cell death-inducing domain, and wherein the first polypeptide monomer and the second polypeptide monomer are configured to oligomerize with each other upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in a cell in which the first polypeptide monomer and the second polypeptide monomer are expressed.

[0030] In some aspects, the ligand binding domains of the first polypeptide monomer and the second polypeptide monomer each comprise a modified ER-LBD, wherein the modified ER-LBD comprises: (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are independently selected for each of the first polypeptide monomer and the second polypeptide monomer, with reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, and positions 517-540, and position 547 of SEQ ID NO: 1. In some aspects, the ligand binding domains of the first polypeptide monomer and the second polypeptide monomer comprise the same additional amino acid substitutions.

[0031] In some embodiments, the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2. In some embodiments, the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to endogenous ligands as a result of one or more additional amino acid substitutions. In some embodiments, the modified ER-LBD has greater selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2.

[0032] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer (e.g., a polypeptide of an inducible cell death system comprising both a first and a second polypeptide monomer), and / or the second polypeptide monomer comprise a L391 substitution. In some aspects, the L391 substitution is L391V. In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a N413D mutation. In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution and a N413D mutation.

[0033] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an H524 substitution. In some aspects, the H524 substitution is an H524L substitution or an H524F substitution. In some aspects, the H524 substitution is an H524L substitution.

[0034] In some embodiments, the H524 substitution is a H524F substitution.

[0035] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an M421L substitution.

[0036] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a S463P substitution.

[0037] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an M421L substitution and an S463P substitution.

[0038] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L384M substitution.

[0039] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L354I substitution.

[0040] In some aspects, the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a Q414E substitution.

[0041] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L354I substitution and a Q414E substitution.

[0042] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, and a H524 substitution. In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a M421L substitution. In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a S463P substitution. In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a Q414E substitution. In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an L391V substitution, an N413D mutation, an H524 substitution, and an L354I substitution.

[0043] In some aspects, the H524 substitution is an H524L substitution or an H524F substitution.

[0044] In some aspects, the modified estrogen receptor ligand binding domain (ER-LBD) comprises an amino acid sequence corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises a G400V amino acid substitution, an M543A amino acid substitution, and an L544A amino acid substitution, with reference to SEQ ID NO: 1, and one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are with reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, and positions 517-540, and position 547 of SEQ ID NO: 1, and wherein the modified ER-LBD has greater sensitivity and / or selectivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2, or compared to endogenous ligands, as a result of the one or more additional amino acid substitutions. In some aspects, the modified ER-LBD further comprises a V595A amino acid substitution. In some aspects, the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0045] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, or second polypeptide monomer are at one or more positions of SEQ ID NO: 1 selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537, 538, 539, 540, and 547.

[0046] In some aspects, the one or more positions include position 343 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 343 of SEQ ID NO: 1 is selected from the group consisting of M343F, M343I, M343L, and M343V.

[0047] In some aspects, the one or more positions include position 344 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 344 of SEQ ID NO: 1 is G344M.

[0048] In some aspects, the one or more positions include position 345 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 345 of SEQ ID NO: 1 is L345S.

[0049] In some aspects, the one or more positions include position 346 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 346 of SEQ ID NO: 1 is selected from the group consisting of L346I, L346M, L346F, and L346V.

[0050] In some aspects, the one or more positions include position 347 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 347 of SEQ ID NO: 1 is selected from the group consisting of T347D, T347E, T347F, T347I, T347K, T347L, T347M, T347N, T347Q, T347R, T347S, and T347V.

[0051] In some aspects, the one or more positions include position 348 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 348 of SEQ ID NO: 1 is N348K.

[0052] In some aspects, the one or more positions include position 349 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 349 of SEQ ID NO: 1 is selected from the group consisting of L349I, L349M, L349F, and L349V.

[0053] In some aspects, the one or more positions include position 350 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 350 of SEQ ID NO: 1 is selected from the group consisting of A350F, A350I, A350L, A350M, and A350V.

[0054] In some aspects, the one or more positions include position 351 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 351 of SEQ ID NO: 1 is selected from the group consisting of D351E, D351F, D351I, D351L, D351M, D351N, D351Q, and D351V.

[0055] In some aspects, the one or more positions include position 352 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 352 of SEQ ID NO: 1 is R352K.

[0056] In some aspects, the one or more positions include position 354 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 of SEQ ID NO: 1 is selected from the group consisting of L354I, L354M, L354F, and L354V.

[0057] In some aspects, the one or more positions include position 380 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 380 of SEQ ID NO: 1 is E380Q.

[0058] In some aspects, the one or more positions include position 384 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 384 of SEQ ID NO: 1 is selected from the group consisting of L384I, L384M, L384F, and L384V.

[0059] In some aspects, the one or more positions include position 386 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 386 of SEQ ID NO: 1 is I386V.

[0060] In some aspects, the one or more positions include position 387 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 387 of SEQ ID NO: 1 is selected from the group consisting of L387I, L387M, L387F, and L387V.

[0061] In some aspects, the one or more positions include position 388 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 388 of SEQ ID NO: 1 is selected from the group consisting of M388I, M388L, and M388F.

[0062] In some aspects, the one or more positions include position 389 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 389 of SEQ ID NO: 1 is I389M.

[0063] In some aspects, the one or more positions include position 391 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 391 of SEQ ID NO: 1 is selected from the group consisting of L391I, L391M, L391F, and L391V.

[0064] In some aspects, the one or more positions include position 392 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 392 of SEQ ID NO: 1 is V392M.

[0065] In some aspects, the one or more positions include position 404 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 404 of SEQ ID NO: 1 is selected from the group consisting of F404I, F404L, F404M, and F404V.

[0066] In some aspects, the one or more positions include position 407 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 407 of SEQ ID NO: 1 is N407D.

[0067] In some aspects, the one or more positions include position 409 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 409 of SEQ ID NO: 1 is L409V.

[0068] In some aspects, the one or more positions include position 413 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 413 of SEQ ID NO: 1 is N413D.

[0069] In some aspects, the one or more positions include position 414 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 414 of SEQ ID NO: 1 is Q414E.

[0070] In some aspects, the one or more positions include position 417 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 417 of SEQ ID NO: 1 is C417S.

[0071] In some aspects, the one or more positions include position 418 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 418 of SEQ ID NO: 1 is selected from the group consisting of V418I, V418L, V418M, and V418F.

[0072] In some aspects, the one or more positions include position 420 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 420 of SEQ ID NO: 1 is selected from the group consisting of G420I, G420M, G420F, and G420V.

[0073] In some aspects, the one or more positions include position 421 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 421 of SEQ ID NO: 1 is selected from the group consisting of M421I, M421L, M421F, and M421V.

[0074] In some aspects, the one or more positions include position 422 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 422 of SEQ ID NO: 1 is V422I.

[0075] In some aspects, the one or more positions include position 424 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 424 of SEQ ID NO: 1 is selected from the group consisting of I424L, I424M, I424F, and I424V.

[0076] In some aspects, the one or more positions include position 428 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 428 of SEQ ID NO: 1 is selected from the group consisting of L428I, L428M, L428F, and L428V.

[0077] In some aspects, the one or more positions include position 463 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 463 of SEQ ID NO: 1 is S463P.

[0078] In some aspects, the one or more positions include position 517 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 517 of SEQ ID NO: 1 is M517A.

[0079] In some aspects, the one or more positions include position 521 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 521 of SEQ ID NO: 1 is selected from the group consisting of G521A, G521F, G521I, G521L, G521M, and G521V.

[0080] In some aspects, the one or more positions include position 522 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 522 of SEQ ID NO: 1 is selected from the group consisting of M522I, M522L, and M522V.

[0081] In some aspects, the one or more positions include position 524 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 524 of SEQ ID NO: 1 is selected from the group consisting of H524A, H524I, H524L, H524F, and H524V.

[0082] In some aspects, the one or more positions include position 525 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 525 of SEQ ID NO: 1 is selected from the group consisting of L525F, L525I, L525M, L525N, L525Q, L525S, L525T, and L525V.

[0083] In some aspects, the one or more positions include position 526 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 526 of SEQ ID NO: 1 is Y526L.

[0084] In some aspects, the one or more positions include position 527 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 527 of SEQ ID NO: 1 is S527N.

[0085] In some aspects, the one or more positions include position 528 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 528 of SEQ ID NO: 1 is selected from the group consisting of M528F, M528I, and M528V.

[0086] In some aspects, the one or more positions include position 533 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 533 of SEQ ID NO: 1 is selected from the group consisting of V533F and V533W.

[0087] In some aspects, the one or more positions include position 534 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 534 of SEQ ID NO: 1 is selected from the group consisting of V534Q and V534R.

[0088] In some aspects, the one or more positions include position 536 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 536 of SEQ ID NO: 1 is selected from the group consisting of L536F, and L536M, L536R, and L536Y.

[0089] In some aspects, the one or more positions include position 537 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 537 of SEQ ID NO: 1 is selected from the group consisting of Y537E and Y537S.

[0090] In some aspects, the one or more positions include position 538 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 538 of SEQ ID NO: 1 is selected from the group consisting of D538G and D538K.

[0091] In some aspects, the one or more positions include position 539 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 539 of SEQ ID NO: 1 is selected from the group consisting of L539A and L539R.

[0092] In some aspects, the one or more positions include position 540 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 540 of SEQ ID NO: 1 is selected from the group consisting of L540A and L540F.

[0093] In some aspects, the one or more positions include position 547 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 547 of SEQ ID NO: 1 is H547A.

[0094] In some aspects, the one or more additional amino acid substitutions are two amino acid substitutions. In some aspects, each of the two amino acid substitutions is at a position of SEQ ID NO:1 selected from 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525. In some aspects, the two amino acid substitutions are at positions 345 and 348 of SEQ ID NO:1, wherein the amino acid substitution at position 345 of SEQ ID NO:1 is L345S and the amino acid substitution at position 348 of SEQ ID NO:1 is N348K. In some aspects, the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO:1, wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 389 of SEQ ID NO:1 is I389M. In some aspects, the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO:1, and wherein the amino acid substitution at position 421 of SEQ ID NO:1 is M421I and the amino acid substitution at position 392 of SEQ ID NO:1 is V392M. In some aspects, the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F. In some aspects, the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 384 of SEQ ID NO:1 is L384M. In some aspects, the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 387 of SEQ ID NO:1 is L387M. In some aspects, the two amino acid substitutions are at positions 387 and 391, and wherein the amino acid substitution at position 387 of SEQ ID NO:1 is L387M and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F.In some aspects, the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO:1, and wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, and the amino acid substitution at position 387 of SEQ ID NO:1 is L387M. In some aspects, the two amino acid substitutions are at positions 384 and 391 of SEQ ID NO:1, and wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F.

[0095] In some aspects, the one or more additional amino acid substitutions are three amino acid substitutions, in some aspects, each of the three amino acid substitutions is at a position of SEQ ID NO:1 selected from 343, 347, 351, 354, 388, 391, 404, 414, 418, 463, 521, 524, and 525. In some aspects, the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F. In some aspects, the three amino acid substitutions are at positions 414, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L.

[0096] In some aspects, the one or more additional amino acid substitutions are four amino acid substitutions, in some aspects, each of the four amino acid substitutions is at a position of SEQ ID NO:1 selected from 343, 347, 351, 354, 384, 388, 391, 404, 413, 418, 463, 521, 524, and 525. In some aspects, the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391F, and the amino acid substitution at position 418 of SEQ ID NO:1 is V418I. In some aspects, the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO:1, and wherein the amino acid substitution at position 343 of SEQ ID NO:1 is M343I, the amino acid substitution at position 388 of SEQ ID NO:1 is M388I, the amino acid substitution at position 521 of SEQ ID NO:1 is G521I, and the amino acid substitution at position 404 of SEQ ID NO:1 is F404L. In some aspects, the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO:1, and wherein the amino acid substitution at position 524 of SEQ ID NO:1 is H524V, the amino acid substitution at position 347 of SEQ ID NO:1 is T347R, the amino acid substitution at position 351 of SEQ ID NO:1 is D351Q, and the amino acid substitution at position 525 of SEQ ID NO:1 is L525N. In some aspects, the four amino acid substitutions are at positions 354, 384, 391, and 463 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, and the amino acid substitution at position 463 of SEQ ID NO:1 is S463P.In some aspects, the four amino acid substitutions are at positions 384, 391, 413, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F.

[0097] In some aspects, the one or more additional amino acid substitutions are five amino acid substitutions. In some aspects, each of the five amino acid substitutions is at a position of SEQ ID NO:1 selected from 354, 384, 391, 409, 413, 414, 421, 463, and 524. In some aspects, the five amino acid substitutions are at positions 384, 409, 413, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L. In some aspects, the five amino acid substitutions are at positions 391, 413, 414, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F. In some aspects, the five amino acid substitutions are at positions 391, 414, 421, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F. In some aspects, the five amino acid substitutions are at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L.In some aspects, the five amino acid substitutions are at positions 354, 409, 421, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L.

[0098] In some aspects, the one or more additional amino acid substitutions are six amino acid substitutions. In some aspects, each of the six amino acid substitutions is at a position of SEQ ID NO:1 selected from 354, 384, 391, 409, 413, 414, 421, 463, and 524. In some aspects, the six amino acid substitutions are at positions 384, 391, 413, 421, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L. In some aspects, the six amino acid substitutions are at positions 409, 413, 414, 421, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L. In some aspects, the six amino acid substitutions are at positions 354, 391, 409, 413, 414, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L.

[0099] In some aspects, the one or more additional amino acid substitutions are seven amino acid substitutions, hi some aspects, each of the seven amino acid substitutions is at a position of SEQ ID NO: 1 selected from 354, 384, 391, 409, 413, 414, 421, 463, 517, and 524. In some aspects, the seven amino acid substitutions are at positions 354, 384, 409, 413, 421, 463, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F. In some aspects, the seven amino acid substitutions are at positions 354, 391, 413, 421, 463, 517, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L. In some aspects, the seven amino acid substitutions are at positions 354, 391, 413, 414, 421, 517, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F.

[0100] In some embodiments, the one or more additional amino acid substitutions are eight amino acid substitutions at positions 384, 391, 409, 413, 421, 463, 517, and 524 of SEQ ID NO:1, and wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F.

[0101] In some aspects, the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an N413D mutation, an H524 substitution, and an S463P substitution.

[0102] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 90 or 103.

[0103] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L384M substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 91 or 104.

[0104] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 92 or 105.

[0105] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 93 or 106.

[0106] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 94 or 107.

[0107] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an L391V substitution, an L409V substitution, a Q414E substitution, an N413D substitution, an S463P substitution, an L354I substitution, and an H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 95 or 108.

[0108] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, an L384M substitution, and an H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 96 or 109.

[0109] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 97 or 110.

[0110] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a L384M substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 98 or 111.

[0111] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 99 or 112.

[0112] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an N413D substitution, an S463P substitution, an L354I substitution, an L384M substitution, and an H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 100 or 113.

[0113] In some aspects, the one or more additional amino acid substitutions in the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, and an H524L substitution. In some aspects, the polypeptide, first polypeptide monomer, and / or second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 101 or 114.

[0114] Also provided herein is an inducible cell death system comprising a first polypeptide and a second polypeptide monomer, wherein the first and second polypeptide monomers each comprise a ligand-binding domain and a cell death-inducing domain, wherein the first and second polypeptide monomers are configured to oligomerize upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in a cell in which the first and second polypeptide monomers are expressed, and wherein the ligand-binding domain comprises a modified estrogen receptor ligand-binding domain (ER-LBD) comprising an amino acid sequence corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a (b) additional amino acid substitutions, wherein the additional amino acid substitutions are, with reference to SEQ ID NO: 1: (i) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) additional amino acid substitutions, wherein the additional amino acid substitutions are, with reference to SEQ ID NO: 1: (i) a L384M substitution, a L391V substitution, a N413D substitution, a M421L substitution, a S463P substitution, and a H524L substitution. (ii) an L391V substitution, an N413D substitution, a Q414E substitution, an S463P substitution, and an H524F substitution; (iii) an L354I substitution, an L391V substitution, an N413D substitution, a Q414E substitution, an M421L substitution, an M517A substitution, and an H524F substitution; or (iv) an L354I substitution, an L391V substitution, an L409V substitution, an N413D substitution, a Q414E substitution, and an H524L substitution.

[0115] In some embodiments, the ligand is a non-endogenous ligand. Exemplary non-endogenous ligands are provided herein. In some embodiments, the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. In some embodiments, the non-endogenous ligand includes a tamoxifen metabolite. In some embodiments, the non-endogenous ligand is endoxifen. In some embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating a cell death-inducing signal at a concentration of 0.25 nM endoxifen or less and / or at a concentration of 0.04 nM 4-OHT or less. In some embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating a cell death-inducing signal at a concentration of 2.5 nM endoxifen or less and / or at a concentration of 0.4 nM 4-OHT or less. In some aspects, the first polypeptide monomer and the second polypeptide monomer are capable of generating an oligomerization and / or cell death-inducing signal at a concentration of at least 0.001 pM 4-OHT, hi some aspects, the first polypeptide monomer and the second polypeptide monomer are capable of generating an oligomerization and / or cell death-inducing signal at a concentration of at least 0.01 pM 4-OHT.

[0116] In some embodiments, the cell death-inducing domain is selected from the group consisting of caspase (e.g., any one of caspases 1 to 11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, and caspase 9), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspases (SMAC), Omi, Bmf, and Bid. , Bim, p53-upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, and purine nucleoside phosphorylase. In some embodiments, the cell death-inducing domain comprises a caspase domain or a derivative or functional fragment thereof. In some embodiments, the caspase is selected from any one of caspases 1 to 11, such as caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or a derivative or functional fragment thereof. In some embodiments, the caspase is caspase 9 or a functional fragment thereof. In some embodiments, the cell death induction domain comprises an amino acid sequence derived from caspase 9 set forth in SEQ ID NO: 48 or 125. In some embodiments, the caspase domain or a derivative or functional fragment thereof, e.g., inducible Casp-9, does not comprise a caspase activation and recruitment domain (CARD) domain sequence.

[0117] In some aspects, the cell death-inducing domain is a transcription factor comprising a nucleic acid binding domain and a transcription effector domain, wherein the transcription factor is configured to generate a cell death-inducing signal by inducing expression of a caspase domain or a derivative or functional fragment thereof, optionally wherein the caspase (e.g., any one of caspases 1 to 11) is selected from the group consisting of caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or a derivative or functional fragment thereof, diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with a death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TNF). RADD), TNF receptor (TNF-R), APAF-1, Granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53 upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, or purine nucleoside phosphorylase.

[0118] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a N413D substitution, a S463P substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1.

[0119] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1.

[0120] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1.

[0121] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include an L391V substitution, an L409V substitution, a Q414E substitution, an N413D substitution, an S463P substitution, an M517A substitution, and an H524L substitution, with reference to SEQ ID NO: 1.

[0122] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an M421L substitution, an L354I substitution, and an H524L substitution, with reference to SEQ ID NO: 1.

[0123] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO:1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO:1.

[0124] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, an M421L substitution, and an H524L substitution, with reference to SEQ ID NO: 1.

[0125] Also provided herein are modified estrogen receptor ligand binding domains (ER-LBDs) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1.

