ERT2 Mutants and Methods of Use Thereof

Abstract

Provided herein are mutants of the estrogen receptor alpha ligand binding domain (ER-LBD), and chimeric proteins comprising such mutant ER-LBDs. Methods of regulating the transcription and localization of such chimeric proteins are also provided.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 171,227, filed April 6, 2021, which is incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing, which has been submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on April 4, 2022, is named STB-028WO_SL.txt and is 212,560 bytes in size. [Background technology]

[0003] background 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 the ER in a tissue-selective manner. Tamoxifen and its active metabolites are also utilized as tools to control nuclear localization in research settings. For example, an ER ligand-binding domain variant known as ERT2 has been widely used as a fusion protein with Cre recombinase to regulate Cre recombinase-based gene editing in animal model systems. The ability to manipulate nuclear localization using synthetic ligands would also be useful in therapeutic applications, such as for the regulation of therapeutic genes. Thus, ERT2-based systems with improved sensitivity and / or selectivity to synthetic ligands would be useful for employing ERT2-based gene regulation in clinical settings. Summary of the Invention

[0004] overview In some embodiments, provided herein are modified estrogen receptor ligand binding domains (ER-LBDs) that have 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, gene switches, polynucleotide molecules encoding the modified ER-LBDs and chimeric proteins described herein, cells encoding the polynucleotide molecules described herein or expressing the modified ER-LBDs and chimeric proteins described herein, and methods of using the modified ER-LBDs, chimeric proteins, polynucleotide molecules, gene switches, or cells described herein.

[0005] The modified ER-LBD and chimeric proteins described herein have higher sensitivity and / or selectivity to non-endogenous ligands (e.g., 4-hydroxytamoxifen, also referred to as "4-OHT") compared to ERT2. ERT2 is a ligand-binding domain of ER that contains a G400V amino acid substitution, an M543A amino acid substitution, and an L544A amino acid substitution (see SEQ ID NO:2). ERT2 can also contain a V595A amino acid substitution in addition to G400V / M543A / L544A (see SEQ ID NO:3). The average peak plasma concentration after a typical clinical dose of tamoxifen is in the nanomolar range (e.g., about 40 ng / mL). However, the sensitivity of wild-type ERT2 to tamoxifen metabolites (e.g., endoxifen and 4-OHT) is too low for its use to regulate gene expression at nanomolar concentrations of metabolites. In addition, ERT2 can respond to endogenous ligands such as estradiol. Thus, the improved sensitivity and / or selectivity of modified ER-LBDs and chimeric proteins comprising modified ER-LBDs to non-endogenous ligands enables the use of ER-based systems to control gene regulation.

[0006] Provided herein is a modified estrogen receptor ligand binding domain (ER-LBD) comprising an amino acid sequence corresponding to amino acids 282-595 of SEQ ID NO:1, the modified ER-LBD comprising a G400V amino acid substitution, an M543A amino acid substitution, and an L544A amino acid substitution, and one or more additional amino acid substitutions, the one or more additional amino acid substitutions being within a region of SEQ ID NO:1 selected from the group consisting of positions 343-354, 380-392, 404-463, and 517-540, and 547, and the modified ER-LBD has greater sensitivity and / or selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acids of SEQ ID NO:2, or compared to endogenous ligands, as a result of the one or more additional amino acid substitutions. In some embodiments, the modified ER-LBD further comprises a V595A amino acid substitution. In some embodiments, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0007] In some aspects, the one or more additional amino acid substitutions are at one or more positions of SEQ ID NO:1 selected from the group consisting of 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.

[0008] 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.

[0009] In some embodiments, the one or more positions include position 344 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 344 of SEQ ID NO: 1 is G344M.

[0010] In some embodiments, the one or more positions include position 345 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 345 of SEQ ID NO: 1 is L345S.

[0011] 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.

[0012] 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.

[0013] In some embodiments, the one or more positions include position 348 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 348 of SEQ ID NO: 1 is N348K.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] In some embodiments, the one or more positions include position 352 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 352 of SEQ ID NO: 1 is R352K.

[0018] 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.

[0019] In some embodiments, the one or more positions include position 380 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 380 of SEQ ID NO: 1 is E380Q.

[0020] 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.

[0021] In some embodiments, the one or more positions comprises position 386 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 386 of SEQ ID NO: 1 is I386V.

[0022] 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.

[0023] 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.

[0024] In some embodiments, the one or more positions include position 389 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 389 of SEQ ID NO: 1 is I389M.

[0025] 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.

[0026] In some embodiments, the one or more positions include position 392 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 392 of SEQ ID NO: 1 is V392M.

[0027] 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.

[0028] In some embodiments, the one or more positions comprises position 407 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 407 of SEQ ID NO: 1 is N407D.

[0029] In some embodiments, the one or more positions include position 409 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 409 of SEQ ID NO: 1 is L409V.

[0030] In some embodiments, the one or more positions include position 413 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 413 of SEQ ID NO: 1 is N413D.

[0031] In some embodiments, the one or more positions comprises position 414 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 414 of SEQ ID NO: 1 is Q414E.

[0032] In some embodiments, the one or more positions include position 417 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 417 of SEQ ID NO: 1 is C417S.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] In some embodiments, the one or more positions include position 422 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 422 of SEQ ID NO: 1 is V422I.

[0037] 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.

[0038] 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.

[0039] In some embodiments, the one or more positions comprises position 463 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 463 of SEQ ID NO: 1 is S463P.

[0040] In some embodiments, the one or more positions include position 517 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 517 of SEQ ID NO: 1 is M517A.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] In some embodiments, the one or more positions include position 526 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 526 of SEQ ID NO: 1 is Y526L.

[0046] In some embodiments, the one or more positions comprises position 527 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 527 of SEQ ID NO: 1 is S527N.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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 of SEQ ID NO: 1 is selected from the group consisting of Y537E and Y537S.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] In some embodiments, the one or more positions include position 547 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 547 of SEQ ID NO: 1 is H547A.

[0056] In some embodiments, the one or more additional amino acid substitutions are two amino acid substitutions. In some embodiments, each of the two amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525. In some embodiments, the two amino acid substitutions are at positions 345 and 348 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 387 and 391, where 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 embodiments, the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO:1, where 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 embodiments, the two amino acid substitutions are at positions 384 and 391 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 391 of SEQ ID NO:1 is L391F.

[0057] In some embodiments, the one or more additional amino acid substitutions are three amino acid substitutions. In some embodiments, each of the three amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 343, 347, 351, 354, 388, 391, 404, 414, 418, 463, 521, 524, and 525. In some embodiments, the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO:1, 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, 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.

[0058] In some embodiments, the one or more additional amino acid substitutions are four amino acid substitutions. In some embodiments, each of the four amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 343, 347, 351, 354, 384, 388, 391, 404, 413, 418, 463, 521, 524, and 525. In some embodiments, the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO:1, 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 embodiments, the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO:1, where 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 embodiments, the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO:1, where 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, 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 embodiments, the four amino acid substitutions are at positions 384, 391, 413, and 524 of SEQ ID NO:1, 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.

[0059] In some embodiments, the one or more additional amino acid substitutions are five amino acid substitutions. In some embodiments, each of the five amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, and 524. In some embodiments, the five amino acid substitutions are at positions 384, 409, 413, 463, and 524 of SEQ ID NO:1, 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, 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 embodiments, the five amino acid substitutions are at positions 391, 414, 421, 463, and 524 of SEQ ID NO:1, 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 embodiments, the five amino acid substitutions are at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1, 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 embodiments, the five amino acid substitutions are at positions 354, 409, 421, 463, and 524 of SEQ ID NO:1, 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.

[0060] In some embodiments, the one or more additional amino acid substitutions are six amino acid substitutions. In some embodiments, each of the six amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, and 524. In some embodiments, the six amino acid substitutions are at positions 384, 391, 413, 421, 463, and 524 of SEQ ID NO:1, 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 embodiments, the six amino acid substitutions are at positions 409, 413, 414, 421, 463, and 524 of SEQ ID NO:1, 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 embodiments, the six amino acid substitutions are at positions 354, 391, 409, 413, 414, and 524 of SEQ ID NO:1, 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.

[0061] In some embodiments, the one or more additional amino acid substitutions are seven amino acid substitutions. In some embodiments, each of the seven amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, 517, and 524. In some embodiments, the seven amino acid substitutions are at positions 354, 384, 409, 413, 421, 463, and 524 of SEQ ID NO:1, 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 embodiments, the seven amino acid substitutions are at positions 354, 391, 413, 421, 463, 517, and 524 of SEQ ID NO:1, 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 embodiments, the seven amino acid substitutions are at positions 354, 391, 413, 414, 421, 517, and 524 of SEQ ID NO:1, 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.

[0062] In some embodiments, the one or more additional amino acid substitutions are eight amino acid substitutions. In some embodiments, the eight amino acid substitutions are at positions 384, 391, 409, 413, 421, 463, 517, and 524 of SEQ ID NO:1, where 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.

[0063] Also provided herein is a chimeric protein 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 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 chimeric protein comprises a chimeric transcription factor, and the polypeptide of interest comprises a nucleic acid binding domain and a transcription regulator domain. In some aspects, the transcription regulator domain is a transcription activator. In some embodiments, 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; 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 DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64).

[0064] Also provided herein is an isolated polynucleotide molecule comprising a nucleotide sequence that encodes a modified ER-LBD as described herein, or that encodes a chimeric protein as described herein.

[0065] Also provided herein is a heterologous construct comprising a promoter operably linked to a polynucleotide molecule described herein.

[0066] Also provided herein are plasmids that contain the heterologous constructs described herein.

[0067] Also provided herein are cells comprising a heterologous construct described herein or a plasmid described herein.

[0068] Also provided herein is a gene switch for regulating transcription of a gene of interest, the gene switch comprising: (a) a chimeric protein as described herein, where the chimeric protein binds to a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to a gene of interest; and (b) a non-endogenous ligand, where binding of the non-endogenous ligand to the modified ER-LBD induces the chimeric protein to regulate transcription of the gene of interest.

[0069] In some embodiments, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0070] In some embodiments, 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 coactivator molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme.

[0071] In some embodiments, the gene of interest encodes a cytokine selected from the group consisting of IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha; and / or

[0072] In some embodiments, the gene of interest encodes an IL12p70 fusion protein comprising the amino acid sequence of (SEQ ID NO:58).

[0073] Also provided herein is a method of modulating transcription of a gene of interest, comprising transforming a cell with a target expression cassette comprising (i) a heterologous construct encoding a chimeric protein described herein, and (ii) a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to the gene of interest, and inducing the chimeric protein to modulate transcription of the gene of interest 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 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. In some aspects, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0074] Also provided herein are methods of modulating localization of a chimeric protein comprising transforming a cell with a heterologous construct encoding a 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 prior to inducing nuclear localization. In some aspects, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0075] In some aspects, 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.

[0076] In some embodiments, the non-endogenous ligand is administered at a concentration such that the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha of SEQ ID NO:1.

[0077] Provided herein is a modified ER-LBD comprising an amino acid sequence corresponding to amino acids 282-595 of SEQ ID NO:1 and one or more additional amino acid substitutions within a region of SEQ ID NO:1 selected from the group consisting of positions 343-354, 380-392, 404-463, and 517-540, and 547. In some embodiments, the modified ER-LBD described herein further comprises a G400V amino acid substitution, a M543A amino acid substitution, and a L544A amino acid substitution. In some embodiments, the modified ER-LBD further comprises a G400V amino acid substitution, a M543A amino acid substitution, a L544A, and a V595A amino acid substitution.

[0078] In some embodiments, the modified ER-LBD comprises a G400V, an M543A, and an L544A amino acid substitution, and one or more additional amino acid substitutions. 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 selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 2.

[0079] In some embodiments, the modified ER-LBD comprises a G400V, an M543A, an L544A, and a V595A amino acid substitution, and one or more additional amino acid substitutions. In some embodiments, the modified ER-LBD has a higher sensitivity to non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the modified ER-LBD has a higher selectivity for non-endogenous ligands compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO: 3.

[0080] In some embodiments, the modified ER-LBD of the disclosure has greater sensitivity to a non-endogenous ligand as compared to an endogenous ligand as a result of one or more additional amino acid substitutions.

[0081] In some aspects, the modified ER-LBD of the present disclosure has greater sensitivity to non-endogenous ligands as compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0082] In some aspects, the modified ER-LBD of the disclosure has greater selectivity for non-endogenous ligands as compared to an ER-LBD comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3.

[0083] In some aspects, the one or more additional amino acid substitutions are at one or more positions of SEQ ID NO:1 selected from the group consisting of 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.

[0084] 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.

[0085] In some embodiments, the one or more positions include position 344 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 344 is G344M.

[0086] In some embodiments, the one or more positions comprises position 345 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 345 is L345S.

[0087] In some aspects, the one or more positions comprises 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.

[0088] 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 is selected from the group consisting of T347D, T347E, T347F, T347I, T347K, T347L, T347M, T347N, T347Q, T347R, T347S, and T347V.

[0089] In some embodiments, the one or more positions include position 348 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 348 is N348K.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] In some embodiments, the one or more positions include position 352 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 352 is R352K.

[0094] 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.

[0095] In some embodiments, the one or more positions include position 380 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 380 is E380Q.

[0096] 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.

[0097] In some embodiments, the one or more positions comprises position 386 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 386 is I386V.

[0098] In some aspects, the one or more positions comprises 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.

[0099] 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.

[0100] In some embodiments, the one or more positions include position 389 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 389 is I389M.

[0101] 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.

[0102] In some embodiments, the one or more positions include position 392 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 392 is V392M.

[0103] 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.

[0104] In some embodiments, the one or more positions include position 407 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 407 is N407D.

[0105] In some embodiments, the one or more positions comprises position 409 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 409 is L409V.

[0106] In some embodiments, the one or more positions include position 413 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 413 is N413D.

[0107] In some embodiments, the one or more positions comprises position 414 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 414 is Q414E.

[0108] In some embodiments, the one or more positions include position 417 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 417 is C417S.

[0109] 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.

[0110] 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.

[0111] 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.

[0112] In some embodiments, the one or more positions include position 422 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 422 is V422I.

[0113] 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.

[0114] In some embodiments, the one or more positions comprises position 428 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 428 is selected from the group consisting of L428I, L428M, L428F, and L428V.

[0115] In some embodiments, the one or more positions comprises position 463 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 463 is S463P.

[0116] In some embodiments, the one or more positions include position 517 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 517 is M517A.

[0117] 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.

[0118] 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.

[0119] 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.

[0120] 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.

[0121] In some embodiments, the one or more positions include position 526 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 526 is Y526L.

[0122] In some embodiments, the one or more positions comprises position 527 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 527 is S527N.

[0123] 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.

[0124] 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.

[0125] 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.

[0126] In some embodiments, the one or more positions comprises position 536 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 536 is selected from the group consisting of L536F, and L536M, L536R, and L536Y.

[0127] 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.

[0128] 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.

[0129] 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.

[0130] In some aspects, the one or more positions comprise 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.

[0131] In some embodiments, the one or more positions comprises position 547 of SEQ ID NO: 1. In some embodiments, the amino acid substitution at position 547 is H547A.

[0132] In some embodiments, the one or more additional amino acid substitutions comprise two amino acid substitutions, hi some embodiments, each of the two amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525.

[0133] In some embodiments, the two amino acid substitutions are at positions 345 and 348 of SEQ ID NO: 1. In some embodiments, 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.

[0134] In some embodiments, the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO: 1. In some embodiments, 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.

[0135] In some embodiments, the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO: 1. In some embodiments, 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.

[0136] In some embodiments, the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO: 1. In some embodiments, 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.

[0137] In some embodiments, the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO: 1. In some embodiments, 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.

[0138] In some embodiments, the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO: 1. In some embodiments, 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.

[0139] 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.

[0140] In some embodiments, the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO: 1. In some embodiments, 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.

[0141] In some embodiments, the two amino acid substitutions are at positions 384 and 391 of SEQ ID NO: 1. In some embodiments, 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.

[0142] In some embodiments, the one or more additional amino acid substitutions comprise three amino acid substitutions, hi some embodiments, the three amino acid substitutions are each at a position of SEQ ID NO:1 selected from the group consisting of 343, 347, 351, 354, 388, 391, 404, 418, 521, 524, and 525.

[0143] In some embodiments, the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO: 1. In some embodiments, 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.

[0144] In some embodiments, the one or more additional amino acid substitutions comprises four amino acid substitutions.

[0145] In some embodiments, the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO: 1. In some embodiments, 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.

[0146] In some embodiments, the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO: 1. In some embodiments, 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.

[0147] In some embodiments, the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO: 1. In some embodiments, 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.

[0148] In some embodiments, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0149] Also provided herein is a chimeric protein comprising a polypeptide of interest fused to a modified ER-LBD as described herein. In some embodiments, the polypeptide of interest comprises a nucleic acid binding domain. In some embodiments, the nucleic acid binding domain comprises a zinc finger (ZF) domain. In some embodiments, the chimeric protein is a transcription factor and the polypeptide of interest comprises a transcription regulator domain.

[0150] Also provided is an isolated polynucleotide molecule that encodes a modified ER-LBD as described herein, or a chimeric protein as described herein.

[0151] Also provided is a heterologous construct comprising a promoter operably linked to a polynucleotide molecule encoding a modified ER-LBD described herein or a chimeric protein described herein.

[0152] Also provided are heterologous constructs comprising the heterologous constructs described herein.

[0153] Also provided is a cell (such as an isolated cell or a population of cells) that comprises a heterologous construct described herein or a plasmid described herein.

[0154] Also provided is a gene switch for regulating transcription of a gene of interest. In some embodiments, the gene switch comprises a chimeric protein comprising a modified ER-LBD as described herein, a transcription 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. In some embodiments, the non-endogenous ligand of the gene switch is selected from the group consisting of 4-hydroxytamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0155] Also provided herein are methods for regulating transcription of a gene of interest. In some aspects, the methods include (a) transforming a cell with a heterologous construct encoding a chimeric protein comprising (i) a modified ER-LBD and a transcription 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) inducing the chimeric protein to regulate transcription of the gene of interest by contacting the transformed cell with a non-endogenous ligand.

[0156] In some aspects, the method of modulating transcription is a method of activating transcription.

[0157] In some aspects, the method of modulating transcription is a method of repressing transcription.

[0158] In some aspects, the target expression cassette is encoded by a heterologous construct encoding a chimera.

[0159] In some aspects, the target expression cassette is encoded by a heterologous construct that is different from the heterologous construct encoding the chimera.

[0160] Also provided are methods for regulating the localization of a polypeptide of interest. In some embodiments, the methods include: (a) transforming a cell with a heterologous construct encoding a chimeric protein comprising a polypeptide of interest fused to a modified ER-LBD as described herein; (b) culturing the transformed cell under conditions suitable for expression of the chimeric protein; and (c) inducing nuclear localization of the chimeric protein by contacting the transformed cell with a non-endogenous ligand.

[0161] In some aspects, the transformed cell of any of the methods described herein is in a human or animal. In some aspects, contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal.

[0162] In some aspects, the non-endogenous ligand in step (c) of the foregoing method is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0163] In some embodiments, the non-endogenous ligand is administered at a concentration such that the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha. [Brief description of the drawings]

[0164] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description and accompanying drawings.