[0126] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO:1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO:1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO:1.

[0127] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1.

[0128] Also provided herein are modified estrogen receptor ligand binding domains (ER-LBDs) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include a L391V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1.

[0129] Also provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises (a) a G400V amino acid substitution, an M543A amino acid substitution, an L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and (b) one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions include an L391V substitution, an L409V substitution, an N413D substitution, an S463P substitution, an M517A substitution, and an H524L substitution, with reference to SEQ ID NO: 1.

[0130] Also provided herein is a modified ER-LBD comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 90-114.

[0131] In some embodiments of the modified ER-LBD, the modified ER-LBD has greater sensitivity and / or selectivity to a non-endogenous ligand compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2, or compared to an endogenous ligand as a result of one or more additional amino acid substitutions. In some embodiments, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen. In some embodiments, the endogenous ligand is estradiol. In some embodiments, the modified ER-LBD further comprises a V595A amino acid substitution.

[0132] Also provided herein are chimeric proteins comprising a polypeptide of interest fused to a modified ER-LBD described herein. In some aspects, the polypeptide of interest comprises a nucleic acid binding domain. In some aspects, the nucleic acid binding domain comprises a zinc finger domain. In some aspects, the nucleic acid binding domain comprises a zinc finger domain. In some aspects, the zinc finger domain comprises the sequence MSRPGERPFQCRICMRNFSNMSNLTRHTRTHTGEKPFQCRICMRNFSDRSVLRRHLRTHTGSQKPFQCRICMRNFSDPSNLARHTRTHTGEKPFQCRICMRNFSDRSSLRRHLRTHTGSQKPFQCRICMRNFSQSGTLHRHTRTHTGEKPFQCRICMRNFSQRPNLTRHLRTHLRGS (SEQ ID NO: 62). In some aspects, the zinc finger domain comprises the sequence set forth in SEQ ID NO: 57 or SEQ ID NO: 84. In some aspects, the chimeric protein comprises a chimeric transcription factor, wherein the polypeptide of interest comprises a nucleic acid binding domain and a transcription regulator domain. In some embodiments, the transcriptional regulator domain is a transcriptional activator selected from the group consisting of a herpes simplex virus protein 16 (VP16) activation domain, an activation domain comprising four tandem copies of VP16, a VP64 activation domain, the p65 activation domain of NFκB (p65), an Epstein-Barr virus R transactivator (Rta) activation domain, a tripartite activator comprising VP64, p65, and Rta activation domains (VPR activation domain), a tripartite activator comprising VP64, p65, and HSF1 activation domains (VPH activation domain), and the histone acetyltransferase core domain of human E1A-associated protein p300 (p300 HAT core activation domain).In some embodiments, the transcription regulator domain is a p65 transcription activator comprising the amino acid sequence DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64).

[0133] Also provided herein is an isolated polynucleotide comprising a nucleotide sequence encoding a modified ER-LBD, as described herein. Also provided herein is an isolated polynucleotide comprising a nucleotide sequence encoding a chimeric protein, a polypeptide, a first polypeptide monomer, and / or a second polypeptide monomer, as described herein.

[0134] Also provided herein is a heterologous construct comprising a promoter operably linked to a polynucleotide as described herein.

[0135] Also provided herein is a plasmid or vector comprising a heterologous construct as described herein.

[0136] Also provided herein are cells comprising a heterologous construct as described herein or a plasmid or vector as described herein.

[0137] Also provided herein is a molecular switch for generating a cell death-inducing signal in a cell, comprising: (a) an inducible cell death system described herein, an isolated polynucleotide described herein, a heterologous construct described herein, a plasmid or vector described herein, or a cell described herein, wherein the inducible cell death system is capable of generating a cell death-inducing signal in the cell; and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD generates a cell death-inducing signal in the cell.

[0138] Also provided herein is a molecular switch for generating a cell death-inducing signal in a cell, comprising: (a) an inducible cell death system described herein, an isolated polynucleotide described herein, a heterologous construct described herein, a plasmid or vector described herein, or a cell described herein, wherein the inducible cell death system is capable of generating a cell death-inducing signal in the cell upon oligomerization of the first and second polypeptide monomers; and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD induces oligomerization of the first and second polypeptide monomers, thereby generating a cell death-inducing signal in the cell.

[0139] Exemplary non-endogenous ligands are provided herein. In some aspects, the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0140] Also provided herein is a method of inducing oligomerization of a chimeric protein, comprising: (i) transforming a cell with a heterologous construct encoding any one of the inducible cell death systems described herein, the isolated polynucleotide described herein, the heterologous construct described herein, the plasmid or vector described herein, or the cell described herein, wherein the inducible cell death system is capable of generating a cell death-inducing signal in the cell upon oligomerization of the first and second polypeptide monomers; and (ii) contacting the transformed cell with a non-endogenous ligand of the modified estrogen receptor ligand-binding domain (ER-LBD).

[0141] Also provided herein are methods of modulating transcription of a gene of interest, comprising transforming a cell with (i) a heterologous construct encoding a chimeric protein as described herein, and (ii) a target expression cassette comprising a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to the gene of interest, and contacting the transformed cell with a non-endogenous ligand, thereby inducing the chimeric protein to modulate transcription of the gene of interest. In some aspects, the method further comprises culturing the transformed cell under conditions suitable for expression of the chimeric protein prior to inducing the chimeric protein to modulate transcription. In some aspects, modulating transcription comprises activating transcription of the gene of interest. In some aspects, the target expression cassette is encoded by a heterologous construct encoding a chimeric protein described herein, or the target expression cassette is encoded by a second heterologous construct. Exemplary non-endogenous ligands are provided herein. In some aspects, the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.In some embodiments, the gene of interest is selected from a caspase domain (e.g., any one of caspases 1-11) or a derivative or functional fragment thereof, optionally wherein the caspase is selected from caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or a derivative or functional fragment thereof, diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, caspase 1, caspase 2, caspase 3, caspase 4, caspase 5 ... second mitochondrial-derived activator of apoptosis (SMAC), Omi, Bmf, Bid, Bim, p53 up-regulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, or purine nucleoside phosphorylase.

[0142] Also provided herein is a method for modulating the localization of a chimeric protein, comprising transforming a cell with a heterologous construct encoding the chimeric protein described herein and inducing nuclear localization of the chimeric protein by contacting the transformed cell with a non-endogenous ligand. In some aspects, the method further comprises culturing the transformed cell under conditions suitable for expression of the chimeric protein before inducing nuclear localization. Exemplary non-endogenous ligands are provided herein. In some aspects, the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0143] In some aspects, the transformed cell is in a human or animal, and wherein contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal.

[0144] In some embodiments, the non-endogenous ligand is administered at a concentration where the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha of SEQ ID NO:1.

[0145] Provided herein are modified ER-LBDs comprising an amino acid sequence corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1) and one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are with reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, and positions 517-540, and position 547 of SEQ ID NO: 1. In some aspects, the modified ER-LBDs described herein further comprise a G400V amino acid substitution, a M543A amino acid substitution, and a L544A amino acid substitution, with reference to SEQ ID NO: 1. In some aspects, the modified ER-LBDs further comprise a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and a V595A amino acid substitution, with reference to SEQ ID NO: 1.

[0146] In some aspects, the modified ER-LBD comprises a G400V, an M543A, and an L544A amino acid substitution, as well as one or more additional amino acid substitutions. In some aspects, the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2. In some aspects, the modified ER-LBD has greater selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2.

[0147] In some aspects, the modified ER-LBD comprises a G400V, an M543A, an L544A, and a V595A amino acid substitution, as well as one or more additional amino acid substitutions. In some aspects, the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 3. In some aspects, the modified ER-LBD has greater selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 3.

[0148] In some aspects, the modified ER-LBD of the present disclosure has greater sensitivity to a non-endogenous ligand compared to an endogenous ligand as a result of one or more additional amino acid substitutions.

[0149] In some aspects, the modified ER-LBD of the present disclosure has greater sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0150] In some aspects, the modified ER-LBD of the present disclosure has greater selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0151] In some aspects, the one or more additional amino acid substitutions are at one or more positions of SEQ ID NO: 1 selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537, 538, 539, 540, and 547.

[0152] In some aspects, the one or more positions include position 343 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 343 is selected from the group consisting of M343F, M343I, M343L, and M343V.

[0153] In some aspects, the one or more positions include position 344 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 344 is G344M.

[0154] In some aspects, the one or more positions include position 345 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 345 is L345S.

[0155] In some aspects, the one or more positions include position 346 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 346 is selected from the group consisting of L346I, L346M, L346F, and L346V.

[0156] In some aspects, the one or more positions comprises position 347 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 347 is selected from the group consisting of T347D, T347E, T347F, T347I, T347K, T347L, T347M, T347N, T347Q, T347R, T347S, and T347V.

[0157] In some aspects, the one or more positions include position 348 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 348 is N348K.

[0158] In some aspects, the one or more positions include position 349 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 349 is selected from the group consisting of L349I, L349M, L349F, and L349V.

[0159] In some aspects, the one or more positions include position 350 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 350 is selected from the group consisting of A350F, A350I, A350L, A350M, and A350V.

[0160] In some aspects, the one or more positions include position 351 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 351 is selected from the group consisting of D351E, D351F, D351I, D351L, D351M, D351N, D351Q, and D351V.

[0161] In some aspects, the one or more positions include position 352 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 352 is R352K.

[0162] In some aspects, the one or more positions include position 354 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 is selected from the group consisting of L354I, L354M, L354F, and L354V.

[0163] In some aspects, the one or more positions include position 380 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 380 is E380Q.

[0164] In some aspects, the one or more positions include position 384 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 384 is selected from the group consisting of L384I, L384M, L384F, and L384V.

[0165] In some aspects, the one or more positions include position 386 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 386 is I386V.

[0166] In some aspects, the one or more positions include position 387 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 387 is selected from the group consisting of L387I, L387M, L387F, and L387V.

[0167] In some aspects, the one or more positions include position 388 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 388 is selected from the group consisting of M388I, M388L, and M388F.

[0168] In some aspects, the one or more positions include position 389 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 389 is I389M.

[0169] In some aspects, the one or more positions include position 391 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 391 is selected from the group consisting of L391I, L391M, L391F, and L391V.

[0170] In some aspects, the one or more positions include position 392 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 392 is V392M.

[0171] In some aspects, the one or more positions include position 404 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 404 is selected from the group consisting of F404I, F404L, F404M, and F404V.

[0172] In some aspects, the one or more positions include position 407 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 407 is N407D.

[0173] In some aspects, the one or more positions include position 409 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 409 is L409V.

[0174] In some aspects, the one or more positions include position 413 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 413 is N413D.

[0175] In some aspects, the one or more positions include position 414 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 414 is Q414E.

[0176] In some aspects, the one or more positions include position 417 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 417 is C417S.

[0177] In some aspects, the one or more positions include position 418 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 418 is selected from the group consisting of V418I, V418L, V418M, and V418F.

[0178] In some aspects, the one or more positions include position 420 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 420 is selected from the group consisting of G420I, G420M, G420F, and G420V.

[0179] In some aspects, the one or more positions include position 421 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 421 is selected from the group consisting of M421I, M421L, M421F, and M421V.

[0180] In some aspects, the one or more positions include position 422 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 422 is V422I.

[0181] In some aspects, the one or more positions include position 424 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 424 is selected from the group consisting of I424L, I424M, I424F, and I424V.

[0182] In some aspects, the one or more positions include position 428 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 428 is selected from the group consisting of L428I, L428M, L428F, and L428V.

[0183] In some aspects, the one or more positions include position 463 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 463 is S463P.

[0184] In some aspects, the one or more positions include position 517 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 517 is M517A.

[0185] In some aspects, the one or more positions include position 521 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 521 is selected from the group consisting of G521A, G521F, G521I, G521L, G521M, and G521V.

[0186] In some aspects, the one or more positions include position 522 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 522 is selected from the group consisting of M522I, M522L, and M522V.

[0187] In some aspects, the one or more positions include position 524 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 524 is selected from the group consisting of H524A, H524I, H524L, H524F, and H524V.

[0188] In some aspects, the one or more positions include position 525 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 525 is selected from the group consisting of L525F, L525I, L525M, L525N, L525Q, L525S, L525T, and L525V.

[0189] In some aspects, the one or more positions include position 526 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 526 is Y526L.

[0190] In some aspects, the one or more positions include position 527 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 527 is S527N.

[0191] In some aspects, the one or more positions include position 528 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 528 is selected from the group consisting of M528F, M528I, and M528V.

[0192] In some aspects, the one or more positions comprises position 533 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 533 is selected from the group consisting of V533F and V533W.

[0193] In some aspects, the one or more positions comprises position 534 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 534 is selected from the group consisting of V534Q and V534R.

[0194] In some aspects, the one or more positions include position 536 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 536 is selected from the group consisting of L536F, and L536M, L536R, and L536Y.

[0195] In some aspects, the one or more positions comprises position 537 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 537 is selected from the group consisting of Y537E and Y537S.

[0196] In some aspects, the one or more positions include position 538 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 538 is selected from the group consisting of D538G and D538K.

[0197] In some aspects, the one or more positions include position 539 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 539 is selected from the group consisting of L539A and L539R.

[0198] In some aspects, the one or more positions include position 540 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 540 is selected from the group consisting of L540A and L540F.

[0199] In some aspects, the one or more positions include position 547 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 547 is H547A.

[0200] In some aspects, the one or more additional amino acid substitutions comprise two amino acid substitutions, each of which is at a position of SEQ ID NO: 1 selected from 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525.

[0201] In some aspects, the two amino acid substitutions are at positions 345 and 348 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 345 of SEQ ID NO: 1 is L345S, and the amino acid substitution at position 348 of SEQ ID NO: 1 is N348K.

[0202] In some aspects, the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 384 of SEQ ID NO: 1 is L384M, and the amino acid substitution at position 389 of SEQ ID NO: 1 is I389M.

[0203] In some aspects, the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 421 of SEQ ID NO: 1 is M421I, and the amino acid substitution at position 392 of SEQ ID NO: 1 is V392M.

[0204] In some aspects, the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 of SEQ ID NO: 1 is L354I, and the amino acid substitution at position 391 of SEQ ID NO: 1 is L391F.

[0205] In some aspects, the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 of SEQ ID NO: 1 is L354I, and the amino acid substitution at position 384 of SEQ ID NO: 1 is L384M.

[0206] In some aspects, the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 of SEQ ID NO: 1 is L354I, and the amino acid substitution at position 387 of SEQ ID NO: 1 is L387M.

[0207] In some embodiments, the two amino acid substitutions are at positions 387 and 391. In some embodiments, the amino acid substitution at position 387 of SEQ ID NO:1 is L387M, and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F.

[0208] In some aspects, the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 384 of SEQ ID NO: 1 is L384M, and the amino acid substitution at position 387 of SEQ ID NO: 1 is L387M.

[0209] In some aspects, the two amino acid substitutions are at positions 384 and 391 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 384 of SEQ ID NO: 1 is L384M, and the amino acid substitution at position 391 of SEQ ID NO: 1 is L391F.

[0210] In some embodiments, the one or more additional amino acid substitutions comprise three amino acid substitutions, hi some embodiments, each of the three amino acid substitutions is at a position in SEQ ID NO: 1 selected from the group consisting of 343, 347, 351, 354, 388, 391, 404, 418, 521, 524, and 525.

[0211] In some aspects, the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 of SEQ ID NO: 1 is L354I, the amino acid substitution at position 384 of SEQ ID NO: 1 is L384M, and the amino acid substitution at position 391 of SEQ ID NO: 1 is L391F.

[0212] In some aspects, the one or more additional amino acid substitutions include four amino acid substitutions.

[0213] In some aspects, the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 354 of SEQ ID NO: 1 is L354I, the amino acid substitution at position 384 of SEQ ID NO: 1 is L384M, the amino acid substitution at position 391 of SEQ ID NO: 1 is L391F, and the amino acid substitution at position 418 of SEQ ID NO: 1 is V418I.

[0214] In some aspects, the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 343 of SEQ ID NO: 1 is M343I, the amino acid substitution at position 388 of SEQ ID NO: 1 is M388I, the amino acid substitution at position 521 of SEQ ID NO: 1 is G521I, and the amino acid substitution at position 404 of SEQ ID NO: 1 is F404L.

[0215] In some aspects, the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO: 1. In some aspects, the amino acid substitution at position 524 of SEQ ID NO: 1 is H524V, the amino acid substitution at position 347 of SEQ ID NO: 1 is T347R, the amino acid substitution at position 351 of SEQ ID NO: 1 is D351Q, and the amino acid substitution at position 525 of SEQ ID NO: 1 is L525N.

[0216] Exemplary non-endogenous ligands are provided herein. In some aspects, the non-endogenous ligand is selected from 4-hydroxytamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0217] Also provided are chimeric proteins comprising a polypeptide of interest fused to a modified ER-LBD as described herein. In some aspects, the polypeptide of interest comprises a nucleic acid binding domain. In some aspects, the nucleic acid binding domain comprises a zinc finger (ZF) domain. In some aspects, the chimeric protein is a transcription factor and the polypeptide of interest comprises a transcription regulator domain.

[0218] Also provided are isolated polynucleotides that encode the modified ER-LBDs described herein or the chimeric proteins described herein.

[0219] Also provided are heterologous constructs comprising a promoter operably linked to a polynucleotide encoding a modified ER-LBD described herein or a chimeric protein described herein.

[0220] Also provided are plasmids and vectors that contain the heterologous constructs described herein.

[0221] Also provided are cells (e.g., isolated cells or populations of cells) comprising the heterologous constructs described herein or the plasmids described herein.

[0222] Also provided are molecular switches for regulating transcription of a gene of interest. In some aspects, the molecular switch comprises a chimeric protein comprising a modified ER-LBD described herein, a transcriptional regulator, and a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD induces the chimeric protein to regulate transcription of the gene of interest. Exemplary non-endogenous ligands are provided herein. In some aspects, the non-endogenous ligand of the molecular switch is selected from 4-hydroxytamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0223] Also provided herein are methods for modulating transcription of a gene of interest. In some aspects, the method comprises: (a) transforming a cell with (i) a heterologous construct encoding a chimeric protein comprising a modified ER-LBD and a transcriptional regulator domain, and (ii) a target expression cassette comprising a gene of interest; (b) culturing the transformed cell under conditions suitable for expression of the chimeric protein; and (c) contacting the transformed cell with a non-endogenous ligand, thereby inducing the chimeric protein to regulate transcription of the gene of interest.

[0224] In some aspects, the method of modulating transcription is a method of activating transcription.

[0225] In some aspects, the method of modulating transcription is a method of repressing transcription.

[0226] In some aspects, the target expression cassette is encoded by a heterologous construct that encodes a chimeric protein.

[0227] In some aspects, the target expression cassette is encoded by a heterologous construct that is different from the heterologous construct encoding the chimeric protein.

[0228] Also provided are methods for modulating the localization of a polypeptide of interest. In some aspects, the methods include (a) transforming a cell with a heterologous construct encoding a chimeric protein comprising the polypeptide of interest fused to a modified ER-LBD described herein; (b) culturing the transformed cell under conditions suitable for expression of the chimeric protein; and (c) contacting the transformed cell with a non-endogenous ligand to induce nuclear localization of the chimeric protein.

[0229] In some aspects, the transformed cell of any of the methods described herein is in a human or animal, hi some aspects, contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal.

[0230] Exemplary non-endogenous ligands are provided herein. In some embodiments, the non-endogenous ligand in step (c) of the previously described methods is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0231] In some embodiments, the non-endogenous ligand is administered at a concentration where the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha. [Brief explanation of the drawings]

[0232] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0233] [Figure 1A] Figures 1A and 1B provide binding energy calculations for the first set of mutations analyzed in silico: Figure 1A provides the binding energy calculation for binding to estradiol, and Figure 1B provides the binding energy calculation for binding to 4-OHT. [Figure 1B] Figures 1A and 1B provide binding energy calculations for the first set of mutations analyzed in silico: Figure 1A provides the binding energy calculation for binding to estradiol, and Figure 1B provides the binding energy calculation for binding to 4-OHT. [Figure 2] Figure 2 provides binding energy calculations of the 4-OHT bond for the second set of mutations analyzed in silico. [Figure 3] Figure 3 provides binding energy calculations for the 4-OHT bond for the third set of mutations analyzed in silico. [Figure 4] Figure 4 provides binding energy calculations for the 4-OHT bond for the fourth set of mutations analyzed in silico. [Figure 5] Figure 5 provides binding energy calculations for the 4-OHT bond for the fifth set of mutations analyzed in silico. [Figure 6] FIG. 6 shows the structural differences in the orientation and docking site of helix 12 between the estradiol-bound and non-endogenous ligand-bound conformations. [Figure 7] FIG. 7 provides binding energy calculations for the agonist-bound versus antagonist-bound conformations for the sixth set of mutations analyzed in silico. [Figure 8A] Figures 8A, 8B, and 8C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT assayed in the first transfection screen. [Figure 8B] Figures 8A, 8B, and 8C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT assayed in the first transfection screen. [Figure 8C] Figures 8A, 8B, and 8C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT assayed in the first transfection screen. [Figure 9A] Figures 9A, 9B, and 9C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a second transfection screen. [Figure 9B] Figures 9A, 9B, and 9C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a second transfection screen. [Figure 9C]Figures 9A, 9B, and 9C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a second transfection screen. [Figure 10A] Figures 10A, 10B, and 10C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a third transfection screen. [Figure 10B] Figures 10A, 10B, and 10C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a third transfection screen. [Figure 10C] Figures 10A, 10B, and 10C show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a third transfection screen. [Figure 11A] Figures 11A and 11B show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in the first transfection screen. [Figure 11B] Figures 11A and 11B show the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in the first transfection screen. [Figure 12] FIG. 12 shows the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a second transfection screen. [Figure 13] FIG. 13 shows the effect of various modified ER-LBDs on reporter expression across various concentrations of 4-OHT, as assayed in a second transfection screen. [Figure 14] Figures 14A and 14B show the scaffold for high-throughput protein engineering of ERT2 (SB04401) and OFF mCherry reporter constructs (SB01066). [Figure 15] Figures 15A and 15B show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen and 4-OHT, as assayed in a combinatorial library screen. [Figure 16A] Figures 16A, 16B, 16C, 16D, and 16E show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen, 4-OHT, and estradiol, as assayed in a validation screen. SB03422 is a wild-type ER-LBD and is included as a benchmark for performance. [Figure 16B] Figures 16A, 16B, 16C, 16D, and 16E show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen, 4-OHT, and estradiol, as assayed in a validation screen. SB03422 is a wild-type ER-LBD and is included as a benchmark for performance. [Figure 16C] Figures 16A, 16B, 16C, 16D, and 16E show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen, 4-OHT, and estradiol, as assayed in a validation screen. SB03422 is a wild-type ER-LBD and is included as a benchmark for performance. [Figure 16D] Figures 16A, 16B, 16C, 16D, and 16E show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen, 4-OHT, and estradiol, as assayed in a validation screen. SB03422 is a wild-type ER-LBD and is included as a benchmark for performance. [Figure 16E]Figures 16A, 16B, 16C, 16D, and 16E show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen, 4-OHT, and estradiol, as assayed in a validation screen. SB03422 is a wild-type ER-LBD and is included as a benchmark for performance. [Figure 17] Figures 17A and 17B show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen and 4-OHT, as assayed in NK cells. Arrows indicate estimated pharmacologically relevant concentrations of 4-OHT or endoxifen in humans. [Figure 18] Figures 18A and 18B show the effect of various modified ER-LBDs on IL-12 expression across various concentrations of endoxifen, as assayed in NK cells. [Figure 19] Figure 19 illustrates an exemplary mechanism of action of an inducible cell death system, including ER-mediated transcription induction via nuclear localization (top panel) and ER-mediated suicide switch killing via dimerization (bottom panel). [Figure 20] FIG. 20 shows suicide switch-induced killing for constructs with modified ER-LBD variants in HEK293T cells with 1 μM 4-OHT for 48 hours. [Figure 21] FIG. 21 shows suicide switch-induced killing for constructs with modified ER-LBD variants in HEK293T cells over a 48 hour period at the indicated concentrations of 4-OHT. [Figure 22] FIG. 22 shows suicide switch-induced killing for constructs with modified ER-LBD variants in transduced primary T cells over 5 days under the indicated treatment conditions. [Figure 23] FIG. 23 shows suicide switch-induced killing for constructs with modified ER-LBD variants in transduced primary T cells over the indicated time periods and under the indicated treatment conditions. [Figure 24]Figure 24 shows the results of a time course experiment for the indicated drug conditions assessing the safety-switch activity of exemplary ERT2 mutations in HEK cells. [Figure 25] Figure 25 shows the results of a time course experiment for the indicated drug conditions assessing the safety-switch activity of exemplary ERT2 mutations in HEK cells. [Figure 26A] FIG. 26A depicts the constructs and experimental method for assessing the activity of an exemplary ERT2 mutant transcriptional switch. [Figure 26B] Figure 26B shows the log 10-fold activation across a range of endoxifen concentrations, normalized to the virus-free control. [Figure 26C] Figure 26C shows induced mCherry expression plotted against basal activity for each of the ERT2 transcriptional switch constructs tested. [Figure 27] FIG. 27 shows the results of an experiment assessing the activity of an exemplary ERT2 mutant transcriptional switch, where the readout is mCherry gMFI after background subtraction over a range of estradiol and endoxifen concentrations. [Figure 28A] FIG. 28A depicts the constructs and experimental methodology for evaluating an exemplary ERT2 mutant transcriptional switch to induce expression of an IL-12 payload. [Figure 28B] Figures 28B and 28C show the results of experiments evaluating an exemplary ERT2 mutant transcriptional switch to induce expression of an IL-12 payload. [Figure 28C] Figures 28B and 28C show the results of experiments evaluating an exemplary ERT2 mutant transcriptional switch to induce expression of an IL-12 payload. [Figure 29A] FIG. 29A shows the experimental workflow for assessing suicide switch activity for a number of additional ERT2 mutant safety switch constructs. [Figure 29B-1] Figure 29B shows the results of experiments assessing the suicide switch activity of additional ERT2 mutation safety-switch constructs. [Figure 29B-2]Figure 29B shows the results of experiments assessing the suicide switch activity of additional ERT2 mutation safety-switch constructs. DETAILED DESCRIPTION OF THE INVENTION

[0234] Terms used in the claims and specification, unless otherwise specified, are defined as set forth below.

[0235] The term "in vivo" refers to a process that occurs in a living organism.

[0236] 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.

[0237] The term percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, have a specified percentage of the same nucleotides or amino acid residues, 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 alternatively, over the entire length of the two sequences being compared.

[0238] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered 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 for the test sequence relative to the reference sequence, based on the designated program parameters.

[0239] Optimal alignment of sequences for comparison can be achieved, 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 search for similarity 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).

[0240] 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 / ).

[0241] 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.

[0242] The term "therapeutically effective amount" is an amount that is effective for ameliorating symptoms of a disease. A therapeutically effective amount can also be a "prophylactically effective amount," since prevention can be considered treatment.

[0243] 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.

[0244] The term "about" is used herein to provide literal support for the exact term it precedes, allowing for approximation of that term. For example, a particular range or similarity may be presented with a numerical range preceded by the term "about." If the degree of approximation is not otherwise clear from the context, "about" means either within plus or minus 10% of the provided value, or approximating to the nearest significant numerical value, and in all cases encompasses the provided value. In some embodiments, the term "about" refers to up to ±10%, ±5%, or ±1% of the specified value.

[0245] Inducible cell death system The ability to induce cell death using synthetic ligands, particularly those approved for clinical use, would be beneficial in therapeutic applications, such as in the fields of cell therapy and gene therapy. Cell therapy and gene therapy show great clinical promise; however, they carry the risk of off-target toxicity that can result in organ damage and patient death. To address potential toxicity concerns, it would be desirable to engineer cell therapy and gene therapy products with a "safety" switch, e.g., an inducible cell death system.

[0246] Synthetic ligands that bind to ER have been developed to treat ER-positive cancers, such as ER-positive breast cancer. For example, the active metabolite of the drug tamoxifen induces nuclear translocation of ER and antagonizes ER in a tissue-selective manner.

[0247] Tamoxifen and its active metabolites have also been used as tools to control nuclear localization in research settings. For example, an ER ligand-binding domain variant known as ERT2 has been used as a fusion protein with Cre recombinase to regulate Cre recombinase-based gene editing in animal model systems.

[0248] As shown herein, engineered ERT2 mutants with additional amino acid substitutions can be implemented in an inducible cell death system to induce potent cell killing. Accordingly, provided herein is an inducible cell death system comprising a modified estrogen receptor ligand-binding domain (ER-LBD). The inducible cell death system described herein can comprise a polypeptide, wherein the polypeptide comprises a ligand-binding domain and a cell death-inducing domain. In some embodiments, the polypeptide herein is configured to regulate a cell death induction pathway in a cell in which the polypeptide is expressed upon contact of the ligand-binding domain with a ligand. In some embodiments, the polypeptide herein is configured to generate a cell death-inducing signal in a cell in which the polypeptide is expressed upon contact of the ligand-binding domain with a ligand.

[0249] FIG. 19 shows an exemplary inducible cell death system, including ER-mediated transcription induction via nuclear localization (top panel) and ER-mediated suicide switch killing via dimerization (bottom panel).

[0250] In some embodiments, the polypeptides herein are configured to regulate expression of a polynucleotide and / or additional polypeptide of a cell death-inducing pathway in a cell in which the polypeptide is expressed upon contact of the ligand-binding domain with a ligand. In exemplary embodiments of polypeptides comprising a modified ER-LBD and a cell death-inducing domain disclosed herein, the cell death-inducing domain is a transcription factor comprising a nucleic acid-binding domain disclosed herein (e.g., the ZF10-1 DNA-binding domain depicted in FIG. 19, top panel) and a transcription effector domain, also referred to herein as a transcriptional regulator (e.g., p65 depicted in FIG. 19, top panel). Without wishing to be bound by theory, upon contact of a ligand with the ligand-binding domain (e.g., where contact includes binding of the ligand to the ligand-binding domain), the polypeptide may translocate to the nucleus and activate expression of a death-inducing payload.

[0251] Inducible cell death systems include systems in which the polypeptide is or comprises a first polypeptide monomer, and the inducible cell death system further comprises a second polypeptide monomer. "Polypeptide monomer" refers to a protein, protein subunit, and / or protein domain that is configured to generate a cell death-inducing signal in a cell in which the first and second polypeptide monomers are expressed upon contact with a ligand of the ER-LBD (e.g., where contacting comprises binding of the ligand to the ligand-binding domains of the first and second polypeptide monomers). In some embodiments, the first and second polypeptide monomers are configured to oligomerize with each other upon contact with a ligand of the ligand-binding domain (e.g., where contacting comprises binding of the ligand to the ligand-binding domains of the first and second polypeptide monomers), thereby generating a cell death-inducing signal in a cell in which the first and second polypeptide monomers are expressed. Without wishing to be bound by theory, binding of the ligand-binding domains of the first and second polypeptide monomers to a ligand can induce oligomerization of the polypeptide monomers, where the oligomerization activates the death-inducing domain to generate a death-inducing signal. See, e.g., Figure 19 (lower panel, depicting caspase-9 as an exemplary death-inducing domain).

[0252] Such inducible cell death systems can beneficially improve the safety profile of cell and gene therapy products.

[0253] Exemplary cell death-inducing domains and / or death-inducing payloads may be derived from proteins, such as caspases (e.g., any one of caspases 1 to 11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, etc.), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspases (SMA), and the like. C), Omi, Bmf, Bid, Bim, p53 upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related apoptosis-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, and / or purine nucleoside phosphorylase. Exemplary sequences can be found in Table A.

[0254] (Table A) TIFF2026504440000002.tif248164TIFF2026504440000003.tif253164TIFF202 6504440000004.tif254164TIFF2026504440000005.tif253164TIFF20265044400 00006.tif253164TIFF2026504440000007.tif253164TIFF2026504440000008.t if233164TIFF2026504440000009.tif253164TIFF2026504440000010.tif252164 TIFF2026504440000011.tif253164TIFF2026504440000012.tif253164TIFF202 6504440000013.tif253164TIFF2026504440000014.tif253164TIFF20265044400 00015.tif253164TIFF2026504440000016.tif253164TIFF2026504440000017.t if253164TIFF2026504440000018.tif253164TIFF2026504440000019.tif177164

[0255] The cell death-inducing domain can include or be derived from caspase-9, e.g., the amino acid sequence set forth in SEQ ID NO: 39 or 123. Derivatives of caspase-9 include inducible caspase-9 ("iCasp-9"), which is capable of inducing apoptosis due to drug-based dimerization, e.g., the amino acid sequence set forth in SEQ ID NO: 48 or 125. In some embodiments, the caspase domain or a derivative or functional fragment thereof, e.g., inducible caspase-9, does not include a caspase activation and recruitment domain (CARD) domain sequence.

[0256] The cell death induction domain can include BAX, for example, the amino acid sequence shown in SEQ ID NO:32.

[0257] Regulatable Cell Survival Polypeptides The inducible cell death system may comprise a regulatable cell survival polypeptide that contains a modified estrogen receptor ligand binding domain (ER-LBD).

[0258] Exemplary cell survival polypeptides include one or more of XIAP, Bcl-2, Bcl-Xl, Bcl-w, Bcl-2-related protein A1 (BCL2A1), Mcl-1, FLICE-like inhibitory protein (c-FLIP), and adenoviral E1B-19K protein. Cell survival polypeptides can include XIAP. Cell survival polypeptides can include, for example, wild-type XIAP having the amino acid sequence SEQ ID NO: 107. Cell survival polypeptides can include modified XIAP. Modified XIAP can include one or more amino acid substitutions with reference to SEQ ID NO: 107. Modified XIAP can include one or more amino acid substitutions within positions 305-325 with reference to SEQ ID NO: 107. Modified XIAP can include one or more amino acid substitutions at positions 305, 306, 308, or 325 with reference to SEQ ID NO: 107. The modified XIAP may include one or more amino acid substitutions, including each of 305, 306, 308, and 325, with reference to SEQ ID NO: 107. The modified XIAP may include one or more amino acid substitutions, including each of 305, 306, 308, and 325, with reference to SEQ ID NO: 107, including T308S, G306S, G305M, and P325S. The modified XIAP may include one or more amino acid substitutions, including each of 305, 306, 308, and 325, with reference to SEQ ID NO: 107, including T308D, G306S, G305M, and P325S. The modified XIAP may include an amino acid substitution at position 305 of SEQ ID NO: 107. The modified XIAP may include an amino acid substitution at position 305 of SEQ ID NO: 107 that is G305M. The modified XIAP may include an amino acid substitution at position 306 of SEQ ID NO: 107. The modified XIAP may comprise an amino acid substitution at position 306 of SEQ ID NO: 107 that is G306S. The modified XIAP may comprise an amino acid substitution at position 308 of SEQ ID NO: 107 that is T308S or T308D. The modified XIAP may comprise an amino acid substitution at position 308 of SEQ ID NO: 107 that is T308S. The modified XIAP may comprise an amino acid substitution at position 308 of SEQ ID NO: 107 that is T308D. The modified XIAP may comprise an amino acid substitution at position 325 of SEQ ID NO: 107.The modified XIAP can include an amino acid substitution at position 325 of SEQ ID NO: 107, which is P325S.

[0259] Chimeric proteins In some aspects, the disclosure provides chimeric proteins comprising a polypeptide of interest fused to a modified ER-LBD, wherein the polypeptide of interest can comprise a cell death-inducing domain, and the chimeric protein is configured to generate a cell death-inducing signal in a cell in which the polypeptide is expressed upon contact of the ligand-binding domain with a ligand.

[0260] The polypeptide of interest can include a pro-apoptotic factor, such as a pro-apoptotic transcription factor. The polypeptide of interest can include a pro-cell survival factor and / or an inhibitor of a pro-cell survival factor. The polypeptide of interest can include polypeptide monomers that are generally inactive in monomeric form, but are active upon oligomerization.

[0261] The modified ER-LBD may be capable of inducing oligomerization and / or nuclear localization upon binding to a non-endogenous ligand. Thus, fusion of a modified ER-LBD to a polypeptide of interest may allow for control of the cellular localization and / or oligomerization of the polypeptide of interest.

[0262] In some embodiments, a polypeptide of interest may be fused directly or indirectly to the modified ER-LBD, e.g., via a linker. One or more linkers may be used between various domains of the chimeric protein, such as between the ER-LBD and the polypeptide of interest. For example, a polypeptide linker may include one or more of the amino acid sequences GGGGSGGGGSGGGGSVDGF (SEQ ID NO: 4) and ASGGGGSAS (SEQ ID NO: 5).

[0263] In some embodiments, the polypeptide of interest comprises at least one nucleic acid binding domain. In some embodiments, the nucleic acid binding domain is a zinc finger domain. In some embodiments, the chimeric protein comprises a transcriptional regulator, such as a transcriptional activator or a transcriptional repressor. The inclusion of a nucleic acid binding domain may allow target nucleic acid binding by the chimeric protein to be inducible by a non-endogenous ligand (e.g., 4-OHT or endoxifen).

[0264] In some embodiments, the nucleic acid binding domain comprises a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ZF protein domain is modular in design and composed of a zinc finger array (ZFA). In some embodiments, the transcription effector domain comprises a herpes simplex virus protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16; a VP64 activation domain; the p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains (VPR activation domain); a tripartite activator comprising VP64, p65, and HSF1 activation domain (VPH activation domain); a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (p300 activation domain). HAT core activation domain; Kruppel-associated box (KRAB) repression domain; repressor element silencing transcription factor (REST) ​​repression domain; WRPW motif (SEQ ID NO: 82) of hairy-related basic helix-loop-helix repressor protein (which motif is known as the WRPW (SEQ ID NO: 82) repression domain); DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1 alpha chromoshadow repression domain.

[0265] In some embodiments, ZF protein domains are modular in design and are composed of zinc finger arrays (ZFAs). 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 with specificity 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, a ZFA is an array, string, or chain of ZF motifs arranged in tandem. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 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.

[0266] 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 ZFAs. 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.