[0165] [Figure 1A] Figures 1A and 1B provide binding energy calculations for the first set of mutations analyzed in silico, with Figure 1A providing binding energy calculations for binding to estradiol and Figure 1B providing binding energy calculations for binding to 4-OHT. [Figure 1B] See legend to Figure 1A. [Diagram 2] We provide binding energy calculations of the 4-OHT bond for the second set of mutations analyzed in silico. [Diagram 3] We provide binding energy calculations of the 4-OHT bond for the third set of mutations analyzed in silico. [Figure 4] We provide binding energy calculations of the 4-OHT bond for the fourth set of mutations analyzed in silico. [Diagram 5] We provide binding energy calculations of the 4-OHT bond for the fifth set of mutations analyzed in silico. [Figure 6] The orientation of helix 12 and the structural differences in the docking site between the estradiol-bound and non-endogenous ligand-bound conformations are shown. [Figure 7] For the sixth set of mutations analyzed in silico, binding energy calculations for the agonist-bound versus antagonist-bound conformations are provided. [Figure 8A] 8A, 8B, and 8C 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 8B] See legend to Figure 8A. [Figure 8C] See legend to Figure 8A. [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] See legend to Figure 9A. [Figure 9C] See legend to Figure 9A. [Figure 10A] 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 tertiary transfection screen. [Figure 10B] See legend to Figure 10A. [Figure 10C] See legend to Figure 10A. [Figure 11A] 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] See legend to Figure 11A. [Figure 12] FIG. 1 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. 1 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 14A] Figures 14A and 14B show the framework for high throughput protein engineering of ERT2 (SB04401) and OFF mCherry reporter constructs (SB01066). [Figure 14B] See legend to Figure 14A. [Figure 15] Figures 15A and 15B show the effect of various modified ER-LBDs on reporter expression across different concentrations of endoxifen and 4-OHT as assayed in a combinatorial library screen. [Figure 16-1]Figures 16A, 16B, 16C, and 16D show the effect of various modified ER-LBDs on reporter expression across various concentrations of endoxifen, 4-OHT, and estradiol assayed in a validation screen. SB03422 is a wild-type ER-LBD and is included as a performance benchmark. [Figure 16-2] See description of Figure 16-1. [Figure 17] Figures 17A and 17B show the effect of various modified ER-LBDs on reporter expression across different concentrations of endoxifen and 4-OHT as assayed in NK cells. Arrows indicate the putative 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0166] Detailed Description Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0167] The term "in vivo" refers to a process that takes place within a living organism.

[0168] The term "mammal" as used herein includes both humans and non-humans, including, but not limited to, humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0169] 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 have a certain percentage of nucleotides or amino acid residues that are the same when compared and aligned for maximum correspondence, as measured 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, for example, 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.

[0170] For sequence comparison, typically, one sequence acts as a reference sequence with which test sequence is compared.When using sequence comparison algorithm, test and reference sequences are input into computer, coordinates of subsequences are designated as necessary, and sequence algorithm program parameters are designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on designated program parameters.

[0171] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the 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).

[0172] One example of a suitable algorithm for determining percent sequence identity and sequence similarity is the BLAST algorithm 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 / ).

[0173] 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.

[0174] The term "therapeutically effective amount" is an amount effective for ameliorating symptoms of a disease. A therapeutically effective amount can be a "prophylactically effective amount" since prevention can be considered a treatment.

[0175] 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.

[0176] Modified Estrogen Receptor Ligand Binding Domain (ER-LBD) The present disclosure provides a modified estrogen receptor ligand binding domain (ER-LBD) (human estrogen receptor, UniProt ID number: P03372) comprising an amino acid sequence corresponding to amino acids 282-595 of SEQ ID NO:1, including amino acid substitutions G400V, M543A, and L544A, or amino acid substitutions G400V, M543A, L544A, and V595A, and including one or more additional amino acid substitutions to ligand binding residues within a region of SEQ ID NO:1 selected from positions 343-354, 380-392, 404-463, and 517-540, and 547. 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 or SEQ ID NO:3, and / or (c) greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO:2 or SEQ ID NO:3.

[0177] 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 or SEQ ID NO:3, and / or (c) greater selectivity for non-endogenous ligands compared to the ER-LBD of SEQ ID NO:2 or 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: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: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: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.

[0178] "Ligand-binding residues" refers to residues located in the ligand-binding pocket of the estrogen receptor (ER) or ER ligand-binding domain, including pockets for binding endogenous ligands (e.g., estradiol) and pockets for binding non-endogenous ligands such as 4-OHT. 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 designated helix 12 and are involved in endogenous ligand binding.

[0179] Higher sensitivity to a non-endogenous ligand compared to sensitivity to a non-endogenous ligand means that the modified ER-LBD binds to a non-endogenous ligand (e.g., endoxifen) with higher affinity compared to its binding affinity to an endogenous ligand (e.g., estradiol).

[0180] Higher sensitivity to a non-endogenous ligand compared to sensitivity of an ER-LBD that does not contain one or more amino acid substitutions (e.g., an ER-LBD that contains the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3) means that the modified ER-LBD binds to a non-endogenous ligand (e.g., endoxifen) with higher affinity compared to the affinity of binding to the non-endogenous ligand of an ER-LBD that does not contain one or more additional amino acid substitutions. In some embodiments, the higher 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 a non-endogenous ligand compared to binding of an ER-LBD that does not contain one or more additional amino acid substitutions. In some embodiments, the higher sensitivity is indicated by more transcriptional regulation (e.g., more transcriptional activation or more transcriptional repression) of a chimeric transcription factor that includes a modified ER-LBD compared to a chimeric transcription factor that includes an ER-LBD that lacks one or more additional amino acid substitutions. 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% more expression of a reporter under 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).

[0181] Higher 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 a 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 higher the selectivity coefficient, the further a competing ligand (e.g., an endogenous ligand) will displace an initial ligand (e.g., a non-endogenous ligand) from the complex formed with the substrate (e.g., a modified ER-LBD). In some embodiments, higher selectivity is indicated by improved transcriptional regulation of the chimeric transcription factor in the presence of a non-endogenous ligand compared to transcriptional regulation in the presence of an 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% more 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).

[0182] In some aspects, the one or more amino acid substitutions to ligand binding residues comprise one or more amino acid substitutions in 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 in helix 12 are at one or more positions selected from 538, 536, 539, 540, 547, 534, 533, and 537.

[0183] "Non-endogenous ligand" may 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, but are not limited to, tamoxifen and its metabolites, such as 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0184] The one or more additional amino acid substitutions may be 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. In some embodiments, the one or more additional amino acid substitutions include 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.

[0185] In some embodiments, the one or more additional amino acid substitutions are selected from one or more of the substitutions listed in Table 1.

[0186] (Table 1) TIFF2024513231000002.tif194142

[0187] In some embodiments, 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 embodiments, the one or more additional mutations include 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.

[0188] In some embodiments, the one or more additional mutations comprise at least two mutations selected from the mutations listed in Table 2.

[0189] (Table 2) TIFF2024513231000003.tif107153

[0190] In some embodiments, provided herein are modified ER-LBD variants having an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the modified ER-LBD described herein, provided that the variant comprises a G400V / MS43A / L544A triple amino acid substitution or a G400V / M543A / L544A / V595A quadruple amino acid substitution and comprises one or more additional amino acid substitutions (e.g., one or more of the amino acid substitutions set forth in Tables 1 and 2) that confer greater sensitivity and / or greater selectivity to non-endogenous ligands.

[0191] Chimeric Proteins In some embodiments, the present disclosure provides a chimeric protein comprising a polypeptide of interest fused to a modified ER-LBD. The modified ER-LBD is capable of nuclear localization upon binding to a non-endogenous ligand. Thus, the fusion of the modified ER-LBD to a polypeptide of interest can allow the control of the cellular localization of the polypeptide of interest.

[0192] In some embodiments, the polypeptide of interest comprises a linker. One or more linkers can be used between various domains of the chimeric protein, such as between the ER-LBD and the polypeptide of interest. For example, the polypeptide linker can include an amino acid sequence such as one or more of GGGGSGGGGSGGGGSVDGF (SEQ ID NO: 4) and ASGGGGSAS (SEQ ID NO: 5).

[0193] 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).

[0194] 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; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising VP64, p65, and Rta activation domains (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); a HAT core activation domain; a Kruppel-associated box (KRAB) repression domain; a repressor element silencing transcription factor (REST) ​​repression domain; a WRPW motif (SEQ ID NO: 82) of the hairy-associated basic helix-loop-helix repressor protein (which motif is known as the WRPW (SEQ ID NO: 82) domain); a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

[0195] In some embodiments, the ZF protein domain is modular in design and consists of a zinc finger array (ZFA). A zinc finger array contains multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA 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, the 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.

[0196] 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 ten ZFAs.

[0197] 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 set forth in the sequence MSRPGERPFQCRICMRNFSNMSNLTRHTRTHTGEKPFQCRICMRNFSDRSVLRRHLRTHTGSQKPFQCRICMRNFSDPSNLARHTRTHTGEKPFQCRICMRNFSDRSSLRRHLRTHTGSQKPFQCRICMRNFSQSGTLHRHTRTHTGEKPFQCRICMRNFSQRPNLTRHLRTHLRGS (SEQ ID NO:62).

[0198] In some embodiments, the chimeric protein is a chimeric transcription factor and comprises, in addition to a 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.

[0199] As used herein, a "transcriptional regulator domain" refers 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 a promoter of interest), is capable of regulating the transcription of the gene. In some embodiments, the transcriptional effector 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 WRPW motif of hairy-related basic helix-loop-helix repressor protein (the 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.

[0200] In some embodiments, the transcription effector domain comprises a transcription activator. In some embodiments, the transcription activator comprises a transcription 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 transcription regulator domain comprises a p65 transcription activator. In some embodiments, the p65 transcriptional activator comprises the amino acid sequence DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64).

[0201] Genetic Switch Also provided herein is a gene switch for regulating transcription.The gene switch can comprise (a) a chimeric transcription factor that comprises modified ER-LBD and can bind to a chimeric transcription factor responsive promoter (CTF responsive promoter) that is operably linked to a gene of interest, and (b) a non-endogenous ligand that binds to the modified ER-LBD of the chimeric protein.When the non-endogenous ligand binds to the modified ER-LBD, the chimeric protein can regulate the transcription of the gene of interest.

[0202] In some embodiments, 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 coactivator molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme. In some embodiments, the gene of interest encodes a cytokine. In some embodiments, the gene of interest encodes a cytokine selected from the group consisting of IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha. In some embodiments, the gene of interest encodes an IL12p70 fusion protein comprising the amino acid sequence of (SEQ ID NO:58).

[0203] In some embodiments, the non-endogenous ligand is selected from 4-hydroxy-tamoxifen (4-OHT), N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen.

[0204] In certain embodiments, the non-endogenous ligand is 4-hydroxytamoxifen (4-OHT, also known as afimoxifen).