[0267] An exemplary ZF protein domain is set forth in the sequence SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGSQKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS (SEQ ID NO: 6). In some embodiments, the ZF protein domain is a ZF5-7 DNA binding domain. An exemplary ZF5-7 DNA binding domain is shown in the sequence MSRPGERPFQCRICMRNFSNMSNLTRHTRTHTGEKPFQCRICMRNFSDRSVLRRHLRTHTGSQKPFQCRICMRNFSDPSNLARHTRTHTGEKPFQCRICMRNFSDRSSLRRHLRTHTGSQKPFQCRICMRNFSQSGTLHRHTRTHTGEKPFQCRICMRNFSQRPNLTRHLRTHLRGS (SEQ ID NO: 62).

[0268] In some embodiments, the chimeric protein is a chimeric transcription factor and comprises, in addition to the modified ER-LBD, a nucleic acid binding domain and a transcription regulator domain. In some aspects, the nucleic acid binding domain and the transcription regulator domain are part of the same naturally occurring protein. In some aspects, the nucleic acid binding domain and the transcription regulator domain are heterologous and do not naturally occur within the same protein.

[0269] As used herein, "transcriptional regulator domain" and "transcriptional effector domain" refer to a polypeptide domain that, when targeted to the promoter region of a gene (e.g., by a nucleic acid binding domain that specifically binds to the promoter of interest), is capable of regulating transcription of the gene. In some embodiments, the transcriptional regulator domain comprises a transcriptional repressor. In some embodiments, the transcriptional repressor comprises a transcriptional repressor domain selected from a 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 (SEQ ID NO: 82), which motif is known as the WRPW (SEQ ID NO: 82) repression domain; a DNA (cytosine 5'-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

[0270] In some embodiments, the transcriptional regulator domain comprises a transcriptional activator. In some embodiments, the transcriptional activator comprises a transcriptional activator domain selected from a herpes simplex virus protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFκB (i.e., p65); an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains (VPR activation domain); a tripartite activator comprising VP64, p65, and HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of human E1A-associated protein p300 (p300 HAT core activation domain). In some embodiments, the transcriptional regulator domain comprises a p65 transcriptional activator. In some embodiments, the p65 transcriptional activator comprises the amino acid sequence DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64).

[0271] Engineered estrogen receptor ligand-binding domain (ER-LBD) The present disclosure provides modified estrogen receptor ligand binding domains (ER-LBDs) comprising an amino acid sequence corresponding to the hormone-binding domain of the reference human estrogen receptor sequence, SEQ ID NO:1 (human estrogen receptor, UniProt ID No:P03372), and comprising the amino acid substitutions G400V, M543A, and L544A, or the amino acid substitutions G400V, M543A, L544A, and V595A, and one or more additional amino acid substitutions to ligand-binding residues selected from positions 343-354, 380-392, 404-463, and 517-540, and 547. Such amino acid substitutions are to be understood with reference to SEQ ID NO:1. In some aspects, the one or more amino acid substitutions result in (a) greater sensitivity to non-endogenous ligands compared to endogenous ligands, (b) greater sensitivity to non-endogenous ligands compared to the ER-LBD of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:3A, or SEQ ID NO:3B, and / or (c) greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:3A, or SEQ ID NO:3B.

[0272] In some embodiments, the one or more additional amino acid substitutions result in greater sensitivity to non-endogenous ligands compared to the ER-LBD of SEQ ID NO:2. In some embodiments, the one or more additional amino acid substitutions result in greater sensitivity to non-endogenous ligands compared to the ER-LBD of SEQ ID NO:3. In some embodiments, the one or more additional amino acid substitutions result in greater sensitivity to non-endogenous ligands compared to the ER-LBD of SEQ ID NO:3A. In some embodiments, the one or more additional amino acid substitutions result in greater sensitivity to non-endogenous ligands compared to the ER-LBD of SEQ ID NO:3B. In some embodiments, the one or more additional amino acid substitutions result in greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO:2. In some embodiments, the one or more additional amino acid substitutions result in greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO:3. In some embodiments, the one or more additional amino acid substitutions result in greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO:3A. In some embodiments, the one or more additional amino acid substitutions confer greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO: 3B.

[0273] In some embodiments, the modified ER-LBD may further comprise other modifications, such as amino acid substitutions, deletions, and / or insertions (with reference to SEQ ID NO: 1). Such other modifications may be within or outside of positions 343-354, 380-392, 404-463, 517-540, and / or 547 with reference to SEQ ID NO: 1. Such other modifications may be within or outside of positions 283-594 with reference to SEQ ID NO: 1.

[0274] "Ligand-binding residues" refers to residues located in the ligand-binding pocket of an estrogen receptor (ER) or ER ligand-binding domain, and includes pockets for binding endogenous ligands (e.g., estradiol) and pockets for binding non-endogenous ligands, such as 4-OHT. In some embodiments, the hormone-binding domain of a reference human estrogen receptor sequence corresponds to positions 282-595 of the human estrogen receptor (SEQ ID NO: 1). It should be understood that a hormone-binding domain does not necessarily require all of amino acid residues 282-595 of SEQ ID NO: 1. It should be understood that, by way of example only, positions 283-594 of SEQ ID NO: 1, or other functional truncations or fragments thereof, can function as a hormone-binding domain.

[0275] Residues within positions 343-354, 380-392, and 404-463, corresponding to SEQ ID NO: 1, are involved in binding to both endogenous and non-endogenous ligands. Residues within positions 517-547, corresponding to SEQ ID NO: 1 (e.g., residues 517-40 and residue 547), are located within a helix referred to as helix 12 and are involved in endogenous ligand binding.

[0276] Greater sensitivity to the non-endogenous ligand compared to sensitivity to the non-endogenous ligand means that the modified ER-LBD binds to the non-endogenous ligand (e.g., endoxifen) with greater affinity compared to its binding affinity to the endogenous ligand (e.g., estradiol).

[0277] Greater sensitivity to a non-endogenous ligand compared to sensitivity to an ER-LBD that does not contain one or more amino acid substitutions (e.g., an ER-LBD comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3) means that the modified ER-LBD binds to the non-endogenous ligand (e.g., endoxifen) with greater affinity compared to the binding affinity of the ER-LBD that does not contain the one or more additional amino acid substitutions to the non-endogenous ligand. In some embodiments, greater sensitivity is at least a 1.5-fold, at least a 2-fold, at least a 3-fold, at least a 4-fold, or at least a 5-fold improvement in binding affinity to the non-endogenous ligand compared to the binding of an ER-LBD that does not contain the one or more additional amino acid substitutions. In some embodiments, greater sensitivity is demonstrated by increased transcriptional regulation (e.g., greater transcriptional activation or greater transcriptional repression) of the chimeric transcription factor comprising the modified ER-LBD compared to a chimeric transcription factor comprising an ER-LBD lacking one or more additional amino acid substitutions. In some embodiments, in transfection transduction assays, a chimeric transcription factor comprising a modified ER-LBD is capable of inducing at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% greater expression of a reporter under the control of a chimeric transcription factor-responsive promoter in response to a non-endogenous ligand (e.g., 4-OHT) compared to expression of a reporter with an ER-LBD lacking one or more additional amino acid substitutions under the same conditions (as measured by % of cells positive for the reporter or as measured by geometric mean fluorescence intensity).

[0278] Greater selectivity for a non-endogenous ligand refers to preferential binding to the non-endogenous ligand (e.g., 4-OHT or endoxifen) compared to the endogenous ligand (e.g., estradiol). Selectivity can be measured using the selectivity coefficient, which is the equilibrium constant for the reaction of displacement of one ligand (e.g., a non-endogenous ligand) in a complex with a substrate (e.g., a modified ER-LBD) by another ligand (e.g., an endogenous ligand). The larger the selectivity coefficient, the further a competing ligand (e.g., an endogenous ligand) will displace the initial ligand (e.g., a non-endogenous ligand) from the complex formed with the substrate (e.g., a modified ER-LBD). In some embodiments, greater selectivity is demonstrated by improved transcriptional regulation of the chimeric transcription factor in the presence of the non-endogenous ligand compared to transcriptional regulation in the presence of the endogenous ligand. In some embodiments, in a transfection transduction assay, a chimeric transcription factor comprising a modified ER-LBD is capable of inducing at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, or at least 35% greater expression of a reporter under the control of a chimeric transcription factor-responsive promoter in response to a non-endogenous ligand (e.g., 4-OHT) compared to expression of the reporter in response to an endogenous ligand (e.g., estradiol) under the same conditions (as measured by % of cells positive for the reporter or as measured by geometric mean fluorescence intensity).

[0279] In some aspects, the one or more amino acid substitutions to ligand-binding residues comprise one or more amino acid substitutions within helix 12. Helix 12 of ER-LBD comprises residue positions 533-547 of SEQ ID NO: 1. In some embodiments, the one or more amino acid substitutions within helix 12 are at one or more positions selected from 538, 536, 539, 540, 547, 534, 533, and 537.

[0280] "Non-endogenous ligand" can refer, for example, to a synthetic estrogen receptor binding ligand that is not naturally expressed by an organism that expresses an estrogen receptor. Non-endogenous estrogen receptor binding ligands include, without limitation, tamoxifen and its metabolites, such as 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0281] The one or more additional amino acid substitutions may be at one or more positions in SEQ ID NO: 1 selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537, 538, 539, 540, and 547. In some embodiments, the one or more additional amino acid substitutions comprise substitutions at one of the positions listed above, two of the positions listed above, three of the positions listed above, four of the positions listed above, or five of the positions listed above.

[0282] In some embodiments, the one or more additional amino acid substitutions are selected from one or more of the substitutions listed in Table 1.

[0283] (Table 1) TIFF2026504440000020.tif190128

[0284] In some aspects, the one or more additional mutations include at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, or at least eight mutations. In some aspects, the one or more additional mutations comprise 2-10 mutations, 2-9 mutations, 2-8 mutations, 2-7 mutations, 2-6 mutations, 2-5 mutations, 2-4 mutations, 2-3 mutations, 3-10 mutations, 3-9 mutations, 3-8 mutations, 3-7 mutations, 3-6 mutations, 3-5 mutations, 3-4 mutations, 4-10 mutations, 4-9 mutations, 4-8 mutations, 4-7 mutations, 4-6 mutations, 4-5 mutations, 5-10 mutations, 5-9 mutations, 5-8 mutations, 5-7 mutations, 5-6 mutations, 6-10 mutations, 6-9 mutations, 6-8 mutations, 6-7 mutations, 7-10 mutations, 7-9 mutations, 7-8 mutations, 8-10 mutations, 8-9 mutations, or 9-10 mutations.

[0285] In some embodiments, the one or more additional mutations comprise at least two mutations selected from the mutations listed in Table 2.

[0286] (Table 2) TIFF2026504440000021.tif97165

[0287] In some embodiments, the one or more additional amino acid substitutions comprise an L391V substitution and an N413D mutation. In some embodiments, the one or more additional amino acid substitutions comprise an L391V substitution, an N413D mutation, and an H524 substitution. In some embodiments, the one or more additional amino acid substitutions comprise an L391V substitution, an N413D mutation, an H524 substitution, and an M421L substitution. In some embodiments, the one or more additional amino acid substitutions comprise an L391V substitution, an N413D mutation, an H524 substitution, and an S463P substitution. In some embodiments, the one or more additional amino acid substitutions comprise an L391V substitution, an N413D mutation, an H524 substitution, and a Q414E substitution. In some embodiments, the one or more additional amino acid substitutions comprise an L391V substitution, an N413D mutation, an H524 substitution, and an L354I substitution. The H524 substitution can be an H524F substitution or an H524L substitution.

[0288] In certain embodiments, the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution; and (b) additional amino acid substitutions, wherein the additional amino acid substitutions are: (i) a L384M substitution, a L391V substitution, a N413D substitution, a M421L substitution, a S463P substitution, and a H524L substitution. (ii) an L391V substitution, an N413D substitution, a Q414E substitution, an S463P substitution, and an H524F substitution; (iii) an L354I substitution, an L391V substitution, an N413D substitution, a Q414E substitution, an M421L substitution, an M517A substitution, and an H524F substitution; or (iv) an L354I substitution, an L391V substitution, an L409V substitution, an N413D substitution, a Q414E substitution, and an H524L substitution.

[0289] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L384M, L391V, N413D, M421L, S463P, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 41 or 116.

[0290] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, N413D, Q414E, S463P, and H524F comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 45.

[0291] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L354I, L391V, N413D, Q414E, M421L, M517A, and H524F comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 50.

[0292] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L354I, L391V, L409V, N413D, Q414E, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 52.

[0293] In some embodiments, the one or more additional amino acid substitutions comprise an N413D mutation, an H524 substitution, and an S463P substitution. The H524 substitution can be an H524F substitution or an H524L substitution. In some embodiments, the one or more additional amino acid substitutions comprise an N413D mutation, an H524L substitution, and an S463P substitution.

[0294] In some embodiments, the modified ER-LBD comprises the additional amino acid substitutions L391V, L409V, Q414E, N413D, S463P, M517A, and H524L. In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, L409V, Q414E, N413D, S463P, M517A, and H524L comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 90 or 103.

[0295] In some embodiments, the modified ER-LBD comprises the additional amino acid substitutions L409V, N413D, S463P, M421L, L384M, and H524L. In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L409V, N413D, S463P, M421L, L384M, and H524L comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 91 or 104.

[0296] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, L409V, N413D, S463P, M517A, M421L, L354I, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 92 or 105.

[0297] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, Q414E, N413D, S463P, M421L, L354I, L384M, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 93 or 106.

[0298] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, L409V, N413D, S463P, M517A, M421L, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 94 or 107.

[0299] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, L409V, Q414E, N413D, S463P, L354I, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 95 or 108.

[0300] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, L409V, N413D, S463P, M421L, L354I, L384M, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 96 or 109.

[0301] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, Q414E, N413D, S463P, M517A, M421L, L354I, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 97 or 110.

[0302] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, N413D, S463P, M517A, L384M, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 98 or 111.

[0303] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions L391V, L409V, N413D, S463P, M517A, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 99 or 112.

[0304] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions N413D, S463P, L354I, L384M, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 100 or 113.

[0305] In some embodiments, the modified ER-LBD comprising the additional amino acid substitutions N413D, S463P, M421L, L354I, and H524L comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 101 or 114.

[0306] molecular switch Also provided herein is a molecular switch for a cell death-inducing signal in a cell, which can include (a) any of the inducible cell death systems described herein (including any of the isolated polynucleotides, heterologous constructs, plasmids, vectors, or cells described herein), and (b) a non-endogenous ligand that binds to the modified ER-LBD of the chimeric protein.

[0307] Upon binding of a non-endogenous ligand to the modified ER-LBD, the chimeric protein generally generates a cell death-inducing signal.

[0308] The molecular switch can include (a) an inducible cell death system comprising a polypeptide, wherein the polypeptide comprises a ligand-binding domain and a cell death-inducing domain, wherein the polypeptide is configured to generate a cell death-inducing signal in a cell in which the polypeptide is expressed upon contact of the ligand-binding domain with a ligand, and wherein the ligand-binding domain comprises a modified ER-LBD (such as, for example, any of the modified ER-LBDs described herein); and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD generates a cell death-inducing signal in the cell.

[0309] The molecular switch can include (a) an inducible cell death system comprising a first polypeptide monomer and a second polypeptide monomer, wherein the first polypeptide monomer and the second polypeptide monomer each comprise a ligand-binding domain and a cell death-inducing domain, wherein the first polypeptide monomer and the second polypeptide monomer are configured to oligomerize with each other upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in a cell in which the first polypeptide monomer and the second polypeptide monomer are expressed, and wherein the ligand-binding domain comprises a modified ER-LBD (such as any of the modified ER-LBDs described herein); and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD induces oligomerization of the first and second polypeptide monomers, thereby generating a cell death-inducing signal in the cell.

[0310] Also provided herein is an inducible cell death system comprising a cell death-inducing domain that is a transcription factor comprising a nucleic acid binding domain and a transcription effector domain, wherein the transcription factor is configured to generate a cell death-inducing signal by inducing expression of a gene of interest. The transcription factor configured to generate a cell death-inducing signal may comprise (a) a chimeric transcription factor comprising a modified ER-LBD and capable of binding to a chimeric transcription factor-responsive promoter (CTF-responsive promoter) operably linked to the gene of interest, and (b) a non-endogenous ligand that binds to the modified ER-LBD of the chimeric protein. Upon binding of the non-endogenous ligand to the modified ER-LBD, the chimeric protein can regulate transcription of the gene of interest.

[0311] In some embodiments, the gene of interest encodes a polypeptide selected from a caspase domain or a derivative or functional fragment thereof, optionally wherein the caspase is any one of caspases 1-11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or a derivative or functional fragment thereof, diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, or a death domain. The protein is selected from Fas-associated protein (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53 up-regulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, or purine nucleoside phosphorylase.

[0312] In some embodiments, the non-endogenous ligand is selected from 4-hydroxytamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0313] In certain embodiments, the non-endogenous ligand is 4-hydroxytamoxifen (4-OHT, also referred to as afimoxifen). In certain embodiments, the non-endogenous ligand includes a tamoxifen metabolite, such as 4-OHT, endoxifen, or a combination of 4-OHT and endoxifen. In certain embodiments, the non-endogenous ligand is endoxifen. In certain embodiments, the molecular switch is capable of generating a cell death-inducing signal at a concentration of 0.25 nM endoxifen or less and / or at a concentration of 0.04 nM 4-OHT or less. In certain embodiments, the molecular switch is capable of generating a cell death-inducing signal at a concentration of 2.5 nM endoxifen or less and / or at a concentration of 0.4 nM 4-OHT or less. In certain embodiments, the molecular switch is capable of generating a cell death-inducing signal at a concentration of at least 0.001 pM 4-OHT. In certain embodiments, the molecular switch is capable of generating a cell death-inducing signal at a concentration of 4-OHT of at least 0.01 pM.

[0314] In certain embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating a cell death-inducing signal at a concentration of 0.25 nM endoxifen or less and / or at a concentration of 0.04 nM 4-OHT or less. In certain embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating a cell death-inducing signal at a concentration of 2.5 nM endoxifen or less and / or at a concentration of 0.4 nM 4-OHT or less. In certain embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating a cell death-inducing signal at a concentration of at least 0.001 pM 4-OHT. In certain embodiments, the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating a cell death-inducing signal at a concentration of at least 0.01 pM 4-OHT.

[0315] Isolated Polynucleotides and Heterologous Constructs Also provided herein are isolated polynucleotides and heterologous constructs encoding an inducible modified ER-LBD or chimeric protein (e.g., any of the polypeptides, first polypeptide monomers, and / or second polypeptide monomers of the inducible cell death system herein). In some aspects, the present disclosure provides an isolated polynucleotide comprising a nucleotide sequence encoding a modified ER-LBD or chimeric protein as described herein. In some aspects, the present disclosure provides a heterologous construct comprising a promoter operably linked to a polynucleotide encoding a modified ER-LBD or chimeric protein.

[0316] In some aspects, the present disclosure further provides isolated polynucleotides and / or heterologous constructs comprising target gene expression cassettes.