[0205] In certain embodiments, the non-endogenous ligand is endoxifen.

[0206] Isolated polynucleotide molecules and heterologous constructs Also provided herein are isolated polynucleotide molecules and heterologous constructs encoding the modified ER-LBD or chimeric proteins described herein. In some aspects, the disclosure provides isolated polynucleotide molecules comprising a nucleotide sequence encoding the modified ER-LBD or chimeric proteins described herein. In some aspects, the disclosure provides heterologous constructs comprising a promoter operably linked to a polynucleotide molecule encoding the modified ER-LBD or chimeric protein.

[0207] In some aspects, the disclosure further provides isolated polynucleotides and / or heterologous constructs comprising target gene expression cassettes.

[0208] 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 to be 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 molecule that is normally contained in a cell that contains the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0209] Isolated polynucleotide molecules 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.

[0210] In some embodiments, the isolated polynucleotide molecule is selected from DNA, cDNA, RNA, mRNA, and naked plasmid (linear or circular).

[0211] 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 it may contain up to 5 point mutations per each 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 replaced with another nucleotide, or up to 5% of the number of nucleotides of the total nucleotides in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the 5' or 3' terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, either individually between the residues in the reference sequence, or interspersed in one or more consecutive groups within the reference sequence. 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.

[0212] In some aspects, the chimeric protein encoded by the polynucleotide molecule is a chimeric transcription factor, and the polynucleotide molecule 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.

[0213] The term "expression cassette" refers to a recombinantly or synthetically produced polynucleotide having a set of nucleic acid elements that allow for transcription of a particular polynucleotide in a target cell. An expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the expression cassette portion of an expression vector includes, among other sequences, a nucleic acid sequence to be transcribed and a promoter. In some aspects, the disclosure provides an expression cassette that includes a polynucleotide encoding a modified ER-LBD or a chimeric protein that includes a modified ER-LBD.

[0214] The isolated polynucleotide molecules and heterologous constructs comprising the modified ER-LBD described herein are engineered polynucleotide molecules. An "engineered polynucleotide" is a polynucleotide that does not occur in nature. However, it should be understood that while an engineered polynucleotide does not occur in nature as a whole, it may contain nucleotide sequences that occur in nature. 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 nucleotide sequences, bacterial nucleotide sequences, human nucleotide sequences, and / or viral nucleotide sequences. The term "engineered polynucleotide" includes recombinant and synthetic nucleic acids. A "recombinant polynucleotide" refers to a molecule that is constructed by linking nucleotide molecules and, in some embodiments, is 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 modified 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. Patent 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 WO1998 / 039352, U.S. Patent 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 distinct molecules (e.g., multiple distinct independently replicating molecules).

[0215] The engineered polynucleotides of the present disclosure may 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, DGet al. Nature Methods, 343-345, 2009, and Gibson, DGet al. Nature Methods, 901-903, 2010, each of which is incorporated herein by reference). GIBSON ASSEMBLY® typically uses three enzyme 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 the complementary sequence for annealing. The polymerase activity then fills in the gaps on the annealed region. DNA ligase then seals the nicks and covalently links the DNA fragments together. The overlapping sequences of the adjacent fragments are much longer than those used in Golden Gate Assembly, thus resulting in a high rate of correct assembly. In some embodiments, engineered nucleic acid constructs are produced using IN-FUSION® Cloning (Clontech).

[0216] In some embodiments, the polynucleotide molecules described herein are included in a heterologous construct. The term "vector" or "expression vector" is synonymous with "heterologous construct" and refers to a polynucleotide molecule used to introduce and induce expression of one or more genes operably associated with the construct in a target cell. The term includes the construct as a self-replicating nucleic acid structure as well as the vector integrated into the genome of the host cell into which it is introduced. The heterologous constructs described herein include an expression cassette. In some aspects, a heterologous construct is provided herein that includes an expression cassette comprising a promoter operably linked to a polynucleotide molecule encoding a modified ER-LBD or a chimeric protein comprising a modified ER-LBD.

[0217] As used herein, a "promoter" refers to a regulatory region of a nucleic acid sequence where the initiation and rate of transcription of the remainder of the nucleic acid sequence is controlled. A promoter may also contain a subregion to which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, may bind. A promoter may be constitutive, inducible, repressible, tissue-specific, or any combination thereof. A promoter drives the expression or transcription of a nucleic acid sequence that it 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.

[0218] A promoter may be a promoter naturally associated with a 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, as well as synthetic promoters or enhancers that are not "naturally occurring", such as those that contain different elements of transcriptional regulation and / or mutations that modify expression through methods of genetic engineering known in the art. In addition to synthetically producing promoter and enhancer nucleic acid sequences, the sequences may be produced using recombinant cloning and / or nucleic acid amplification techniques, including polymerase chain reaction (PCR) (see, e.g., U.S. Pat. Nos. 4,683,202 and 5,928,906).

[0219] 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. The signal can be an endogenous or usually exogenous condition (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a 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. Activation of transcription can include acting directly on the promoter to drive transcription, or acting indirectly on the promoter by inactivating a repressor that is preventing the promoter from driving transcription. Conversely, inactivation of transcription can include acting directly on the promoter to prevent transcription, or acting indirectly on the promoter by activating a repressor that then acts on the promoter.

[0220] As used herein, a promoter is "responsive to" or "regulated by" a local tumor condition (e.g., inflammation or hypoxia) or signal when 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 control element (PEACE), zinc finger DNA binding domain (DBD), interferon gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121-34, incorporated herein by reference), interferon stimulated response element (ISRE) (Han, KJ et al. J Biol Chem. 2004 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.

[0221] 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) is also shown (B: binding, D: dissociation, nd: undetermined) (A: activation, DA: deactivation, DR: derepression) (see Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components of inducible promoters include those presented in Table 4.

[0222] (Table 3) TIFF2024513231000004.tif204148TIFF2024513231000005.tif210153

[0223] (Table 4) TIFF2024513231000006.tif199148

[0224] 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 the 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.

[0225] In some aspects, the disclosure provides a heterologous construct comprising a promoter operably linked to a polynucleotide molecule encoding a modified ER-LBD or chimeric protein described herein.

[0226] In some embodiments, the promoter operably linked to the polynucleotide molecule encoding the modified ER-LBD or chimeric protein is a constitutive promoter, an inducible promoter, or a synthetic promoter.

[0227] In some embodiments, the promoter operably linked to the polynucleotide molecule 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.

[0228] (Table 5) TIFF2024513231000007.tif171148TIFF2024513231000008.tif236151TIFF2024513231000009.tif236151TIFF20245132310 00010.tif235152TIFF2024513231000011.tif234151TIFF2024513231000012.tif235149TIFF2024513231000013.tif146148

[0229] Expression system further comprising a target expression cassette In some aspects, the chimeric protein is a chimeric transcription factor, and the disclosure further provides a target expression cassette comprising a chimeric transcription factor-responsive (CTF-responsive) promoter.

[0230] "Target expression cassette" refers to an expression cassette that includes a gene having expression controllable by a chimeric transcription factor. Expression is controlled by the chimeric transcription factor upon the presence of a non-endogenous ligand (e.g., 4-OHT or endoxifen).

[0231] In some aspects, the present disclosure provides a polynucleotide molecule encoding a gene of interest operably linked to a chimeric transcription factor responsive promoter (CTF responsive promoter). The CTF responsive promoter is a synthetic inducible promoter that responds to a chimeric transcription factor comprising a modified ER-LBD and is inducible in response to a non-endogenous ligand such as 4-OHT.

[0232] In some embodiments, the CTF responsive promoter comprises a core promoter sequence and a binding domain that binds a chimeric transcription factor described herein.

[0233] 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 set forth in the sequence: cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO:37).

[0234] The core promoter sequence may include a minimal promoter. Examples of minimal promoters include minP, minCMV, YB_TATA, and minTK.

[0235] 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 is a first heterologous construct comprising an expression cassette comprising a polynucleotide molecule 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.

[0236] In some embodiments, the engineered polynucleotide or construct of the present disclosure is configured to produce multiple polypeptides. For example, the polynucleotide can be configured to produce two different polypeptides. The polynucleotide molecule can be configured to produce a polypeptide comprising a chimeric protein as described herein and a polypeptide of interest expressed under the control of a promoter responsive to the chimeric protein.

[0237] In some embodiments, the ER-LBD or chimeric protein described herein and the gene of interest that can be transcriptionally regulated by the ER-LBD or chimeric protein can be encoded by the same polynucleotide molecule or heterologous construct.

[0238] In some embodiments, the engineered nucleic acid may be multicistronic, i.e., more than one distinct polypeptide (e.g., multiple exogenous polynucleotides) may be produced from a single transcription product. The engineered nucleic acid may be multicistronic through the use of various linkers, for example, a polynucleotide sequence encoding a first exogenous polynucleotide may be linked to a nucleotide sequence encoding a second exogenous polynucleotide in a 5' to 3' orientation, such as first gene:linker:second gene. The linker polynucleotide sequence may 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 distinct polypeptides encoded by the first and second genes during translation. The linker can encode a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, and following expression, the cleavable linker polypeptide is cleaved such that the separate polypeptides encoded by the first and second genes are produced. The cleavable linker can include a polypeptide sequence, such as a flexible linker (e.g., a Gly-Ser-Gly sequence), which further facilitates cleavage.

[0239] The linker can encode an internal ribosome entry site (IRES) such 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.

[0240] The linker may be a combination of linkers, such as a Furin-2A linker, which can produce a separate polypeptide through 2A ribosomal skipping followed by further cleavage of the Furin site, which allows 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.

[0241] In general, a multicistronic system can express any number of genes or portions thereof using any number or combination of linkers (e.g., an engineered nucleic acid can encode a first, second, and third polypeptide molecule, each separated by a linker, such that separate polypeptides encoded by the first, second, and third polypeptides are produced).

[0242] A "linker" as used herein may refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, or to a multicistronic linker as described above.

[0243] Post-transcriptional regulatory elements In some embodiments, the engineered nucleic acid of the present disclosure comprises a post-transcriptional regulatory element (PRE). PREs can enhance gene expression through 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, the WPRE comprises the alpha, beta, and gamma components of a WPRE element. In some embodiments, the WPRE comprises the alpha component of a WPRE element. Examples of WPRE sequences include SEQ ID NO:38 and SEQ ID NO:39.