[0317] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, such as DNA or RNA, that has been removed from its natural environment. For example, a polynucleotide encoding a modified ER-LBD or chimeric protein contained in a heterologous construct is considered isolated. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide also includes a polynucleotide contained in a cell that normally contains the polynucleotide, but where the polynucleotide is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0318] Isolated polynucleotides include, but are not limited to, cDNA polynucleotides, RNA polynucleotides, RNAi oligonucleotides (e.g., siRNA, miRNA, antisense oligonucleotides, shRNA, etc.), mRNA polynucleotides, circular plasmids, linear DNA fragments, vectors, minicircles, ssDNA, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs), and oligonucleotides.

[0319] In some embodiments, the isolated polynucleotide is selected from DNA, cDNA, RNA, mRNA, and naked plasmid (linear or circular).

[0320] By a nucleic acid or polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur either individually among residues in the reference sequence or in one or more consecutive groups within the reference sequence, at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions. As a practical matter, whether any particular polynucleotide sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence of the present invention can be conventionally determined using known computer programs.

[0321] In some aspects, the chimeric protein encoded by the polynucleotide is a polypeptide of an inducible cell death system described herein. In some aspects, the chimeric protein encoded by the polynucleotide is a first polypeptide monomer of an inducible cell death system described herein. In some aspects, the chimeric protein encoded by the polynucleotide is a second polypeptide monomer of an inducible cell death system described herein. In some aspects, the chimeric protein encoded by the polynucleotide is a first polypeptide monomer and a second polypeptide monomer of an inducible cell death system described herein. In some aspects, the chimeric protein encoded by the polynucleotide is a chimeric transcription factor, and the polynucleotide further comprises a target expression cassette comprising a gene of interest operably linked to a chimeric transcription factor-responsive (CTF-responsive) promoter. In some embodiments, the target expression cassette is present in the same heterologous construct as the chimeric protein. In some embodiments, the chimeric protein and the target expression cassette are present in separate heterologous constructs.

[0322] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide with a set of nucleic acid elements that allow for transcription of a particular polynucleotide in a target cell. Expression cassettes can be incorporated into plasmids, chromosomes, mitochondrial DNA, plastid DNA, viruses, or nucleic acid fragments. Typically, the expression cassette portion of an expression vector contains, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some aspects, the present disclosure provides expression cassettes containing a polynucleotide encoding a modified ER-LBD or a chimeric protein containing a modified ER-LBD.

[0323] The isolated polynucleotides and heterologous constructs comprising the modified ER-LBD described herein are engineered polynucleotides. An "engineered polynucleotide" is a polynucleotide that does not occur in nature. However, it should be understood that while an engineered polynucleotide does not entirely occur in nature, it can contain naturally occurring nucleotide sequences. In some embodiments, an engineered polynucleotide contains nucleotide sequences from different organisms (e.g., from different species). For example, in some embodiments, an engineered polynucleotide contains murine, bacterial, human, and / or viral nucleotide sequences. The term "engineered polynucleotide" includes recombinant nucleic acids and synthetic nucleic acids. A "recombinant polynucleotide" refers to a molecule constructed by linking nucleotide molecules and, in some embodiments, capable of replicating in a living cell. A "synthetic polynucleotide" refers to a molecule that is amplified or synthesized chemically or by other means. Synthetic polynucleotides include those that are chemically or otherwise modified but are capable of base-pairing with naturally occurring nucleotide molecules. Modifications include, but are not limited to, one or more modified internucleotide linkages and non-naturally occurring nucleic acids. Modifications are described in further detail in U.S. Patent No. 6,673,611 and U.S. Application Publication No. 2004 / 0019001, each of which is incorporated by reference in its entirety. The modified internucleotide linkages can be phosphorodithioate or phosphorothioate linkages. The non-natural nucleic acids can be locked nucleic acids (LNAs), peptide nucleic acids (PNAs), glycol nucleic acids (GNAs), phosphorodiamidate morpholino oligomers (PMOs or "morpholinos"), and threose nucleic acids (TNAs). Non-natural nucleic acids are described in further detail in International Application No. 1998 / 039352, U.S. Application Publication No. 2013 / 0156849, and U.S. Patent Nos. 6,670,461, 5,539,082, and 5,185,444, each of which is incorporated by reference in its entirety. Recombinant and synthetic polynucleotides also include molecules resulting from the replication of any of the foregoing.The engineered polynucleotides of the disclosure can be encoded by a single molecule (e.g., contained in the same plasmid or other vector) or by multiple different molecules (e.g., multiple different, independently replicating molecules).

[0324] The engineered polynucleotides of the present disclosure can be produced using standard molecular biology methods (see, e.g., Green and Sambrook, Molecular Cloning, A Laboratory Manual, 2012, Cold Spring Harbor Press). In some embodiments, the engineered nucleic acid constructs are produced using GIBSON ASSEMBLY® cloning (see, e.g., Gibson, D.G. et al. Nature Methods, 343-345, 2009; and Gibson, D.G. et al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzymatic activities in a single-tube reaction: a 5' exonuclease, a Y-extension activity of a DNA polymerase, and a DNA ligase activity. The 5' exonuclease activity bites back the 5'-end sequence, exposing complementary sequences for annealing. The polymerase activity then fills in gaps in the annealed region. DNA ligase then seals the nicks and covalently joins the DNA fragments together. The overlapping sequences of adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a higher percentage of correct assembly. In some embodiments, engineered nucleic acid constructs are produced using IN-FUSION® Cloning (Clontech).

[0325] In some embodiments, the polynucleotides described herein are comprised in a heterologous construct. The term "vector" or "expression vector" is synonymous with "heterologous construct" and refers to a polynucleotide used to introduce and direct expression of one or more genes operably associated with the construct in a target cell. This term includes the construct as a self-replicating nucleic acid structure as well as a vector integrated into the genome of a host cell into which it is introduced. The heterologous constructs described herein comprise an expression cassette. In some aspects, provided herein are heterologous constructs comprising an expression cassette comprising a promoter operably linked to a polynucleotide encoding a modified ER-LBD or a chimeric protein comprising a modified ER-LBD.

[0326] As used herein, 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 include small regions to which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors. Promoters 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 regulates. As used herein, a promoter is considered to be "operably linked" when it is in the correct functional location and orientation relative to the nucleic acid sequence that it regulates to control ("drive") the transcription initiation and / or expression of that sequence.

[0327] A promoter may be a promoter naturally associated with a given gene or sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment of a given gene or sequence. Such a promoter may be referred to as "endogenous." In some embodiments, a coding nucleic acid sequence may be placed under the control of a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with the coded sequence in its natural environment. Such promoters may 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 of transcriptional regulation and / or mutations that alter expression through methods of genetic engineering known in the art. In addition to producing promoter and enhancer nucleic acid sequences synthetically, the sequences may 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).

[0328] As used herein, an "inducible promoter" refers to a promoter that is characterized by modulating (e.g., initiating or activating) transcriptional activity when in the presence of, affected by, or contacted by a signal. This signal can be an endogenous or usually exogenous condition (e.g., light), compound (e.g., a chemical or non-chemical compound), or protein (e.g., a chimeric transcription factor described herein) that contacts the inducible promoter in such a way that it is active in modulating transcriptional activity from the inducible promoter. Activating transcription can 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, inactivating transcription can 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.

[0329] As used herein, a promoter is "responsive to" or "regulated by" a local tumor condition (e.g., inflammation or hypoxia) or signal if, in the presence of that condition or signal, transcription from the promoter is activated, inactivated, increased, or decreased. 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 modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, but are not limited to, phloretin tunable regulatory 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 Apr 9;279(15):15652-61, incorporated herein by reference), NF-kappa B 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 repeats of the same nucleotide sequence encoding the response element) to generally increase the sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2x, 3x, 4x, 5x, etc. to indicate the number of repeats present.

[0330] Non-limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF-beta responsive promoters) are listed in Table 3, which shows the promoter and transcription factor design, and the effect of the transcription factor (TF) and inducer molecule on 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). Non-limiting examples of inducible promoter components include those presented in Table 4.

[0331] (Table 3) TIFF2026504440000022.tif206166TIFF2026504440000023.tif165166

[0332] (Table 4) TIFF2026504440000024.tif62166TIFF2026504440000025.tif123166

[0333] 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 containing 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.

[0334] In some aspects, the present disclosure provides a heterologous construct comprising a promoter operably linked to a polynucleotide encoding a modified ER-LBD or chimeric protein described herein.

[0335] In some embodiments, the promoter operably linked to the polynucleotide encoding the modified ER-LBD or chimeric protein is a constitutive promoter, an inducible promoter, or a synthetic promoter.

[0336] In some embodiments, the promoter operably linked to the polynucleotide encoding the modified ER-LBD or chimeric protein is a constitutive promoter. Examples of constitutive promoters are shown in Table 5. 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.

[0337] (Table 5) TIFF2026504440000026.tif254164TIFF2026504440000027.tif253164TIFF20265044400 00028.tif253164TIFF2026504440000029.tif254164TIFF2026504440000030.tif132164

[0338] In some embodiments, the engineered polynucleotides or constructs of the present disclosure are configured to produce multiple polypeptides. For example, the polynucleotides can be configured to produce two different polypeptides. The polynucleotides can be configured to produce polypeptides of an inducible cell death system comprising a modified ER-LBD as described herein, e.g., polypeptides comprising a first polypeptide monomer and / or a second polypeptide monomer, each comprising a modified ER-LBD and a cell death-inducing domain.

[0339] In some embodiments, the polypeptides of the inducible cell death system comprising a modified ER-LBD described herein, e.g., a first polypeptide monomer and / or a second polypeptide monomer, each comprising a modified ER-LBD and a cell death-inducing domain, can be encoded by the same polynucleotide or heterologous construct.

[0340] In some embodiments, the engineered nucleic acid can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polypeptides, such as a first polypeptide monomer and a second polypeptide monomer, each comprising a modified ER-LBD and a cell death-inducing domain) can be produced from a single transcript. The engineered nucleic acid can be multicistronic through the use of various linkers; for example, a polynucleotide sequence encoding a first exogenous polynucleotide can be linked to a nucleotide sequence encoding a second exogenous polynucleotide, such as in a 5' to 3' orientation of the first gene:linker:second gene. The linker polynucleotide sequence can encode one or more 2A ribosomal skipping elements, such as T2A. Other 2A ribosomal skipping elements include, but are not limited to, E2A, P2A, and F2A. The 2A ribosomal skipping elements allow for the production of separate polypeptides encoded by the first and second genes, which are produced during translation. The linker can encode a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, where, following expression, the cleavable linker polypeptide is cleaved to produce separate polypeptides encoded by the first and second genes. The cleavable linker can include polypeptide sequences that further facilitate cleavage, such as flexible linkers (e.g., Gly-Ser-Gly sequences).

[0341] The linker can encode an internal ribosome entry site (IRES) so that separate polypeptides encoded by the first and second genes are produced during translation. The linker can encode a splice acceptor, such as a viral splice acceptor.

[0342] The linker can be a combination of linkers, such as a furin-2A linker, that can produce separate polypeptides through 2A ribosomal skipping followed by further cleavage of the furin site, allowing 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.

[0343] Generally, 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 polypeptide molecules, each separated by a linker, such that separate polypeptides encoded by the first, second, and third polypeptides are produced). "Linker," as used herein, may refer to a polypeptide that connects a first polypeptide sequence and a second polypeptide sequence, or to a multicistronic linker as described above.

[0344] Expression systems further comprising a target expression cassette In some aspects, the chimeric protein is a chimeric transcription factor, and the present disclosure further provides a target expression cassette comprising a chimeric transcription factor-responsive (CTF-responsive) promoter. For example, a chimeric transcription factor having a modified ER-LDB may be used to regulate the expression of a cell death-promoting / apoptotic factor (e.g., a polypeptide comprising a cell death-inducing domain), a cell survival polypeptide, an inhibitor of a proapoptotic factor, and / or an inhibitor of a cell survival polypeptide.

[0345] A "target expression cassette" refers to an expression cassette containing a gene with expression controllable by a chimeric transcription factor, where expression is controlled by the chimeric transcription factor upon the presence of a non-endogenous ligand (e.g., 4-OHT or endoxifen).

[0346] In some aspects, the present disclosure provides a polynucleotide encoding a gene of interest operably linked to a chimeric transcription factor-responsive promoter (CTF-responsive promoter), which is a synthetic inducible promoter that is responsive to a chimeric transcription factor comprising a modified ER-LBD and is inducible in response to a non-endogenous ligand, such as 4-OHT.

[0347] In some embodiments, the CTF-responsive promoter comprises a core promoter sequence and a binding domain that binds a chimeric transcription factor described herein.

[0348] A binding domain can comprise one or more zinc finger binding sites. A binding domain can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more zinc finger binding sites. In some embodiments, a binding domain comprises one zinc finger binding site. In some embodiments, a binding domain comprises two zinc finger binding sites. In some embodiments, a binding domain comprises three zinc finger binding sites. In some embodiments, a binding domain comprises four zinc finger binding sites. An exemplary binding domain containing a zinc finger binding site is shown in the sequence: CGGGTTTCGTAACAATCGCATGAGGATTCGCAACGCCTTCGGCGTAGCCGATGTCGCGCTCCCGTCTCAGTAAAGGTCGGCGTAGCCGATGTCGCGCAATCGGACTGCCTTCGTACGGCGTAGCCGATGTCGCGCGTATCAGTCGCCTCGGAACGGCGTAGCCGATGTCGCGCATTCGTAAGAGGCTCACTCTCCCTTACACGGAGTGGATAACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 37).

[0349] The core promoter sequence may include a minimal promoter. Examples of minimal promoters include minP, minCMV, YB_TATA, and minTK.

[0350] In some aspects, the chimeric protein comprising a modified ER-LBD is a chimeric transcription factor, and the heterologous construct further comprises a target expression cassette comprising a chimeric transcription factor-responsive promoter. In some aspects, provided herein are a first heterologous construct comprising an expression cassette comprising a polynucleotide encoding a chimeric transcription factor comprising a modified ER-LBD, and a second heterologous construct comprising a target expression cassette comprising a chimeric transcription factor-responsive (CTF-responsive) promoter.

[0351] 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 alpha, beta, and gamma components of a WPRE element. In some embodiments, a WPRE comprises the alpha component of a WPRE element. Exemplary WPRE sequences include SEQ ID NO: 38 and SEQ ID NO: 39.

[0352] Engineered cells Also provided herein are cells comprising one or more polynucleotides or constructs of the present disclosure, and methods for producing the cells. These cells are referred to herein as "engineered cells." These cells typically comprise one or more engineered nucleic acids and do not occur in nature. In some embodiments, the cells are isolated cells that recombinantly express one or more engineered polynucleotides. In some embodiments, the engineered polynucleotides are expressed from one or more vectors or selected loci from the genome of the cell. In some embodiments, the cells are engineered to comprise a polynucleotide comprising a promoter operably linked to a nucleotide sequence.

[0353] The engineered cells of the present disclosure can include an engineered polynucleotide integrated into the genome of the cell. The engineered cells can include an engineered polynucleotide that is capable of expression without integration into the genome of the cell, for example, engineered using a transient expression system, such as a plasmid or mRNA.

[0354] Engineered cell types The engineered cells of the present disclosure can be human cells. The engineered cells can be human primary cells. The engineered primary cells can be any somatic cell. The engineered primary cells can be any stem cell. In some embodiments, the engineered cells are derived from a subject. In some embodiments, the engineered cells are allogeneic with respect to the subject.

[0355] The engineered cells of the present disclosure can be isolated from a subject, such as a subject known or suspected of having cancer. Cell isolation methods are known to those of skill 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, negative isolation, magnetic isolation, and combinations thereof. The engineered cells can be allogeneic with respect to the subject being treated. The allogeneic modified cells can be HLA-matched to the subject being treated. The engineered cells can be cultured cells, such as 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.

[0356] In some embodiments, the engineered cells of the present disclosure are selected from T cells (e.g., CD8+ T cells, CD4+ T cells, or 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 (e.g., M1 macrophages or M2 macrophages), monocytes, dendritic cells, erythrocytes, platelet cells, neurons, oligodendrocytes, astrocytes, placode-derived cells, Schwann cells, cardiomyocytes, endothelial cells, nodal myocytes, microglial cells, hepatocytes, cholangiocytes, beta 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.

[0357] In some embodiments, the engineered cells of the present disclosure are T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma-delta T cells). In some embodiments, the engineered cells of the present disclosure are cytotoxic T lymphocytes (CTLs). In some embodiments, the engineered cells of the present disclosure are regulatory T cells. In some embodiments, the engineered cells of the present disclosure are natural killer T (NKT) cells. In some embodiments, the engineered cells of the present disclosure are natural killer cells (NK). In some embodiments, the engineered cells of the present disclosure are B cells. In some embodiments, the engineered cells of the present disclosure are tumor-infiltrating lymphocytes (TILs). In some embodiments, the engineered cells of the present disclosure are innate lymphoid cells. In some embodiments, the engineered cells of the present disclosure are mast cells. In some embodiments, the engineered cells of the present disclosure are eosinophils. In some embodiments, the engineered cells of the present disclosure are basophils. In some embodiments, the engineered cells of the present disclosure are neutrophils. In some embodiments, the engineered cells of the present disclosure are myeloid cells. In some embodiments, the engineered cells of the present disclosure are macrophages, e.g., M1 macrophages or M2 macrophages. In some embodiments, the engineered cells of the present disclosure are monocytes. In some embodiments, the engineered or isolated cells of the present disclosure are dendritic cells. In some embodiments, the engineered cells of the present disclosure are erythrocytes. In some embodiments, the engineered cells of the present disclosure are platelet cells. In some embodiments, the cells of the present disclosure are neurons. In some embodiments, the cells of the present disclosure are oligodendrocytes. In some embodiments, the cells of the present disclosure are astrocytes. In some embodiments, the cells of the present disclosure are placode-derived cells. In some embodiments, the engineered cells of the present disclosure are Schwann cells. In some embodiments, the engineered cells of the present disclosure are cardiomyocytes. In some embodiments, the engineered cells of the present disclosure are endothelial cells. In some embodiments, the engineered cells of the present disclosure are nodule cells. In some embodiments, the engineered cells of the present disclosure are microglial cells. In some embodiments, the engineered cells of the present disclosure are hepatocytes. In some embodiments, the engineered cells of the present disclosure are cholangiocytes.In some embodiments, the engineered cells of the present disclosure are beta cells. In some embodiments, the engineered cells of the present disclosure are human embryonic stem cells (ESCs). In some embodiments, the engineered cells of the present disclosure are ESC-derived cells. In some embodiments, the engineered cells of the present disclosure are pluripotent stem cells. In some embodiments, the engineered cells of the present disclosure are mesenchymal stromal cells (MSCs). In some embodiments, the engineered cells of the present disclosure are induced pluripotent stem cells (iPSCs). In some embodiments, the engineered cells of the present disclosure are iPSC-derived cells. In some embodiments, the engineered cells are autologous. In some embodiments, the engineered cells are allogeneic. In some embodiments, the engineered cells of the present disclosure are CD34+ cells, CD3+ cells, CD8+ cells, CD16+ cells, and / or CD4+ T cells.

[0358] In some embodiments, the cell of the present disclosure is a tumor cell selected from 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 prostate tumor cell, a skin tumor cell, a thyroid tumor cell, and a uterine tumor cell.