[0244] Engineered cells Also provided herein are cells comprising one or more polynucleotide molecules or constructs of the present disclosure, and methods of producing the cells. These cells are referred to herein as "engineered cells." These cells, which typically comprise one or more engineered nucleic acids, do not occur in nature. In some embodiments, the cells are isolated cells that recombinantly express one or more engineered 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.

[0245] 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 engineered in a transient expression system, such as a plasmid or mRNA, that is capable of expression without integration into the genome of the cell.

[0246] 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.

[0247] The engineered cells of the present disclosure can be isolated from a subject, such as a subject known or suspected to have cancer. Cell isolation methods are known to those skilled in the art and include, but are not limited to, sorting techniques based on cell surface marker expression, such as FACS sorting, positive isolation techniques, and negative isolation, magnetic isolation, and combinations thereof. The engineered cells can be allogeneic with respect to the subject to which the treatment is being administered. The allogeneic modified cells can be HLA-matched to the subject to which the treatment is being administered. 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.

[0248] 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.

[0249] In some embodiments, an engineered cell of the present disclosure is a T cell (e.g., a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell). In some embodiments, an engineered cell of the present disclosure is a cytotoxic T lymphocyte (CTL). In some embodiments, an engineered cell of the present disclosure is a regulatory T cell. In some embodiments, an engineered cell of the present disclosure is a natural killer T (NKT) cell. In some embodiments, an engineered cell of the present disclosure is a natural killer cell (NK). In some embodiments, an engineered cell of the present disclosure is a B cell. In some embodiments, an engineered cell of the present disclosure is a tumor infiltrating lymphocyte (TIL). In some embodiments, an engineered cell of the present disclosure is an innate lymphoid cell. In some embodiments, an engineered cell of the present disclosure is a mast cell. In some embodiments, an engineered cell of the present disclosure is an eosinophil. In some embodiments, an engineered cell of the present disclosure is a basophil. In some embodiments, an engineered cell of the present disclosure is a neutrophil. In some embodiments, an engineered cell of the present disclosure is a myeloid cell. In some embodiments, the engineered cell of the present disclosure is a macrophage, e.g., an M1 macrophage or an M2 macrophage). In some embodiments, the engineered cell of the present disclosure is a monocyte. In some embodiments, the engineered or isolated cell of the present disclosure is a dendritic cell. In some embodiments, the engineered cell of the present disclosure is a red blood cell. In some embodiments, the engineered cell of the present disclosure is a platelet cell. In some embodiments, the cell of the present disclosure is a neuron. In some embodiments, the cell of the present disclosure is an oligodendrocyte. In some embodiments, the cell of the present disclosure is an astrocyte. In some embodiments, the cell of the present disclosure is a placode-derived cell. In some embodiments, the engineered cell of the present disclosure is a Schwann cell. In some embodiments, the engineered cell of the present disclosure is a cardiomyocyte. In some embodiments, the engineered cell of the present disclosure is an endothelial cell. In some embodiments, the engineered cell of the present disclosure is a nodal cell. In some embodiments, the engineered cell of the present disclosure is a microglial cell.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.

[0250] 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.

[0251] Also provided herein is a method comprising culturing the engineered cells of the present disclosure. The method of culturing the engineered cells described herein is known. Those skilled in the art will recognize that the culture conditions depend on the specific engineered cells of interest. Those skilled in the art will recognize that the culture conditions depend on the specific downstream use of the engineered cells, for example, the specific culture conditions for the subsequent administration of the engineered cells to a subject.

[0252] How to Manipulate Cells Also provided herein are compositions and methods for engineering cells with any of the polynucleotide molecules or constructs described herein.

[0253] 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 to be engineered.

[0254] 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 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 engineered virus-derived nucleic acids. Such engineered virus-derived nucleic acids can also be referred to as recombinant viruses or engineered viruses.

[0255] A viral vector-based delivery platform can encode more than one engineered nucleic acid, gene, or transgene within the same nucleic acid. For example, a nucleic acid from an engineered virus, such as a recombinant virus or an 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 (e.g., a modified ER-LBD) described herein. 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, in addition to a transgene encoding a modified ER-LBD.

[0256] 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, referred to as a trans-acting element or gene, as described herein. For example, a helper-dependent viral vector-based delivery platform can provide additional genes required for viral infectivity and / or virus production on one or more additional separate vectors in addition to a vector encoding a modified ER-LBD. One viral vector, such as one vector delivering an engineered polynucleotide configured to produce a modified ER-LBD and an engineered polynucleotide configured to produce a gene of interest, can deliver more than one engineered polynucleotide. More than one viral vector, 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, can deliver more than one engineered nucleic acid. The number of viral vectors used can depend on the packaging capacity of the viral vector-based vaccine platform described above, but one skilled in the art can select an appropriate number of viral vectors.

[0257] Generally, any of the viral vector-based systems can be used for in vitro production of molecules or, for example, for in vitro delivery, in vivo and ex vivo gene therapy procedures. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as the size of the cargo / payload, the immunogenicity of the viral system, the target cells of interest, the strength and timing of gene expression, and other factors recognized by those skilled in the art.

[0258] 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.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 containing the human ubiquitin C (see, for example, U.S. Pat. No. 6,313,141; Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72(12):9873-9880), other exemplary viral vector-based delivery platforms have been described in the art.

[0259] This sequence may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into a host cell, the infected cell (i.e., engineered cell) can express and, optionally, secrete the modified ER-LBD (or chimeric polypeptide containing 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 in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for introduction (i.e., delivery) of engineered nucleic acids, such as Salmonella typhi vectors, and equivalents, will be apparent to those skilled in the art from the description herein.

[0260] The viral vector-based delivery platform may be a virus that targets tumor cells, referred to herein as an oncolytic virus. 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 another transgene (e.g., an engineered nucleic acid described herein). The transgene can be configured to express the modified ER-LBD (or a chimeric polypeptide that includes the modified ER-LBD), and optionally a gene of interest.

[0261] 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).

[0262] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors are composed 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., a transgene encoding a modified ER-LBD) into the target cell 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.

[0263] The viral vector-based delivery platform can be lentivirus-based. In general, 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. Pat. 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.

[0264] Viral vector-based delivery platform can be adenovirus-based.Generally, adenovirus-based vectors can achieve very high transduction efficiency in many cell types, do not require cell division, achieve high titer and expression levels, and can be produced in large quantities in a relatively simple system.Since adenoviruses do not typically integrate into the genome of the host, adenoviruses can generally be used for the transient expression of transgenes in infected cells. 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. Pat. Nos. 5,585,362, 6,083,716, 7,371,570, 7,348,178, 7,323,177, 7,319,033, 7,318,919, and 7,306,793, and International Patent Application WO 96 / 13597, each of which is incorporated herein by reference for all purposes.

[0265] The viral vector-based delivery platform 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). The AAV system can be used for in vitro production of the modified ER-LBD (or chimeric polypeptides comprising the modified ER-LBD) or can be used in in vivo and ex vivo gene therapy procedures, for example, for in vivo delivery of the modified ER-LBD (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. Nos. 4,797,368, 5,436,146, 6,632,670, 6,642,051, 7,078,387, 7,314,912, 6,498,244, 7,906,111; U.S. Patent Publication Nos. US2003-0138772, US2007 / 0036760, and US2009 / 0197338; Gao, et al., (see, e.g., Gao, et al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al., Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and International Patent Applications WO2010 / 138263 and WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994)). 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, an AAV-based vector comprises 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.

[0266] 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, the cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated ex vivo within the purified particles. Thus, the production of VLPs maintains the separation of the nucleic acid encoding the viral structural proteins and the nucleic acid encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo using methods known to those skilled in the art to produce VLPs. The production of VLPs is described in detail by Seow et al. (Mol Ther. 2009 May;17(5):767-777), which is incorporated herein by reference for all purposes.

[0267] Viral vector-based delivery platforms can be engineered to target (i.e., infect) a range of cells, target a narrow subset of cells, or target specific cells. In general, the envelope protein selected for the viral vector-based delivery platform will determine the tropism of the virus. The virus used in the viral vector-based delivery platform can be pseudotyped to target specific cells of interest. The viral vector-based delivery platform is pantropic and can infect a range of cells. For example, a pantropic viral vector-based delivery platform can include a VSV-G envelope. The viral vector-based delivery platform is amphotropic and can infect mammalian cells. Thus, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0268] Lipid structure delivery system The engineered nucleic acid of the present disclosure (e.g., a nucleic acid encoding a modified ER-LBD or chimeric protein described herein) can be introduced into cells using a lipid-mediated delivery system. Generally, lipid-mediated delivery systems use structures that are composed of an external lipid membrane that encases 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.

[0269] Lipid-based nanoparticles can include, but are not limited to, unilamellar liposomes, multilamellar liposomes, and lipid preparations. As used herein, "liposome" is a general term that encompasses in vitro preparations of lipid vehicles formed by encapsulating a desired cargo, e.g., 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 that generally comprises phospholipids, and an internal medium that generally comprises 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. Liposomes can be multilamellar liposomes. Liposomes can be multivesicular liposomes. Liposomes can be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can 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 stability of liposomes in 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 any purpose.

[0270] Multilamellar liposomes are formed spontaneously 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 a 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.

[0271] The liposomes used according to the present embodiment can be prepared by different methods, as known to those skilled in the art. The preparation of liposomes is described in more detail in WO2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and US Patents 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.

[0272] The liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent Nos. 5,962,016, 5,030,453, 6,680,068, U.S. Patent Application 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each of which is incorporated herein by reference for all purposes.

[0273] 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.

[0274] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment after fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges 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 exosomes, and display membrane proteins from the parent cell on their surface. Exosomes useful for delivery of nucleic acids are known to those skilled in the art and are, for example, exosomes described in more detail in U.S. Pat. No. 9,889,210, which is incorporated herein by reference for any purpose.

[0275] As used herein, the term "extracellular vesicles" or "EVs" refers to cell-derived vesicles that contain a membrane that encloses an internal space. In general, extracellular vesicles include all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. In general, extracellular vesicles range from 20 nm to 1000 nm in diameter and can contain a variety of macromolecular cargo, either within the internal space and / or across the membrane that is presented on the exterior 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 successive 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). Extracellular vesicles can be derived from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0276] 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 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 such as any of the engineered nucleic acids described herein, RNA, or DNA), 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 hereby incorporated by reference in its entirety.

[0277] As used herein, the term "nanovesicles" (also referred to as "microvesicles") refers to small (20-250 nm diameter, more preferably between 30-150 nm diameter) vesicles of cell origin that include a membrane surrounding an internal space and are generated from the producing 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 manipulations of the producing cells include, but are not limited to, continuous extrusion, treatment with alkaline solutions, sonication, or combinations 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 the 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 such as any of the engineered nucleic acids described herein, RNA, or DNA), a sugar (e.g., a monosaccharide, polysaccharide, or glycan), or other molecule. Once it has been derived from the production cell according to the engineered procedure, the nanovesicle can be isolated from the production cell based on its size, density, biochemical parameters, or a combination thereof.

[0278] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that rely on the amphiphilic nature of lipids to form membrane and vesicle-like structures (Riley 2017). Generally, these vesicles deliver cargo / payloads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of a target cell and releasing the cargo into the cytosol. The lipids used in LNP formation can be cationic, anionic, or neutral. The 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. The lipid composition generally includes a mixture 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 attachment of additional moieties. Lipid composition can affect overall LNP size and stability. In one example, lipid composition includes dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or MC3-like molecules. 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. In addition, LNPs can be further engineered or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0279] 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. Micelles typically refer to lipid structures that contain only a lipid monolayer. Micelles are described in detail by Quader et al. (Mol Ther. 2017 Jul 5;25(7):1501-1513), which is incorporated herein by reference for all purposes.

[0280] Nucleic acid vectors, such as expression vectors, directly exposed to serum may have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by free nucleic acid. Similarly, viral delivery systems directly exposed to serum may induce undesirable immune responses and / or neutralization of the viral delivery system. Thus, encapsulation of engineered nucleic acid and / or viral delivery systems may be used to avoid degradation while also avoiding potential off-target effects. In certain instances, the engineered nucleic acid and / or viral delivery system is fully encapsulated within the delivery medium, such as within the aqueous interior of the LNP. Encapsulation of the engineered nucleic acid and / or viral delivery system within the LNP can be performed by 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 the droplet generation device in parallel with the engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like-based LNP. In one example, the droplet generation device can control the size range and size distribution of the LNPs produced. For example, the LNPs can have sizes ranging from 1 to 1000 nanometers in diameter, e.g., sizes of 1, 10, 50, 100, 500, or 1000 nanometers. Following droplet generation, the delivery vehicles (e.g., engineered nucleic acids and / or viral delivery systems) encapsulating the cargo / payload can be further processed or manipulated to prepare them for administration.

[0281] Nanoparticle Delivery Nanomaterials can be used to deliver engineered nucleic acids (e.g., nucleic acids encoding modified ER-LBD or chimeric proteins as described herein). Nanomaterial vehicles are importantly made from non-immunogenic materials and generally can avoid eliciting immunity against the delivery vector itself. These materials include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in detail by Riley et al. (Recent Advances in Nanomaterials for Gene Delivery-A Review. Nanomaterials 2017,7(5),94), which is incorporated herein by reference for all purposes.

[0282] Genome Editing Genome editing systems can be used to engineer host genomes to encode engineered nucleic acids, such as the nucleic acids encoding modified ER-LBDs of the present disclosure. In general, "genome editing systems" refer to any system for integrating exogenous genes 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.

[0283] A transposon system can be used to integrate engineered nucleic acids, such as those of the present disclosure, into a host genome. A transposon generally comprises a terminal inverted repeat (TIR) ​​flanked by a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans, with the TIR flanking cargo. The transposon system can be a retrotransposon system or a DNA transposon system. Generally, the transposon system randomly integrates the cargo / payload (e.g., engineered nucleic acid) into the host genome. Examples of transposon systems include systems that use 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, each of which is incorporated by reference for all purposes. 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 by reference for all purposes.

[0284] Nuclease genome editing systems can be used to engineer host genomes to encode engineered nucleic acids such as the isolated polynucleotides or heterologous constructs of the present disclosure. Although not wishing to be bound by theory, nuclease-mediated gene editing systems used to introduce exogenous genes generally utilize the cell's natural DNA repair mechanisms, in particular the homologous recombination (HR) repair pathway. Briefly, following damage to genomic DNA (typically a double-strand break), cells can eliminate the damage by using another source of DNA with identical or substantially identical sequences at both its 5' and 3' ends as a template during DNA synthesis to repair the damage. In a natural situation, HDR can use other chromosomes present in the cell as templates. In gene editing systems, exogenous polynucleotides are introduced into cells and used as homologous recombination templates (HRT or HR templates). Generally, any additional exogenous sequence not naturally found in a chromosome, with the lesion contained within the HRT (e.g., a gene or part of a gene) between the 5' and 3' complementary ends, 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).

[0285] In some instances, the HR template may be linear. Examples of linear HR templates include, but are not limited to, linearized plasmid vectors, ssDNA, synthetic DNA, and PCR amplified DNA. In certain instances, the HR template may be circular, such as a plasmid. Circular templates may include supercoiled templates.

[0286] 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 the region of the endogenous genome target locus. The HR arms can, in some cases, be substantially identical to the region of the endogenous genome target locus. While substantially identical HR arms can be used, it can be advantageous for the HR arms to be identical, since the efficiency of the HDR pathway can be affected by HR arms with less than 100% identity.

[0287] 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, respectively. The HR arms can generally be any length, but practical considerations such as the impact of the HR arm length and overall template size on the overall editing efficiency can also be taken into account. The HR arms can be identical or substantially identical to the region of the endogenous genome target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genome target locus immediately adjacent to the cleavage site. Each HR arm can be identical or substantially identical to the region of the endogenous genome target locus within a certain distance from 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 from each other.

[0288] 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.

[0289] 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. The CRISPR system is described in detail by M. Adli ("The CRISPR tool kit for genome editing and beyond" Nature Communications; volume 9 (2018), Article number: 1911), which is incorporated by reference for all 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 that has a CRISPR RNA (crRNA) domain and a transactivating 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). Although the Cas9 system is exemplified herein, other CRISPR systems can be used, such as the Cpf1 system. The nuclease can include its derivatives, such as Cas9 functional mutants, e.g., Cas9 "nickase" mutants, which generally mediate only a single strand cleavage of a defined nucleotide sequence, as opposed to the complete double-stranded cleavage typically produced by the Cas9 enzyme.

[0290] In general, the components of the CRISPR system interact with each other to form a ribonucleoprotein (RNP) complex to 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 in vitro (i.e., "complexed") to form an RNP complex. The in vitro produced RNP 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 complex can be delivered to a cell by a variety of 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 particular example, the in vitro produced RNP complex can be delivered to a cell using Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation system, Miltenyi CliniMACS electroporation system, Neon electroporation system, and BTX electroporation system.CRISPR nucleases, such as Cas9, can be produced in vitro (i.e., synthesized and purified) using various protein production techniques known in the art.CRISPR RNAs, such as sgRNAs, can be produced in vitro (i.e., synthesized and purified) using various RNA production techniques known to those skilled in the art, such as in vitro transcription or chemical synthesis.

[0291] The RNP complex produced in vitro can be complexed with different ratios of nuclease to gRNA.The RNP complex produced in vitro can also be used in different amounts in CRISPR-mediated editing system.For example, depending on the number of cells that are desired to be edited, the total amount of RNP added can be adjusted, such as reducing the amount of RNP complex added when editing a large number of cells in reaction.

[0292] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) can be encoded by a polynucleotide separately, 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 an exemplary multi-cistronic system below) and introduced into a cell. After expression of the CRISPR components encoded by each polynucleotide in the cell (e.g., translation of nuclease and transcription of CRISPR RNA), an RNP complex can be formed in the cell and then directed to site-specific cleavage.

[0293] Some RNPs can be engineered to have moieties that facilitate delivery of the RNP into the nucleus. For example, the Cas9 nuclease can have a nuclear localization signal (NLS) domain, such 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.

[0294] 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.

[0295] In some CRISPR systems, more than one CRISPR composition can be provided, each of which targets the same gene or common genomic locus in more than one target nucleotide sequence separately. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance from each other. In some CRISPR systems, more than one CRISPR composition can be provided, each of which targets opposite strands of the same gene or common genomic locus separately. For example, two separate CRISPR "nickase" compositions can be provided to direct cleavage at the same gene or common genomic locus in opposite strands.

[0296] 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 are composed of the DNA binding domain of 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 monomer of the DNA binding domain, different artificial TALENs can be created to target different 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 US 12 / 965,590, US 8,450,471, US 8,440,431, US 8,440,432, US 10,172,880, and US 13 / 738,381, all of which are incorporated herein by reference in their entirety. ZFN-based editing systems are described in more detail in U.S. Pat. Nos. 6,453,242, 6,534,261, 6,599,692, 6,503,717, 6,689,558, 7,030,215, 6,794,136, 7,067,317, 7,262,054, 7,070,934, 7,361,635, 7,253,273, and U.S. Patent Publication Nos. 2005 / 0064474, 2007 / 0218528, and 2005 / 0267061, all of which are incorporated by reference in their entireties for all purposes.

[0297] Other engineered delivery systems A variety of additional means for introducing an engineered nucleic acid (e.g., an isolated polynucleotide molecule 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.

[0298] Electroporation can be used to deliver polynucleotides to recipient entities. Electroporation is a method of internalizing a cargo / payload into an internal compartment of a target cell or entity through the application of an electric field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing a cell or target entity between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). A transient set voltage is then applied to disrupt, i.e., permeabilize, the lipid membrane of the cell, thereby allowing the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example 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, concentration of cargo, 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. A variety of 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.

[0299] 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.

[0300] Compositions and methods for delivery of naked plasmids or engineered mRNA, such as mRNA, in vivo 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 by reference herein for all purposes.

[0301] How to use Methods of using the modified ER-LBD, chimeric proteins, or cells described herein are also encompassed by the disclosure.