[0359] Also provided herein are methods comprising culturing the engineered cells of the present disclosure. 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 purpose of the engineered cells. 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.

[0360] How to manipulate cells Also provided herein are compositions and methods for engineering cells with any of the polynucleotides or constructs described herein.

[0361] Generally, cells are engineered through the introduction (i.e., delivery) of one or more polynucleotides of the present disclosure. 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 specific cell type being engineered.

[0362] Viral-mediated delivery Viral vector-based delivery platforms can be used to engineer cells. Generally, viral vector-based delivery platforms engineer cells through introduction (i.e., delivery) into host cells. For example, viral vector-based delivery platforms can engineer cells through the introduction of any of the engineered nucleic acids described herein. Viral vector-based delivery platforms can be nucleic acids, and as such, engineered nucleic acids can also include nucleic acids derived from engineered viruses. Such engineered viral nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0363] Viral vector-based delivery platforms can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid. For example, a nucleic acid derived from an engineered virus, e.g., a recombinant virus or engineered virus, can encode one or more transgenes, including, but not limited to, any of the engineered nucleic acids described herein. The one or more transgenes can be configured to express a polypeptide described herein (e.g., an inducible cell death system comprising a modified ER-LBD). In addition to a transgene encoding a modified ER-LBD, a viral vector-based delivery platform can encode one or more genes, such as viral genes required for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerase, viral transcriptase, etc.), referred to as cis-acting elements or genes.

[0364] A viral vector-based delivery platform can include more than one viral vector, such as a separate viral vector encoding an engineered nucleic acid, gene, or transgene, as described herein and referred to as a trans-acting element or gene. For example, a helper-dependent viral vector-based delivery platform can provide, in addition to a vector encoding a modified ER-LBD, additional genes required for viral infectivity and / or viral production on one or more additional separate vectors. One viral vector can deliver more than one engineered polynucleotide, such as an engineered polynucleotide configured to produce a modified ER-LBD and one vector delivering an engineered polynucleotide configured to produce a gene of interest. More than one viral vector can deliver more than one engineered nucleic acid, such as a first vector delivering an engineered polynucleotide configured to produce a modified ER-LBD and a second vector delivering an additional engineered polynucleotide. The number of viral vectors used can depend on the packaging capacity of the viral vector-based vaccine platform mentioned above, but one skilled in the art can select an appropriate number of viral vectors.

[0365] Generally, any viral vector-based system can be used for in vitro production of molecules or for in vitro delivery, for example, in in vivo and ex vivo gene therapy procedures. The selection of a suitable viral vector-based system will depend on various factors, such as cargo / payload size, 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.

[0366] The viral vector-based delivery platform can be an RNA-based virus or a DNA-based virus. Exemplary viral vector-based delivery platforms include, but are 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 lentivirus, 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). 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).

[0367] This sequence may be preceded by one or more sequences that target an intracellular compartment. Upon introduction (i.e., delivery) into a host cell, the infected cell (i.e., the engineered cell) expresses, and in some cases, secretes, the modified ER-LBD (or chimeric polypeptide comprising the modified ER-LBD). Vaccinia vectors and methods useful in 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 by 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 typhimurium vectors, and the like, will be apparent to those skilled in the art from the description herein.

[0368] 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 viruses, oncolytic adenoviruses, oncolytic measles viruses, oncolytic influenza viruses, oncolytic Indiana vesiculoviruses, oncolytic Newcastle disease viruses, oncolytic vaccinia viruses, oncolytic polioviruses, oncolytic myxoma viruses, oncolytic reoviruses, oncolytic mumps viruses, oncolytic Maraba viruses, oncolytic rabies viruses, oncolytic rotaviruses, oncolytic hepatitis viruses, oncolytic rubella viruses, oncolytic dengue viruses, oncolytic chikungunya viruses, oncolytic respiratory syncytial viruses, oncolytic lymphocytic choriomeningitis viruses, oncolytic morbilliviruses, oncolytic lentiviruses, oncolytic replicating retroviruses, oncolytic rhabdoviruses, oncolytic Seneca Valley viruses, oncolytic Sindbis viruses, and any variants or derivatives thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus that includes an additional transgene (e.g., an engineered nucleic acid described herein). The transgene can be configured to express the modified ER-LBD (or a chimeric polypeptide comprising the modified ER-LBD), and optionally, a gene of interest.

[0369] 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).

[0370] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for replication and packaging of the vector, which are then used to integrate one or more engineered nucleic acids (e.g., transgenes encoding modified ER-LBDs) 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.

[0371] The viral vector-based delivery platform can be lentivirus-based. Generally, lentivirus vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. The lentivirus-based delivery platform can be HIV-based, such as the ViraPower system (ThermoFisher) or the pLenti system (Cell Biolabs). The lentivirus-based delivery platform can be SIV- or FIV-based. Other exemplary lentiviral-based delivery platforms are described in more 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; and 6,955,919, each of which is incorporated herein by reference for all purposes.

[0372] The viral vector-based delivery platform 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 in a relatively simple system. Generally, adenovirus can be used for transient expression of transgenes in infected cells, because adenoviruses typically do not integrate into the host genome. Adenovirus-based delivery platforms are described in more 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 more detail in U.S. Patent 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 No. 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0373] Viral vector-based delivery platforms can be adeno-associated virus (AAV)-based. Adeno-associated virus ("AAV") vectors can 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 modified ER-LBD (or chimeric polypeptides comprising modified ER-LBD) or can be used in in vivo and ex vivo gene therapy procedures, for example, for in vivo delivery of modified ER-LBD (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 Publications US2003-0138772, US2007 / 0036760, and US2009 / 0197338; Gao, et al. 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 more 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.

[0374] 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. Then, after purification, a cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated ex vivo within the purified particles. Thus, VLP production maintains the separation of nucleic acids encoding viral structural proteins and nucleic acids encoding cargo / payload. Viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translational expression systems. Purified viral particles can be denatured and reassembled in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. VLP production is described in more detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777).

[0375] 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 will determine 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 range of cells. For example, a pantropic viral vector-based delivery platform can include a VSV-G envelope. Viral vector-based delivery platforms are amphotropic and can infect mammalian cells. Therefore, those skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0376] Lipid structure delivery system The engineered nucleic acids of the present disclosure (e.g., nucleic acids encoding the modified ER-LBD or chimeric proteins described herein) can be introduced into cells using a lipid-mediated delivery system. Generally, lipid-mediated delivery systems use a structure composed 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.

[0377] Lipid-based nanoparticles can include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid preparations. As used herein, "liposome" is a generic term that encompasses in vitro preparations of lipid vehicles formed by encapsulating a desired cargo, such as an engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or lipid aggregate. Liposomes can 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 can be unilamellar. Liposomes can be multilamellar. Liposomes can be multivesicular. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can generally be formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids and sterols, such as cholesterol. The selection of lipids is generally guided by consideration of the desired purpose, for example, criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in the bloodstream. A variety of methods are available for preparing liposomes, as described, for example, in Szokan 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, each of which is incorporated herein by reference for all purposes.

[0378] Multilamellar liposomes are spontaneously formed when lipids, including phospholipids, are suspended in an excess of aqueous solution, such that multiple lipid layers are separated by aqueous medium. Water and dissolved solutes are trapped in the closed structure between the lipid bilayers after self-rearrangement of the lipid components. The 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 in 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, encapsulated in the liposome, complexed with the liposome, or otherwise associated with the liposome, so that it can be delivered to the target entity. Lipophilic molecules or molecules with lipophilic regions can also be dissolved in or associated with the lipid bilayer.

[0379] The liposomes used in accordance with the present embodiments can be produced by different methods, as will be known to those skilled in the art. The preparation of liposomes is described in further detail in WO2016 / 201323, International Application Nos. 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.

[0380] 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. 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.

[0381] Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372, WO 93 / 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; WO 91 / 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.

[0382] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. Exosome sizes range from 30 to 100 nm in diameter. Their surface consists of a lipid bilayer from the plasma membrane of the donor cell, they contain cytosol from the cell that produced the exosome, and display membrane proteins from the parent cell on their surface. Exosomes useful for nucleic acid delivery are known to those skilled in the art, for example, exosomes described in more detail in U.S. Pat. No. 9,889,210, incorporated herein by reference for all purposes.

[0383] 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 and / or across the membrane displayed on the outer surface of the extracellular vesicle. The 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, vesicles derived from cells by 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.

[0384] As used herein, the term "exosome" refers to a small (20-300 nm diameter, more preferably 40-200 nm diameter) cell-derived vesicle that includes a membrane surrounding an internal space and is generated from a cell by direct plasma membrane budding or by fusion of a late endosome with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecules. Exosomes are derived from producing cells and can be isolated from producing 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 the producing cells. Exosomes and the preparation of exosomes are described in further detail in WO2016 / 201323, which is incorporated herein by reference in its entirety.

[0385] As used herein, the term "nanovesicles" (also referred to as "microvesicles") refers to small (20-250 nm diameter, more preferably 30-150 nm diameter) vesicles of cell origin that comprise a membrane surrounding an interior space and are generated from cells by direct or indirect manipulation, such that the nanovesicles are not produced by the producing cells without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Suitable manipulation of producing cells includes, but is not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or a combination thereof. The production of nanovesicles may, in some cases, result in the destruction of the producing cells. Preferably, the population of nanovesicles is substantially free of vesicles derived from producing cells by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles comprise lipids or fatty acids and polypeptides, and optionally comprise a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. Once derived from the production cells according to the engineered method, the nanovesicles can be isolated from the production cells based on their size, density, biochemical parameters, or a combination thereof.

[0386] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that rely on the amphiphilic properties of lipids to form membrane- and 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. 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 mixtures of defined 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 chemical groups that facilitate the attachment of additional moieties. The lipid composition can affect the overall LNP size and stability. In one example, the lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or an MC3-like molecule. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. LNPs can also 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.

[0387] Micelles are generally spherical synthetic lipid structures formed using single-chain lipids, where the hydrophilic heads of the single-chain lipids form the outer layer or membrane, and the hydrophobic tails of the single-chain lipids form the micellar core. Micelle typically refers to a lipid structure that contains only a lipid monolayer. Micelles are described in more detail by Quader et al. (Mol Ther. 2017 Jul 5;25(7):1501-1513).

[0388] Nucleic acid vectors, such as expression vectors, directly exposed to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by released nucleic acids. Similarly, viral delivery systems directly exposed to serum can induce 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, such as within 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, such as 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 agents, such that the delivery vector and desired agent are fully encapsulated 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 LNPs produced. 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 in size. After 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.

[0389] Nanoparticle Delivery Nanomaterials can be used to deliver engineered nucleic acids (e.g., nucleic acids encoding the modified ER-LBD or chimeric proteins described herein). Importantly, nanomaterial vehicles are made from non-immunogenic materials, generally avoiding eliciting immunity against the delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more 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.

[0390] Genome editing A genome editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as a nucleic acid encoding a modified ER-LBD 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.

[0391] Transposon systems can be used to integrate engineered nucleic acids, such as the engineered nucleic acids of the present disclosure, into a host genome. Transposons generally contain terminal inverted repeats (TIRs) flanking a cargo / payload nucleic acid and a transposase. Transposon systems can provide transposons in cis or trans, with the TIR flanking cargo. Transposon systems can be retrotransposon or DNA transposon systems. Generally, transposon systems randomly integrate cargo / payload (e.g., engineered nucleic acids) into the host genome. Examples of transposon systems include systems using transposons of the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which are described in more 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. Another example of a transposon system includes the PiggyBac transposon system, which is described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is incorporated herein by reference for all purposes.

[0392] Nuclease genome editing systems can be used to engineer a host genome to encode an engineered nucleic acid, such as an isolated polynucleotide or heterologous construct of the present disclosure. While not wishing to be bound by theory, nuclease-mediated gene editing systems used to introduce exogenous genes generally utilize a cell's natural DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. Briefly, following damage to genomic DNA (typically a double-strand break), the cell can resolve the damage by using another DNA source with identical or substantially identical sequences at both its 5' and 3' ends as a template during DNA synthesis to repair the damage. In natural situations, 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, any additional exogenous sequence not naturally found in a chromosome, with the 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, such as any of the engineered nucleic acids described herein).

[0393] In some examples, the HR template may be linear. Examples of linear HR templates include, but are not limited to, linear plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain examples, the HR template may be circular, such as a plasmid. The circular template may include a supercoiled template.

[0394] The identical or substantially identical sequences found at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms) with respect to the exogenous sequence to be introduced. The HR arms can be identical (i.e., 100% identical) to a region of the endogenous genomic target locus. In some cases, the HR arms can be substantially identical to a region of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for the HR arms to be identical, as the efficiency of the HDR pathway can be affected by HR arms with less than 100% identity.

[0395] Each HR arm, i.e., the 5' and 3' HR arms, can be the same size or different sizes. Each HR arm can be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. The HR arms can generally be any length, although practical considerations, such as the effect of HR arm length and overall template size on overall editing efficiency, can also be taken into account. The 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 specific distance of the cleavage site, such as 1 base pair, less than or equal to 10 base pairs, less than or equal to 50 base pairs, or less than or equal to 100 base pairs.

[0396] Nuclease genome editing systems can use various 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.

[0397] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as the engineered nucleic acid described herein. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond,” Nature Communications; volume 9 (2018), Article number: 1911), which is incorporated herein by reference for all that it teaches. Generally, a CRISPR-mediated gene editing system includes a CRISPR-associated (Cas) nuclease and an RNA that directs cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which is composed of a Cas9 nuclease and an RNA having a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity to a target sequence (“defined nucleotide sequence”), e.g., a genomic sequence, through base-pair hybridization, and an RNA domain that hybridizes to the tracrRNA. The tracrRNA can interact with a nuclease (e.g., Cas9), thereby facilitating its recruitment to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is exemplified herein, other CRISPR systems can be used, such as the Cpfl system. Nucleases can include derivatives thereof, such as Cas9 functional mutants, for example, Cas9 "nickase" mutants, which generally mediate only a single-stranded cleavage of a defined nucleotide sequence, as opposed to the complete double-stranded cleavage typically produced by the Cas9 enzyme.

[0398] Generally, the components of a CRISPR system interact with each other to form a ribonucleoprotein (RNP) complex and mediate sequence-specific cleavage. In some CRISPR systems, each component can be produced separately and used to form an RNP complex. In some CRISPR systems, each component can be produced separately in vitro and contacted with each other (i.e., "complexed") in vitro to form an RNP complex. The in vitro-produced RNPs can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, for example, the cytosol and / or nucleus of a T cell. The in vitro-produced 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-produced 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 to those skilled 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.

[0399] The RNP complex produced in vitro can be complexed with different ratios of nuclease and gRNA.The RNP complex produced in vitro can also be used in different amounts in CRISPR-mediated editing system.For example, the total amount of RNP that is added can be adjusted depending on the number of cells that are desired to be edited, for example, when editing a large number of cells in reaction, the amount of RNP complex that is added can be reduced.

[0400] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) can be encoded by a separate polynucleotide, and each polynucleotide can be introduced into a cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multi-cistronic vector; see the description of exemplary multi-cistronic systems below) and introduced into a cell. After expression of the CRISPR components encoded by each polynucleotide in the cell (e.g., translation of a nuclease and transcription of a CRISPR RNA), an RNP complex can form within the cell and then direct site-specific cleavage.

[0401] Some RNPs can be engineered to have moieties that facilitate delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain so that when the Cas9 RNP complex is delivered into the cytosol of a cell, or after translation of Cas9 and subsequent RNP formation, the NLS can facilitate further transport of the Cas9 RNP into the nucleus.

[0402] 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 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, such as adenovirus, retrovirus, lentivirus, or any of the other viral-based delivery methods described herein.

[0403] In some CRISPR systems, more than one CRISPR composition can be provided, each of which separately targets the same gene or common genomic locus at more than one target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a specific distance from each other. In some CRISPR systems, two or more CRISPR compositions can be provided, each of which separately targets opposite strands of the same gene or common genomic locus. For example, two separate CRISPR "nickase" compositions can be provided to direct cleavage at the same gene or common genomic locus on opposite strands.

[0404] In general, the features of the CRISPR-mediated editing system described herein can be applied to other nuclease-based genome editing systems. TALENs are engineered site-specific nucleases that consist of the DNA binding domain of a TALE (transcription activator-like effector) and the catalytic domain of the restriction endonuclease Fokl. By varying the amino acids present in the highly variable residue region of the DNA binding domain monomer, different artificial TALENs can be created to target various nucleotide sequences. The DNA binding domain then directs the nuclease to the target sequence and creates a double-strand break. TALEN-based systems are described in more detail in U.S. Patent 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 more 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; 2005 / 0267061, all of which are incorporated by reference in their entirety for all purposes.

[0405] Other engineered delivery systems A variety of additional means for introducing an engineered nucleic acid (e.g., an isolated polynucleotide encoding a modified ER-LBD or chimeric protein described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein.

[0406] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of applying an electric field to transiently permeabilize the outer membrane or shell of the target cell or entity, thereby internalizing cargo / payload into the internal compartment of the target cell or entity. Generally, the 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, thereby allowing the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the case of cells, at least some, but 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., number of cells, cargo concentration, recovery conditions, voltage, time, capacitance, 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 efficiency of internalization, and the desired survival rate. 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.

[0407] Other means for introducing an engineered nucleic acid (e.g., an isolated polynucleotide encoding a modified ER-LBD or chimeric protein 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.

[0408] Compositions and methods for delivering engineered mRNA in vivo, such as naked plasmids or 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.

[0409] How to use Methods of using the modified ER-LBD, chimeric proteins, or cells of the inducible cell death systems described herein are also encompassed by the present disclosure.

[0410] In some aspects, the methods include a method of inducing cell death, comprising (i) transforming a cell with a heterologous construct encoding an inducible cell death system (e.g., any one of the inducible cell death systems described herein), and (ii) contacting the transformed cell with a non-endogenous ligand of a modified estrogen receptor ligand-binding domain (ER-LBD).

[0411] In some embodiments, methods include methods of inducing cell death, comprising: (i) transforming a cell with a heterologous construct encoding an inducible cell death system comprising a polypeptide, wherein the polypeptide comprises a ligand-binding domain and a cell death-inducing domain, wherein the polypeptide is configured to generate a cell death-inducing signal in a cell in which the polypeptide is expressed upon contact with a ligand of the ligand-binding domain, and wherein the ligand-binding domain comprises a modified ER-LBD (such as any of the modified ER-LBDs described herein); and (ii) contacting the transformed cell with a non-endogenous ligand of the modified estrogen receptor ligand-binding domain (ER-LBD).

[0412] In some embodiments, methods include methods of inducing cell death, comprising: (i) transforming a cell with an inducible cell death system comprising a first polypeptide monomer and a second polypeptide monomer, wherein the first polypeptide monomer and the second polypeptide monomer each comprise a ligand-binding domain and a cell death-inducing domain, and wherein the first polypeptide monomer and the second polypeptide monomer are configured to oligomerize with each other upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in the cell in which the first polypeptide monomer and the second polypeptide monomer are expressed, and wherein the ligand-binding domain comprises a modified ER-LBD (such as, for example, any of the modified ER-LBDs described herein); and (ii) contacting the transformed cell with a non-endogenous ligand of the modified estrogen receptor ligand-binding domain (ER-LBD).