[0302] In some embodiments, the method comprises modulating transcription of a gene of interest. The method of modulating transcription comprises transforming a cell with a target expression cassette comprising (i) a heterologous construct encoding a chimeric transcription factor comprising a modified ER-LBD and (ii) a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to a gene of interest, culturing the transformed cell under conditions suitable for expression of the chimeric protein, and inducing the chimeric protein to modulate transcription of the gene of interest by contacting the transformed cell with a non-endogenous ligand.

[0303] In some embodiments, the method of regulating transcription is a method of activating transcription. Activating transcription can be accomplished using a chimeric protein.

[0304] In some embodiments, the method includes activating transcription, which can be accomplished, for example, using a chimeric protein that includes a modified ER-LBD, a DNA binding domain, and a transcription activation domain.

[0305] In some embodiments, the method includes repressing transcription. Repressing transcription can be accomplished, for example, using a chimeric protein that includes a modified ER-LBD, a DNA binding domain, and a transcriptional repressor domain.

[0306] 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 suitable 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.

[0307] In some embodiments, the non-endogenous ligand is administered at a concentration such that the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha.

[0308] In vivo methods The methods provided herein also include modifying localization or transcription in vivo, for example, by delivering a non-endogenous ligand to cells expressing the modified ER-LBD or chimeric protein in vivo.

[0309] 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-hydroxy-tamoxifen ("4-OHT"), N-desmethyl tamoxifen, tamoxifen-N-oxide, and endoxifen. In some embodiments, the non-endogenous ligand is administered to the subject at a concentration of about 1 mg per day to 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.

[0310] In some aspects, the methods provided herein also include modulating transcription of a gene of interest in vivo, for example, by delivering to a subject (i) a cell transformed with a chimeric transcription factor described herein and (ii) a non-endogenous ligand. 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.

[0311] In some embodiments, the subject is a human or animal, and contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the non-endogenous ligand to the human or animal.

[0312] 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 molecule 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.

[0313] The methods provided herein also include in vivo delivery of a composition capable of producing any of the modified ER-LBD, chimeric protein, or chimeric transcription factor (and in some embodiments, genes regulated by the chimeric transcription factor) described herein. Compositions capable of in vivo production of modified ER-LBD, chimeric protein, or chimeric transcription factor (and in some embodiments, genes regulated by the chimeric transcription factor) include, but are not limited to, any of the engineered nucleic acids described herein. Compositions capable of in vivo production of inducible transcription factor (and in some embodiments, genes regulated by the inducible transcription factor) can be naked mRNA or naked plasmid.

[0314] Pharmaceutical Compositions The modified ER-LBD, chimeric proteins, and cells of the present disclosure can be formulated in pharmaceutical compositions. These compositions can contain, in addition to one or more of the engineered nucleic acids or engineered cells, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. Such materials must be non-toxic and must not interfere with the efficacy of the active ingredient. The exact nature of the carrier or other materials can depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

[0315] Whether cells, polypeptides, nucleic acids, small molecules, or other pharma- ceutical useful compounds according to the present disclosure are administered to an individual, 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, such as determining dosage, 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 the practitioner. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

[0316] The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition being treated.