[0413] In some aspects, the method includes modulating transcription of a gene of interest. The method of modulating transcription includes transforming a cell with (i) a heterologous construct encoding a chimeric transcription factor comprising a modified ER-LBD and (ii) a target expression cassette comprising a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to a gene of interest; culturing the transformed cell under conditions appropriate for expression of the chimeric protein; and contacting the transformed cell with a non-endogenous ligand, thereby inducing the chimeric protein to modulate transcription of the gene of interest.

[0414] In some embodiments, the method of regulating transcription is a method of activating transcription. Activating transcription can be achieved using a chimeric protein.

[0415] In some embodiments, the method includes activating transcription, which can be achieved, for example, using a chimeric protein comprising a modified ER-LBD, a DNA binding domain, and a transcription activation domain.

[0416] In some embodiments, the method includes repressing transcription, which can be achieved, for example, using a chimeric protein comprising a modified ER-LBD, a DNA binding domain, and a transcriptional repressor domain.

[0417] In some aspects, the method comprises modulating localization of the chimeric protein. The method of modulating localization may comprise transforming a cell with a heterologous construct encoding a chimeric protein comprising a modified ER-LBD domain and a polypeptide of interest, culturing the transformed cell under conditions appropriate for expression of the chimeric protein, and inducing nuclear localization of the chimeric protein by contacting the transformed cell with a non-endogenous ligand. In some embodiments, modulating localization comprises inducing nuclear localization.

[0418] In some embodiments, the non-endogenous ligand is administered at a concentration where the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha.

[0419] In vivo methods The methods provided herein also include in vivo methods for, e.g., inducing cell death, inducing oligomerization of the chimeric proteins provided herein, altering localization and / or modulating transcription in vivo, e.g., by delivering a non-endogenous ligand to cells expressing the modified ER-LBD or chimeric protein in vivo.

[0420] In some embodiments, the transformed cell is in a human or animal, and contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal. In some embodiments, the non-endogenous ligand administered to the subject comprises tamoxifen. Upon oral administration of tamoxifen, the drug is converted to an active tamoxifen metabolite in the liver. In some embodiments, the tamoxifen metabolite is selected from 4-hydroxytamoxifen ("4-OHT"), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. In some embodiments, the non-endogenous ligand is administered to the subject at a concentration between about 1 mg per day and about 100 mg per day. In certain embodiments, the non-endogenous ligand is administered to the subject at a concentration of about 40 mg per day. In some embodiments, administering comprises administering one or more non-endogenous ligands to the human or animal. Exemplary non-endogenous ligands include, for example, tamoxifen, 4-OHT, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. In some embodiments, administering includes administering two or more non-endogenous ligands to a human or animal. In some embodiments, the two or more non-endogenous ligands include endoxifen and 4-OHT.

[0421] In some aspects, the methods provided herein also include modulating transcription of a gene of interest in vivo, for example, by (i) a cell transformed with a chimeric transcription factor described herein and (ii) delivering a non-endogenous ligand to a subject. In some embodiments, the transformed cell comprises a target gene expression cassette comprising a chimeric transcription factor-responsive promoter operably linked to a gene of interest.

[0422] In some embodiments, contacting the human or animal subject and the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the non-endogenous ligand to the human or animal.

[0423] In some aspects, the methods provided herein also include delivering a composition in vivo capable of producing an engineered cell as described herein, e.g., capable of delivering a polynucleotide as described herein to a cell 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 capable of engineering cells in vivo.

[0424] The methods provided herein also include delivering in vivo a composition capable of producing any of the modified ER-LBDs, chimeric proteins, or chimeric transcription factors (and in some embodiments, genes regulated by the chimeric transcription factors) described herein. Compositions capable of in vivo production of modified ER-LBDs, chimeric proteins, or chimeric transcription factors (and in some embodiments, genes regulated by the chimeric transcription factors) include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production of inducible transcription factors (and in some embodiments, genes regulated by the inducible transcription factors) can be naked mRNA or naked plasmids.

[0425] Pharmaceutical Composition The modified ER-LBD, chimeric proteins, and cells of the present disclosure can be formulated into pharmaceutical compositions. These compositions can contain, in addition to one or more of the engineered nucleic acids or engineered cells, pharmaceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those of skill in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other materials can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, or intraperitoneal routes.

[0426] Whether administered to an individual is a cell, 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" (although in some cases, prophylaxis may 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 disease being treated. Prescription of treatment, e.g., determining dosage, etc., is within the responsibility of general practitioners and other physicians, and typically takes into account the disorder being treated, the condition of the individual patient, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols referred to above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0427] The compositions can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated.

[0428] Enumerated Embodiments Embodiment 1: An inducible cell death system comprising a polypeptide, the polypeptide comprising a ligand-binding domain and a cell death-inducing domain, the polypeptide configured to generate a cell death-inducing signal in a cell in which the polypeptide is expressed upon contact with a ligand of the ligand-binding domain; and a. the ligand-binding domain comprises a modified estrogen receptor ligand-binding domain (ER-LBD) corresponding to the hormone-binding domain of the reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD: i. a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and ii. An inducible cell death system comprising one or more additional amino acid substitutions, with reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, positions 517-540, and position 547 of SEQ ID NO:1. Embodiment 2: the polypeptide is or comprises a first polypeptide monomer, and the inducible cell death system further comprises a second polypeptide monomer; and a. The inducible cell death system of embodiment 1, wherein the first polypeptide monomer and the second polypeptide monomer each comprise a ligand-binding domain and a cell death-inducing domain, and wherein the first polypeptide monomer and the second polypeptide monomer are configured to oligomerize with each other upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in a cell in which the first polypeptide monomer and the second polypeptide monomer are expressed. Embodiment 3: The ligand binding domains of the first polypeptide monomer and the second polypeptide monomer each comprise a modified ER-LBD, wherein the modified ER-LBD comprises: i. a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1; and ii. The inducible cell death system of embodiment 2, comprising one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are independently selected for each of the first polypeptide monomer and the second polypeptide monomer with reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, and positions 517-540, and position 547 of SEQ ID NO:1. Embodiment 4: 4. The inducible cell death system of embodiment 2 or 3, wherein the ligand binding domains of the first polypeptide monomer and the second polypeptide monomer comprise the same additional amino acid substitution. Embodiment 5: 5. The inducible cell death system according to any one of embodiments 1 to 4, wherein the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2. Embodiment 6: 6. The inducible cell death system of any one of embodiments 1 to 5, wherein the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to endogenous ligands as a result of one or more additional amino acid substitutions. Embodiment 7: 7. The inducible cell death system according to any one of embodiments 1 to 6, wherein the modified ER-LBD has greater selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2. Embodiment 8: 8. The inducible cell death system of any one of embodiments 1-7, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391 substitution. Embodiment 9: 9. The inducible cell death system of embodiment 8, wherein the L391 substitution is L391V. Embodiment 10: 10. The inducible cell death system of any one of embodiments 1-9, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a N413D mutation. Embodiment 11: 8. The inducible cell death system of any one of embodiments 1-7, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution and a N413D mutation. Embodiment 12: 12. The inducible cell death system of any one of embodiments 1-11, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a H524 substitution. Embodiment 13: 13. The inducible cell death system of embodiment 12, wherein the H524 substitution is a H524L substitution or a H524F substitution. Embodiment 14: 13. The inducible cell death system of embodiment 12, wherein the H524 substitution is a H524L substitution. Embodiment 15: 13. The inducible cell death system of embodiment 12, wherein the H524 substitution is a H524F substitution. Embodiment 16: 16. The inducible cell death system of any one of embodiments 1-15, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a M421L substitution. Embodiment 17: 17. The inducible cell death system of any one of embodiments 1-16, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a S463P substitution. Embodiment 18: 16. The inducible cell death system of any one of embodiments 1-15, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a M421L substitution and a S463P substitution. Embodiment 19: 19. The inducible cell death system of any one of embodiments 1-18, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L384M substitution. Embodiment 20: 20. The inducible cell death system of any one of embodiments 1-19, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L354I substitution. Embodiment 21: 21. The inducible cell death system of any one of embodiments 1-20, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a Q414E substitution. Embodiment 22: 20. The inducible cell death system of any one of embodiments 1-19, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L354I substitution and a Q414E substitution. Embodiment 23: 23. The inducible cell death system of any one of embodiments 1-22, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, and a H524 substitution. Embodiment 24: 23. The inducible cell death system of any one of embodiments 1-22, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a M421L substitution. Embodiment 25: 23. The inducible cell death system of any one of embodiments 1-22, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a S463P substitution. Embodiment 26: 23. The inducible cell death system of any one of embodiments 1-22, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a Q414E substitution. Embodiment 27: 23. The inducible cell death system of any one of embodiments 1-22, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a L354I substitution. Embodiment 28: 28. The inducible cell death system of any one of embodiments 23 to 27, wherein the H524 substitution is a H524L substitution or a H524F substitution. Embodiment 29: 29. The inducible cell death system of any one of embodiments 1-28, wherein the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are at one or more positions in SEQ ID NO: 1 selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537, 538, 539, 540, and 547. Embodiment 30: 30. The inducible cell death system of embodiment 29, wherein the one or more positions include position 343 of SEQ ID NO:1. Embodiment 31: 31. The inducible cell death system of embodiment 30, wherein the amino acid substitution at position 343 of SEQ ID NO:1 is selected from the group consisting of M343F, M343I, M343L, and M343V. Embodiment 32: 30. The inducible cell death system of embodiment 29, wherein the one or more positions include position 344 of SEQ ID NO:1. Embodiment 33: 33. The inducible cell death system of embodiment 32, wherein the amino acid substitution at position 344 of SEQ ID NO:1 is G344M. Embodiment 34: 30. The inducible cell death system of embodiment 29, wherein the one or more positions include position 345 of SEQ ID NO:1. Embodiment 35: 35. The inducible cell death system of embodiment 34, wherein the amino acid substitution at position 345 of SEQ ID NO: 1 is L345S. Embodiment 36: 30. The inducible cell death system of embodiment 29, wherein the one or more positions include position 346 of SEQ ID NO:1. Embodiment 37: 37. The inducible cell death system of embodiment 36, wherein the amino acid substitution at position 346 of SEQ ID NO:1 is selected from the group consisting of L346I, L346M, L346F, and L346V. Embodiment 38: 30. The inducible cell death system of embodiment 29, wherein the one or more positions include position 347 of SEQ ID NO:1. Embodiment 39: 39. The inducible cell death system of embodiment 38, wherein the amino acid substitution at position 347 of SEQ ID NO:1 is selected from the group consisting of T347D, T347E, T347F, T347I, T347K, T347L, T347M, T347N, T347Q, T347R, T347S, and T347V. Embodiment 40: 30. The inducible cell death system of embodiment 29, wherein the one or more positions include position 348 of SEQ ID NO:1. Embodiment 41: 41. The inducible cell death sys...

Claims

1. An inducible cell death system comprising a polypeptide, the polypeptide comprising a ligand-binding domain and a cell death-inducing domain, the polypeptide configured to generate a cell death-inducing signal in a cell in which the polypeptide is expressed upon contact with a ligand of the ligand-binding domain; and The ligand binding domain comprises a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises: (a) with reference to SEQ ID NO: 1, a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution; and (b) one or more additional amino acid substitutions, with reference to one or more regions selected from positions 343-354, positions 380-392, positions 404-463, positions 517-540, and position 547 of SEQ ID NO:1; optionally, the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2; optionally, the modified ER-LBD has greater sensitivity to non-endogenous ligands compared to endogenous ligands as a result of the one or more additional amino acid substitutions; optionally, the modified ER-LBD has greater selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2; Optionally, the ligand binding domains of the first polypeptide monomer and the second polypeptide monomer comprise the same additional amino acid substitution.

2. a. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391 substitution, optionally wherein the L391 substitution is L391V; b. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an N413D mutation; or c. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution and a N413D mutation; d. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a H524 substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; e. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a M421L substitution; f. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a S463P substitution; g. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an M421L substitution and an S463P substitution; h. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L384M substitution; i. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L354I substitution; j. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a Q414E substitution; k. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L354I substitution and a Q414E substitution; l. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, and a H524 substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; m. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a M421L substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; n. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a S463P substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; o. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a Q414E substitution, optionally wherein the H524 substitution is a H524L substitution or a H524F substitution; p. The inducible cell death system of claim 1, wherein the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D mutation, a H524 substitution, and a L354I substitution, and optionally, the H524 substitution is a H524L substitution or a H524F substitution.

3. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are at one or more positions of SEQ ID NO: 1 selected from 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 354, 380, 384, 386, 387, 388, 389, 391, 392, 404, 407, 409, 413, 414, 417, 418, 420, 421, 422, 424, 428, 463, 517, 521, 522, 524, 525, 526, 527, 528, 533, 534, 536, 537, 538, 539, 540, and 547; Depending on the situation, i. the one or more positions include position 343 of SEQ ID NO:1, and optionally the amino acid substitution at position 343 of SEQ ID NO:1 is selected from the group consisting of M343F, M343I, M343L, and M343V; ii. the one or more positions include position 344 of SEQ ID NO:1, and optionally the amino acid substitution at position 344 of SEQ ID NO:1 is G344M; iii. the one or more positions include position 345 of SEQ ID NO:1, and optionally the amino acid substitution at position 345 of SEQ ID NO:1 is L345S; iv. the one or more positions include position 346 of SEQ ID NO:1, and optionally the amino acid substitution at position 346 of SEQ ID NO:1 is selected from the group consisting of L346I, L346M, L346F, and L346V; v. said one or more positions comprises position 347 of SEQ ID NO:1, and optionally said amino acid substitution at position 347 of SEQ ID NO:1 is selected from the group consisting of T347D, T347E, T347F, T347I, T347K, T347L, T347M, T347N, T347Q, T347R, T347S, and T347V; vi. the one or more positions include position 348 of SEQ ID NO:1, and optionally the amino acid substitution at position 348 of SEQ ID NO:1 is N348K; vii. the one or more positions include position 349 of SEQ ID NO:1, and optionally the amino acid substitution at position 349 of SEQ ID NO:1 is selected from the group consisting of L349I, L349M, L349F, and L349V; viii. the one or more positions include position 350 of SEQ ID NO:1, and optionally the amino acid substitution at position 350 of SEQ ID NO:1 is selected from the group consisting of A350F, A350I, A350L, A350M, and A350V; ix. the one or more positions include position 351 of SEQ ID NO:1, and optionally the amino acid substitution at position 351 of SEQ ID NO:1 is selected from the group consisting of D351E, D351F, D351I, D351L, D351M, D351N, D351Q, and D351V; x. the one or more positions include position 352 of SEQ ID NO:1, and optionally the amino acid substitution at position 352 of SEQ ID NO:1 is R352K; xi. the one or more positions include position 354 of SEQ ID NO:1, and optionally the amino acid substitution at position 354 of SEQ ID NO:1 is selected from the group consisting of L354I, L354M, L354F, and L354V; xii. the one or more positions include position 380 of SEQ ID NO:1, and optionally the amino acid substitution at position 380 of SEQ ID NO:1 is E380Q; xiii. the one or more positions include position 384 of SEQ ID NO:1, and optionally the amino acid substitution at position 384 of SEQ ID NO:1 is selected from the group consisting of L384I, L384M, L384F, and L384V; xiv. the one or more positions include position 386 of SEQ ID NO:1, and optionally the amino acid substitution at position 386 of SEQ ID NO:1 is I386V; xv. the one or more positions include position 387 of SEQ ID NO:1, and optionally the amino acid substitution at position 387 of SEQ ID NO:1 is selected from the group consisting of L387I, L387M, L387F, and L387V; xvi. the one or more positions include position 388 of SEQ ID NO:1, and optionally the amino acid substitution at position 388 of SEQ ID NO:1 is selected from the group consisting of M388I, M388L, and M388F; xvii. the one or more positions include position 389 of SEQ ID NO:1, and optionally the amino acid substitution at position 389 of SEQ ID NO:1 is I389M; xviii. the one or more positions include position 391 of SEQ ID NO:1, and optionally, the amino acid substitution at position 391 of SEQ ID NO:1 is selected from the group consisting of L391I, L391M, L391F, and L391V, and optionally, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V; xix. the one or more positions include position 392 of SEQ ID NO:1, and optionally the amino acid substitution at position 392 of SEQ ID NO:1 is V392M; xx. the one or more positions include position 404 of SEQ ID NO:1, and optionally the amino acid substitution at position 404 of SEQ ID NO:1 is selected from the group consisting of F404I, F404L, F404M, and F404V; xxi. the one or more positions include position 407 of SEQ ID NO:1, and optionally the amino acid substitution at position 407 of SEQ ID NO:1 is N407D; xxii. the one or more positions include position 409 of SEQ ID NO:1, and optionally the amino acid substitution at position 409 of SEQ ID NO:1 is L409V; xxiii. the one or more positions include position 413 of SEQ ID NO:1, and optionally the amino acid substitution at position 413 of SEQ ID NO:1 is N413D; xxiv. the one or more positions include position 414 of SEQ ID NO:1, and optionally the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E; xxv. the one or more positions include position 417 of SEQ ID NO:1, and optionally the amino acid substitution at position 417 of SEQ ID NO:1 is C417S; xxvi. the one or more positions include position 418 of SEQ ID NO:1, and optionally the amino acid substitution at position 418 of SEQ ID NO:1 is selected from the group consisting of V418I, V418L, V418M, and V418F; xxvii. the one or more positions include position 420 of SEQ ID NO:1, and optionally the amino acid substitution at position 420 of SEQ ID NO:1 is selected from the group consisting of G420I, G420M, G420F, and G420V; xxviii. the one or more positions include position 421 of SEQ ID NO:1, and optionally the amino acid substitution at position 421 of SEQ ID NO:1 is selected from the group consisting of M421I, M421L, M421F, and M421V; xxix. the one or more positions include position 422 of SEQ ID NO:1, and optionally the amino acid substitution at position 422 of SEQ ID NO:1 is V422I; xxx. the one or more positions include position 424 of SEQ ID NO:1, and optionally the amino acid substitution at position 424 of SEQ ID NO:1 is selected from the group consisting of I424L, I424M, I424F, and I424V; xxxi. the one or more positions include position 428 of SEQ ID NO:1, and optionally the amino acid substitution at position 428 of SEQ ID NO:1 is selected from the group consisting of L428I, L428M, L428F, and L428V; xxxii. the one or more positions include position 463 of SEQ ID NO:1, and optionally the amino acid substitution at position 463 of SEQ ID NO:1 is S463P; xxxiii. the one or more positions include position 517 of SEQ ID NO:1, and optionally the amino acid substitution at position 517 of SEQ ID NO:1 is M517A; xxxiv. the one or more positions include position 521 of SEQ ID NO:1, and optionally the amino acid substitution at position 521 of SEQ ID NO:1 is selected from the group consisting of G521A, G521F, G521I, G521L, G521M, and G521V; xxxv. the one or more positions include position 522 of SEQ ID NO:1, and optionally the amino acid substitution at position 522 of SEQ ID NO:1 is selected from the group consisting of M522I, M522L, and M522V; xxxvi. the one or more positions include position 524 of SEQ ID NO:1, and optionally the amino acid substitution at position 524 of SEQ ID NO:1 is selected from the group consisting of H524A, H524I, H524L, H524F, and H524V; xxxvii. the one or more positions include position 525 of SEQ ID NO:1, and optionally the amino acid substitution at position 525 of SEQ ID NO:1 is selected from the group consisting of L525F, L525I, L525M, L525N, L525Q, L525S, L525T, and L525V; xxxviii. the one or more positions include position 526 of SEQ ID NO:1, and optionally, the amino acid substitution at position 526 of SEQ ID NO:1 is Y526L; xxxix. the one or more positions include position 527 of SEQ ID NO:1, and optionally the amino acid substitution at position 527 of SEQ ID NO:1 is S527N; xl. the one or more positions include position 528 of SEQ ID NO:1, and optionally the amino acid substitution at position 528 of SEQ ID NO:1 is selected from the group consisting of M528F, M528I, and M528V; xli. the one or more positions include position 533 of SEQ ID NO:1, and optionally the amino acid substitution at position 533 of SEQ ID NO:1 is selected from the group consisting of V533F and V533W; xlii. the one or more positions include position 534 of SEQ ID NO:1, and optionally, the amino acid substitution at position 534 of SEQ ID NO:1 is selected from the group consisting of V534Q and V534R; xliii. the one or more positions include position 536 of SEQ ID NO:1, and optionally, the amino acid substitution at position 536 of SEQ ID NO:1 is selected from the group consisting of L536F, and L536M, L536R, and L536Y; xliv. the one or more positions include position 537 of SEQ ID NO:1, and optionally the amino acid substitution at position 537 of SEQ ID NO:1 is selected from the group consisting of Y537E and Y537S; xlv. the one or more positions include position 538 of SEQ ID NO:1, and optionally the amino acid substitution at position 538 of SEQ ID NO:1 is selected from the group consisting of D538G and D538K; xlvi. the one or more positions include position 539 of SEQ ID NO:1, and optionally, the amino acid substitution at position 539 of SEQ ID NO:1 is selected from the group consisting of L539A and L539R; xlvii. the one or more positions include position 540 of SEQ ID NO:1, and optionally, the amino acid substitution at position 540 of SEQ ID NO:1 is selected from the group consisting of L540A and L540F; and / or 10. The inducible cell death system of claim 1, wherein the one or more positions include position 547 of SEQ ID NO:1, and optionally the amino acid substitution at position 547 of SEQ ID NO:1 is H547A.

4. a. the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are two amino acid substitutions, optionally each of the two amino acid substitutions being at positions in SEQ ID NO: 1 selected from 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525; and optionally i. the two amino acid substitutions are at positions 345 and 348 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 345 of SEQ ID NO:1 is L345S and the amino acid substitution at position 348 of SEQ ID NO:1 is N348K; ii. the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 389 of SEQ ID NO:1 is I389M; iii. the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 421 of SEQ ID NO:1 is M421I and the amino acid substitution at position 392 of SEQ ID NO:1 is V392M; iv. the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; v. the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 384 of SEQ ID NO:1 is L384M; vi. the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I and the amino acid substitution at position 387 of SEQ ID NO:1 is L387M; vii. the two amino acid substitutions are at positions 387 and 391, and optionally, the amino acid substitution at position 387 of SEQ ID NO:1 is L387M and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; viii. the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 387 of SEQ ID NO:1 is L387M; or ix. the two amino acid substitutions are at positions 384 and 391 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; b. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are three amino acid substitutions, optionally each of the three amino acid substitutions is at a position in SEQ ID NO: 1 selected from 343, 347, 351, 354, 388, 391, 404, 414, 418, 463, 521, 524, and 525; and optionally i. the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, and the amino acid substitution at position 391 of SEQ ID NO:1 is L391F; ii. the three amino acid substitutions are at positions 414, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; c. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are four amino acid substitutions, optionally each of the four amino acid substitutions being at positions in SEQ ID NO: 1 selected from 343, 347, 351, 354, 384, 388, 391, 404, 413, 418, 463, 521, 524, and 525; and optionally i. the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391F, and the amino acid substitution at position 418 of SEQ ID NO:1 is V418I; ii. the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 343 of SEQ ID NO:1 is M343I, the amino acid substitution at position 388 of SEQ ID NO:1 is M388I, the amino acid substitution at position 521 of SEQ ID NO:1 is G521I, and the amino acid substitution at position 404 of SEQ ID NO:1 is F404L; iii. the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 524 of SEQ ID NO:1 is H524V, the amino acid substitution at position 347 of SEQ ID NO:1 is T347R, the amino acid substitution at position 351 of SEQ ID NO:1 is D351Q, and the amino acid substitution at position 525 of SEQ ID NO:1 is L525N; iv. the four amino acid substitutions are at positions 354, 384, 391, and 463 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, and the amino acid substitution at position 463 of SEQ ID NO:1 is S463P; v. the four amino acid substitutions are at positions 384, 391, 413, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; d. the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are five amino acid substitutions, optionally each of the five amino acid substitutions is at a position in SEQ ID NO: 1 selected from 354, 384, 391, 409, 413, 414, 421, 463, and 524; and optionally i. the five amino acid substitutions are at positions 384, 409, 413, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; ii. the five amino acid substitutions are at positions 391, 413, 414, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; iii. the five amino acid substitutions are at positions 391, 414, 421, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; iv. the five amino acid substitutions are at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; or v. the five amino acid substitutions are at positions 354, 409, 421, 463, and 524 of SEQ ID NO:1, and optionally the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; e. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are six amino acid substitutions, optionally each of the six amino acid substitutions is at a position in SEQ ID NO: 1 selected from 354, 384, 391, 409, 413, 414, 421, 463, and 524; and optionally i. the six amino acid substitutions are at positions 384, 391, 413, 421, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; ii. the six amino acid substitutions are at positions 409, 413, 414, 421, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; iii. the six amino acid substitutions are at positions 354, 391, 409, 413, 414, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; f. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are seven amino acid substitutions, optionally each of the seven amino acid substitutions is at a position in SEQ ID NO: 1 selected from 354, 384, 391, 409, 413, 414, 421, 463, 517, and 524; and optionally i. the seven amino acid substitutions are at positions 354, 384, 409, 413, 421, 463, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; ii. the seven amino acid substitutions are at positions 354, 391, 413, 421, 463, 517, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524L; the seven amino acid substitutions are at positions 354, 391, 413, 414, 421, 517, and 524 of SEQ ID NO:1, optionally wherein the amino acid substitution at position 354 of SEQ ID NO:1 is L354I, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F; or g. the one or more additional amino acid substitutions in the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are eight amino acid substitutions, optionally the eight amino acid substitutions are at positions 384, 391, 409, 413, 421, 463, 517, and 524 of SEQ ID NO:1; and optionally i. The inducible cell death system of any one of claims 1 to 3, wherein the amino acid substitution at position 384 of SEQ ID NO:1 is L384M, the amino acid substitution at position 391 of SEQ ID NO:1 is L391V, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D, the amino acid substitution at position 421 of SEQ ID NO:1 is M421L, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A, and the amino acid substitution at position 524 of SEQ ID NO:1 is H524F.

5. an inducible cell death system comprising a first polypeptide and a second polypeptide monomer, each of the first and second polypeptide monomers comprising a ligand-binding domain and a cell death-inducing domain, the first and second polypeptide monomers being configured to oligomerize upon contact with a ligand of the ligand-binding domain, thereby generating a cell death-inducing signal in a cell in which the first and second polypeptide monomers are expressed; and The ligand-binding domain comprises a modified estrogen receptor ligand-binding domain (ER-LBD) comprising an amino acid sequence corresponding to the hormone-binding domain of a reference human estrogen receptor sequence (SEQ ID NO: 1), wherein the modified ER-LBD comprises: (a) with reference to SEQ ID NO: 1, a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution; and (b) additional amino acid substitutions, with reference to SEQ ID NO: 1; (i) an L384M substitution, an L391V substitution, an N413D substitution, an M421L substitution, an S463P substitution, and an H524L substitution; (ii) an L391V substitution, an N413D substitution, a Q414E substitution, an S463P substitution, and an H524F substitution; (iii) an L354I substitution, an L391V substitution, an N413D substitution, a Q414E substitution, an M421L substitution, an M517A substitution, and an H524F substitution, or (iv) An inducible cell death system comprising additional amino acid substitutions, including L354I, L391V, L409V, N413D, Q414E, and H524L substitutions.

6. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a N413D mutation, a H524 substitution, and a S463P substitution; and optionally the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 90 or 103; b. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L384M substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 91 or 104; c. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 92 or 105; d. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 93 or 106; e. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 94 or 107; f. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a L354I substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 95 or 108; g. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 96 or 109; h. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 97 or 110; i. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a L384M substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 98 or 111; j. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 99 or 112; k. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an N413D substitution, an S463P substitution, an L354I substitution, an L384M substitution, and an H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to SEQ ID NO: 100 or 113; or l. the one or more additional amino acid substitutions of the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer comprise an N413D substitution, an S463P substitution, an M421L substitution, an L354I substitution, and an H524L substitution, and optionally the polypeptide, the first polypeptide monomer, and / or the second polypeptide monomer are at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or more identical to SEQ ID NO: 101 or 114. and optionally the ligand is a non-endogenous ligand, optionally the non-endogenous ligand is selected from 4-hydroxy tamoxifen, N-desmethyl tamoxifen, tamoxifen-N-oxide, and endoxifen, optionally the non-endogenous ligand comprises a tamoxifen metabolite, and optionally the non-endogenous ligand is endoxifen.

7. the first polypeptide monomer and the second polypeptide monomer are a. at a concentration of 0.25 nM endoxifen or less, and / or at a concentration of 0.04 nM 4-OHT or less; b. at a concentration of 2.5 nM endoxifen or less, and / or at a concentration of 0.4 nM 4-OHT or less; c. at a concentration of at least 0.001 pM 4-OHT, or d. The inducible cell death system according to any one of claims 1 to 6, which is capable of oligomerization and / or generating the cell death-inducing signal at a concentration of 4-OHT of at least 0.01 pM.

8. The cell death-inducing domain is a. Caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53-upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related death-inducing ligand (TRAIL), herpes simplex virus derived from a protein selected from thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, and purine nucleoside phosphorylase, optionally wherein the cell death-inducing domain comprises the caspase-9 derived amino acid sequence of SEQ ID NO: 48 or 125, and optionally wherein the caspase domain or functional fragment thereof does not comprise a caspase activation and recruitment domain (CARD) domain sequence; or b. A transcription factor comprising a nucleic acid binding domain and a transcription effector domain, wherein the transcription factor is a caspase domain or a functional fragment thereof, optionally wherein the caspase is selected from caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, or a functional fragment thereof; a caspase domain or a functional fragment thereof; diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-Xs, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), O 8. The inducible cell death system according to claim 1, wherein the transcription factor is a transcription factor that is configured to generate a cell death-inducing signal by inducing expression of any one of the following: mi, Bmf, Bid, Bim, p53-upregulated regulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamarase, hepatic cytochrome P450-2B1, or purine nucleoside phosphorylase.

9. An isolated polynucleotide comprising a nucleotide sequence encoding the polypeptide, first polypeptide monomer, and / or second polypeptide monomer of any one of claims 1 to 8.

10. A heterologous construct comprising a promoter operably linked to the polynucleotide of claim 9.

11. A plasmid or vector comprising the heterologous construct of claim 10.

12. A cell comprising the heterologous construct of claim 10 or the plasmid or vector of claim 11.

13. A molecular switch for generating a cell death-inducing signal in a cell, comprising: (a) an inducible cell death system according to any one of claims 1 to 8, an isolated polynucleotide according to claim 9, a heterologous construct according to claim 10, a plasmid or vector according to claim 11, or a cell according to claim 12, wherein the inducible cell death system is capable of generating a cell death-inducing signal in the cell, and (b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD generates the cell death-inducing signal in the cell, optionally the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen, optionally the non-endogenous ligand comprises a tamoxifen metabolite, optionally the non-endogenous ligand is endoxifen, and optionally a. the first polypeptide monomer and the second polypeptide monomer are capable of oligomerizing and / or producing the cell death-inducing signal at a concentration of 0.25 nM endoxifen or less and / or at a concentration of 0.04 nM 4-OHT or less; b. the first polypeptide monomer and the second polypeptide monomer are capable of oligomerizing and / or producing the cell death-inducing signal at a concentration of 2.5 nM endoxifen or less and / or at a concentration of 0.4 nM 4-OHT or less; c. the first polypeptide monomer and the second polypeptide monomer are capable of oligomerizing and / or producing the cell death-inducing signal at a concentration of 4-OHT of at least 0.001 pM; or d. A molecular switch comprising a non-endogenous ligand, wherein the first polypeptide monomer and the second polypeptide monomer are capable of oligomerization and / or generating the cell death-inducing signal at a concentration of 4-OHT of at least 0.01 pM.

14. 12. A method for inducing oligomerization of a chimeric protein, comprising: (i) transforming a cell with a heterologous construct encoding any one of the inducible cell death system of any one of claims 1 to 8, the isolated polynucleotide of claim 9, the heterologous construct of claim 10, or the plasmid or vector of claim 11; and (ii) contacting the transformed cell with a non-endogenous ligand of the modified estrogen receptor ligand binding domain (ER-LBD); and optionally a. the method further comprises culturing the transformed cells under conditions suitable for expression of the inducible cell death system prior to inducing oligomerization and / or inducing cell death; b. the transformed cell is in a human or animal, and contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal; and / or c. The non-endogenous ligand is selected from 4-hydroxy tamoxifen, N-desmethyl tamoxifen, tamoxifen-N-oxide, and endoxifen, optionally wherein the non-endogenous ligand comprises a tamoxifen metabolite, optionally wherein the non-endogenous ligand is endoxifen, and optionally wherein the non-endogenous ligand is administered at a concentration where the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha of SEQ ID NO:

1.

15. a modified estrogen receptor ligand binding domain (ER-LBD) corresponding to the hormone binding domain of the reference human estrogen receptor sequence (SEQ ID NO: 1), a. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a N413D substitution, a S463P substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; b. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1; c. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; d. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, with reference to SEQ ID NO: 1; e. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1; f. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; g. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, and a H524L substitution, with reference to SEQ ID NO: 1; h. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a L409V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1; i. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M421L substitution, a L354I substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; j. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a Q414E substitution, a N413D substitution, a S463P substitution, a M517A substitution, a M421L substitution, a L354I substitution, and a H524L substitution, with reference to SEQ ID NO: 1; k. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a N413D substitution, a S463P substitution, a M517A substitution, a L384M substitution, and a H524L substitution, with reference to SEQ ID NO: 1; l. the modified ER-LBD comprises (a) a G400V amino acid substitution, a M543A amino acid substitution, a L544A amino acid substitution, and optionally a V595A amino acid substitution, with reference to SEQ ID NO: 1, and (b) one or more additional amino acid substitutions, including a L391V substitution, a L409V substitution, a N413D substitution, a S463P substitution, a M517A substitution, and a H524L substitution, with reference to SEQ ID NO: 1; or m. the modified ER-LBD comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 97.5%, 98%, 99%, or 100% identical to any one of SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, and SEQ ID NO:114; Optionally, the modified ER-LBD has greater sensitivity and / or selectivity to a non-endogenous ligand compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2 or compared to an endogenous ligand as a result of the one or more additional amino acid substitutions, optionally the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen, optionally the endogenous ligand is estradiol, and optionally the modified ER-LBD further comprises a V595A amino acid substitution.

16. 16. A chimeric protein comprising a polypeptide of interest fused to the modified ER-LBD of claim 15, optionally wherein the polypeptide of interest comprises a nucleic acid binding domain, optionally wherein the nucleic acid binding domain comprises a zinc finger domain, optionally wherein the zinc finger domain comprises the sequence set forth in SEQ ID NO:57 or SEQ ID NO:84, optionally wherein the chimeric protein comprises a chimeric transcription factor, and wherein the polypeptide of interest comprises a nucleic acid binding domain and a transcription regulator domain, optionally wherein the transcription regulator domain is a transcription activator, optionally wherein the transcription activator is Herpes simplex virus protein 16 (VP16) activation domain, an activation domain containing four tandem copies of VP16, a VP64 activation domain, the p65 activation domain of NFκB (p65), an Epstein-Barr virus R transactivator (Rta) activation domain, a tripartite activator containing the VP64, the p65, and the Rta activation domain (VPR activation domain), a tripartite activator containing the VP64, the p65, and the HSF1 activation domain (VPH activation domain), and a histone acetyltransferase core domain of human E1A-associated protein p300 (p300 activation domain). The transcriptional activator is selected from the group consisting of a herpes simplex virus protein 16 (VP16) activation domain, an activation domain comprising four tandem copies of VP16, a VP64 activation domain, a p65 activation domain of NFκB (p65), an Epstein-Barr virus R transactivator (Rta) activation domain, a tripartite activator comprising the VP64, the p65, and the Rta activation domain (VPR activation domain), a tripartite activator comprising the VP64, the p65, and the HSF1 activation domain (VPH activation domain), and a histone acetyltransferase core domain of human E1A-associated protein p300 (p300 activation domain).HAT core activation domain), and optionally said transcription activator is a p65 transcription activator comprising the amino acid sequence of DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64).

17. An isolated polynucleotide molecule comprising a nucleotide sequence encoding the modified ER-LBD of claim 15 or the chimeric protein of claim 16.

18. 18. A heterologous construct comprising a promoter operably linked to the polynucleotide molecule of claim 17.

19. A cell comprising the heterologous construct of claim 18.

20. A molecular switch for regulating the transcription of a gene of interest, comprising: a) a chimeric protein according to claim 16 or a heterologous construct encoding said chimeric protein, wherein said chimeric protein binds to a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to said gene of interest; and b) a non-endogenous ligand, wherein binding of the non-endogenous ligand to the modified ER-LBD induces the chimeric protein to regulate transcription of the gene of interest; Depending on the situation, a. the non-endogenous ligand is selected from 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen; b. the gene of interest encodes a polypeptide selected from the group consisting of a cytokine, a chemokine, a homing molecule, a growth factor, a cell death regulator, a co-activator molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme; c. the molecular switch further comprises an additional construct comprising the CTF responsive promoter operably linked to the gene of interest; d. the heterologous construct and the additional construct are contained in a single vector, and / or e. A molecular switch wherein the heterologous construct is contained in a first vector and the additional construct is contained in a second vector.

21. 1. A method for modulating the localization of a chimeric protein, comprising: a) transforming a cell with a heterologous construct encoding the chimeric protein of claim 16; and b) contacting the transformed cell with a non-endogenous ligand to induce nuclear localization of the chimeric protein; Optionally, the method further comprises culturing the transformed cell under conditions suitable for expression of the chimeric protein prior to contacting the transformed cell with the non-endogenous ligand; and / or optionally, the heterologous construct and the additional construct are comprised in a single vector, or the heterologous construct is comprised in a first vector and the additional construct is comprised in a second vector; and / or optionally, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, tamoxifen, and endoxifen; and / or optionally, the non-endogenous ligand is administered at a concentration at which the non-endogenous ligand is substantially inactive against the wild-type estrogen receptor alpha of SEQ ID NO: 1.

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