[0317] Additional Embodiments The following paragraphs provide additional enumerated embodiments. 1. 1. A modified estrogen receptor ligand binding domain (ER-LBD) comprising an amino acid sequence corresponding to amino acids 282-595 of 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, and one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are within a region of SEQ ID NO:1 selected from the group consisting of positions 343-354, 380-392, 404-463, 517-540, and 547, 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. 2. 1. A modified estrogen receptor ligand binding domain (ER-LBD) comprising an amino acid sequence corresponding to amino acids 282-595 of 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, and one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are within a region of SEQ ID NO:1 selected from the group consisting of positions 343-354, 380-392, 404-463, 517-540, and 547, wherein 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. 3. 1. A modified estrogen receptor ligand binding domain (ER-LBD) comprising an amino acid sequence corresponding to amino acids 282-595 of 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, and one or more additional amino acid substitutions, wherein the one or more additional amino acid substitutions are within a region of SEQ ID NO:1 selected from the group consisting of positions 343-354, 380-392, 404-463, 517-540, and 547, wherein the modified ER-LBD has a higher selectivity for a non-endogenous ligand compared to an ER-LBD comprising the amino acids of SEQ ID NO:2. 4. 4. The modified ER-LBD of any one of paragraphs 1-3, wherein the one or more additional amino acid substitutions are at one or more positions of SEQ ID NO:1 selected from the group consisting of 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. 5. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 343 of SEQ ID NO:1. 6. 6. The modified ER-LBD of paragraph 5, 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. 7. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 344 of SEQ ID NO:1. 8. The modified ER-LBD of paragraph 7, wherein the amino acid substitution at position 344 of SEQ ID NO:1 is G344M. 9. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 345 of SEQ ID NO:1. 10. 10. The modified ER-LBD of paragraph 9, wherein the amino acid substitution at position 345 of SEQ ID NO:1 is L345S. 11. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 346 of SEQ ID NO:1. 12. 12. The modified ER-LBD of paragraph 11, 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. 13. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 347 of SEQ ID NO:1. 14. 14. The modified ER-LBD of paragraph 13, 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. 15. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 348 of SEQ ID NO:1. 16. 16. The modified ER-LBD of paragraph 15, wherein the amino acid substitution at position 348 of SEQ ID NO:1 is N348K. 17. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 349 of SEQ ID NO:1. 18. 18. The modified ER-LBD of paragraph 17, 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. 19. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 350 of SEQ ID NO:1. 20. 20. The modified ER-LBD of paragraph 19, 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. twenty one. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 351 of SEQ ID NO:1. twenty two. 22. The modified ER-LBD of paragraph 21, 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. twenty three. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 352 of SEQ ID NO:1. twenty four. 24. The modified ER-LBD of paragraph 23, wherein the amino acid substitution at position 352 of SEQ ID NO:1 is R352K. twenty five. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 354 of SEQ ID NO:1. 26. 26. The modified ER-LBD of paragraph 25, wherein the amino acid substitution at position 354 of SEQ ID NO:1 is selected from the group consisting of L354I, L354M, L354F, and L354V. 27. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 380 of SEQ ID NO:1. 28. 28. The modified ER-LBD of paragraph 27, wherein the amino acid substitution at position 380 of SEQ ID NO:1 is E380Q. 29. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 384 of SEQ ID NO:1. 30. 30. The modified ER-LBD of paragraph 29, wherein the amino acid substitution at position 384 of SEQ ID NO:1 is selected from the group consisting of L384I, L384M, L384F, and L384V. 31. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 386 of SEQ ID NO:1. 32. 32. The modified ER-LBD of paragraph 31, wherein the amino acid substitution at position 386 of SEQ ID NO:1 is I386V. 33. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 387 of SEQ ID NO:1. 34. 34. The modified ER-LBD of paragraph 33, wherein the amino acid substitution at position 387 of SEQ ID NO:1 is selected from the group consisting of L387I, L387M, L387F, and L387V. 35. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 388 of SEQ ID NO:1. 36. 36. The modified ER-LBD of paragraph 35, wherein the amino acid substitution at position 388 of SEQ ID NO:1 is selected from the group consisting of M388I, M388L, and M388F. 37. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 389 of SEQ ID NO:1. 38. 38. The modified ER-LBD of paragraph 37, wherein the amino acid substitution at position 389 of SEQ ID NO:1 is I389M. 39. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 391 of SEQ ID NO:1. 40. 40. The modified ER-LBD of paragraph 39, 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. 41. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 392 of SEQ ID NO:1. 42. 42. The modified ER-LBD of paragraph 41, wherein the amino acid substitution at position 392 of SEQ ID NO:1 is V392M. 43. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 404 of SEQ ID NO:1. 44. 44. The modified ER-LBD of paragraph 43, wherein the amino acid substitution at position 404 of SEQ ID NO:1 is selected from the group consisting of F404I, F404L, F404M, and F404V. 45. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 407 of SEQ ID NO:1. 46. 46. ​​The modified ER-LBD of paragraph 45, wherein the amino acid substitution at position 407 of SEQ ID NO: 1 is N407D. 47. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 409 of SEQ ID NO:1. 48. 48. The modified ER-LBD of paragraph 47, wherein the amino acid substitution at position 409 of SEQ ID NO:1 is L409V. 49. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 413 of SEQ ID NO:1. 50. 50. The modified ER-LBD of paragraph 49, wherein the amino acid substitution at position 413 of SEQ ID NO:1 is N413D. 51. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 414 of SEQ ID NO:1. 52. 52. The modified ER-LBD of paragraph 51, wherein the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E. 53. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 417 of SEQ ID NO:1. 54. 54. The modified ER-LBD of paragraph 53, wherein the amino acid substitution at position 417 of SEQ ID NO:1 is C417S. 55. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 418 of SEQ ID NO:1. 56. 56. The modified ER-LBD of paragraph 55, wherein the amino acid substitution at position 418 of SEQ ID NO:1 is selected from the group consisting of V418I, V418L, V418M, and V418F. 57. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 420 of SEQ ID NO:1. 58. 58. The modified ER-LBD of paragraph 57, 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. 59. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 421 of SEQ ID NO:1. 60. 60. The modified ER-LBD of paragraph 59, 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. 61. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 422 of SEQ ID NO:1. 62. The modified ER-LBD of paragraph 61, wherein the amino acid substitution at position 422 of SEQ ID NO:1 is V422I. 63. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 424 of SEQ ID NO:1. 64. 64. The modified ER-LBD of paragraph 63, wherein the amino acid substitution at position 424 of SEQ ID NO:1 is selected from the group consisting of I424L, I424M, I424F, and I424V. 65. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 428 of SEQ ID NO:1. 66. 66. The modified ER-LBD of paragraph 65, wherein the amino acid substitution at position 428 of SEQ ID NO:1 is selected from the group consisting of L428I, L428M, L428F, and L428V. 67. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 463 of SEQ ID NO:1. 68. 68. The modified ER-LBD of paragraph 67, wherein the amino acid substitution at position 463 of SEQ ID NO:1 is S463P. 69. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 517 of SEQ ID NO:1. 70. 70. The modified ER-LBD of paragraph 69, wherein the amino acid substitution at position 517 of SEQ ID NO:1 is M517A. 71. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 521 of SEQ ID NO:1. 72. 72. The modified ER-LBD of paragraph 71, wherein 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. 73. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 522 of SEQ ID NO:1. 74. 74. The modified ER-LBD of paragraph 73, wherein the amino acid substitution at position 522 of SEQ ID NO:1 is selected from the group consisting of M522I, M522L, and M522V. 75. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 524 of SEQ ID NO:1. 76. 76. The modified ER-LBD of paragraph 75, wherein 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. 77. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 525 of SEQ ID NO:1. 78. 78. The modified ER-LBD of paragraph 77, 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. 79. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 526 of SEQ ID NO:1. 80. 80. The modified ER-LBD of paragraph 79, wherein the amino acid substitution at position 526 of SEQ ID NO:1 is Y526L. 81. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 527 of SEQ ID NO:1. 82. The modified ER-LBD of paragraph 81, wherein the amino acid substitution at position 527 of SEQ ID NO:1 is S527N. 83. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 528 of SEQ ID NO:1. 84. The modified ER-LBD of paragraph 83, wherein the amino acid substitution at position 528 of SEQ ID NO:1 is selected from the group consisting of M528F, M528I, and M528V. 85. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 533 of SEQ ID NO:1. 86. 86. The modified ER-LBD of paragraph 85, wherein the amino acid substitution at position 533 of SEQ ID NO:1 is selected from the group consisting of V533F and V533W. 87. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 534 of SEQ ID NO:1. 88. 88. The modified ER-LBD of paragraph 87, wherein the amino acid substitution at position 534 of SEQ ID NO:1 is selected from the group consisting of V534Q and V534R. 89. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 536 of SEQ ID NO:1. 90. 90. The modified ER-LBD of paragraph 89, wherein 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. 91. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 537 of SEQ ID NO:1. 92. 92. The modified ER-LBD of paragraph 91, wherein the amino acid substitution at position 537 of SEQ ID NO:1 is selected from the group consisting of Y537E and Y537S. 93. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 538 of SEQ ID NO:1. 94. 94. The modified ER-LBD of paragraph 93, wherein the amino acid substitution at position 538 of SEQ ID NO:1 is selected from the group consisting of D538G and D538K. 95. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 539 of SEQ ID NO:1. 96. 96. The modified ER-LBD of paragraph 95, wherein the amino acid substitution at position 539 of SEQ ID NO:1 is selected from the group consisting of L539A and L539R. 97. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 540 of SEQ ID NO:1. 98. 98. The modified ER-LBD of paragraph 97, wherein the amino acid substitution at position 540 of SEQ ID NO:1 is selected from the group consisting of L540A and L540F. 99. The modified ER-LBD of paragraph 4, wherein one or more positions includes position 547 of SEQ ID NO:1. 100. 99. The modified ER-LBD of paragraph 99, wherein the amino acid substitution at position 547 of SEQ ID NO:1 is H547A. 101. The modified ER-LBD of any one of paragraphs 1 to 100, wherein the one or more additional amino acid substitutions is two amino acid substitutions. 102. 102. The modified ER-LBD of paragraph 101, wherein each of the two amino acid substitutions is at a position of SEQ ID NO:1 selected from the group consisting of 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525. 103. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 345 and 348 of SEQ ID NO:1. 104. 104. The modified ER-LBD of paragraph 103, 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. 105. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 384 and 389 of SEQ ID NO:1. 106. 106. The modified ER-LBD of paragraph 105, 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. 107. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO:1. 108. 108. The modified ER-LBD of paragraph 107, 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. 109. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO:1. 110. 109. The modified ER-LBD of paragraph 109, 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. 111. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO:1. 112. 112. The modified ER-LBD of paragraph 111, 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. 113. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO:1. 114. 14. The modified ER-LBD of paragraph 113, 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. 115. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 387 and 391. 116. 116. The modified ER-LBD of paragraph 115, 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. 117. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 384 and 387 of SEQ ID NO:1. 118. 118. The modified ER-LBD of paragraph 117, 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. 119. The modified ER-LBD of paragraph 102, wherein the two amino acid substitutions are at positions 384 and 391 of SEQ ID NO:1. 120. 118. The modified ER-LBD of paragraph 117, 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. 121. 121. The modified ER-LBD of any one of paragraphs 1 to 120, wherein the one or more additional amino acid substitutions are three amino acid substitutions. 122. 122. The modified ER-LBD of paragraph 121, wherein 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, 414, 418, 463, 521, 524, and 525. one two three. The modified ER-LBD of paragraph 122, wherein the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO:1. 124. 124. The modified ER-LBD of paragraph 123, 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. 125. The modified ER-LBD of paragraph 122, wherein the three amino acid substitutions are at positions 414, 463, and 524 of SEQ ID NO:1. 126. 126. The modified ER-LBD of paragraph 125, 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. 127. 127. The modified ER-LBD of any one of paragraphs 1 to 126, wherein the one or more additional amino acid substitutions are four amino acid substitutions. 128. 128. The modified ER-LBD of paragraph 127, wherein each of the four amino acid substitutions is at a position in SEQ ID NO:1 selected from the group consisting of 343, 347, 351, 354, 384, 388, 391, 404, 413, 418, 463, 521, 524, and 525. 129. The modified ER-LBD of paragraph 128, wherein the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO:1. 130. 130. The modified ER-LBD of paragraph 129, 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. 131. The modified ER-LBD of paragraph 128, wherein the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO:1. 132. 132. The modified ER-LBD of paragraph 131, 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. 133. The modified ER-LBD of paragraph 128, wherein the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO:1. 134. 134. The modified ER-LBD of paragraph 133, 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. 135. The modified ER-LBD of paragraph 128, wherein the four amino acid substitutions are at positions 354, 384, 391, and 463 of SEQ ID NO:1. 136. 136. The modified ER-LBD of paragraph 135, 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. 137. The modified ER-LBD of paragraph 128, wherein the four amino acid substitutions are at positions 384, 391, 413, and 524 of SEQ ID NO:1. 138. 138. The modified ER-LBD of paragraph 137, 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. 139. The modified ER-LBD of any one of paragraphs 1 to 138, wherein the one or more additional amino acid substitutions are five amino acid substitutions. 140. The modified ER-LBD of paragraph 139, wherein each of the five amino acid substitutions is at a position in SEQ ID NO:1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, and 524. 141. The modified ER-LBD of paragraph 140, wherein the five amino acid substitutions are at positions 384, 409, 413, 463, and 524 of SEQ ID NO:1. 142. 142. The modified ER-LBD of paragraph 141, 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. 143. The modified ER-LBD of paragraph 140, wherein the five amino acid substitutions are at positions 391, 413, 414, 463, and 524 of SEQ ID NO:1. 144. 144. The modified ER-LBD of paragraph 143, 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. 145. The modified ER-LBD of paragraph 140, wherein the five amino acid substitutions are at positions 391, 414, 421, 463, and 524 of SEQ ID NO:1. 146. 146. The modified ER-LBD of paragraph 145, 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. 147. The modified ER-LBD of paragraph 140, wherein the five amino acid substitutions are at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1. 148. 148. The modified ER-LBD of paragraph 147, 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. 149. The modified ER-LBD of paragraph 140, wherein the five amino acid substitutions are at positions 354, 409, 421, 463, and 524 of SEQ ID NO:1. 150. 150. The modified ER-LBD of paragraph 149, 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. 151. The modified ER-LBD of any one of paragraphs 1 to 150, wherein the one or more additional amino acid substitutions are six amino acid substitutions. 152. The modified ER-LBD of paragraph 151, wherein each of the six amino acid substitutions is at a position in SEQ ID NO:1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, and 524. 153. The modified ER-LBD of paragraph 152, wherein the six amino acid substitutions are at positions 384, 391, 413, 421, 463, and 524 of SEQ ID NO:1. 154. 154. The modified ER-LBD of paragraph 153, 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. 155. The modified ER-LBD of paragraph 152, wherein the six amino acid substitutions are at positions 409, 413, 414, 421, 463, and 524 of SEQ ID NO:1. 156. 156. The modified ER-LBD of paragraph 155, 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. 157. The modified ER-LBD of paragraph 152, wherein the six amino acid substitutions are at positions 354, 391, 409, 413, 414, and 524 of SEQ ID NO:1. 158. 158. The modified ER-LBD of paragraph 157, 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. 159. The modified ER-LBD of any one of paragraphs 1 to 158, wherein the one or more additional amino acid substitutions are seven amino acid substitutions. 160. The modified ER-LBD of paragraph 159, wherein each of the seven amino acid substitutions is at a position in SEQ ID NO:1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, 517, and 524. 161. The modified ER-LBD of paragraph 160, wherein the seven amino acid substitutions are at positions 354, 384, 409, 413, 421, 463, and 524 of SEQ ID NO:1. 162. 162. The modified ER-LBD of paragraph 161, 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. 163. The modified ER-LBD of paragraph 160, wherein the seven amino acid substitutions are at positions 354, 391, 413, 421, 463, 517, and 524 of SEQ ID NO:1. 164. 164. The modified ER-LBD of paragraph 163, 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. 165. The modified ER-LBD of paragraph 160, wherein the seven amino acid substitutions are at positions 354, 391, 413, 414, 421, 517, and 524 of SEQ ID NO:1. 166. 166. The modified ER-LBD of paragraph 165, 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. 167. The modified ER-LBD of any one of paragraphs 1 to 166, wherein the one or more additional amino acid substitutions are eight amino acid substitutions. 168. The modified ER-LBD of paragraph 167, wherein the eight amino acid substitutions are at positions 384, 391, 409, 413, 421, 463, 517, and 524 of SEQ ID NO:1. 169. 169. The modified ER-LBD of paragraph 168, 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. 170. The modified ER-LBD of any one of paragraphs 1 to 169, wherein the modified ER-LBD further comprises a V595A amino acid substitution. 171. The modified ER-LBD of any one of paragraphs 1-170, wherein the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. 172. A chimeric protein comprising a polypeptide of interest fused to a modified ER-LBD according to any one of paragraphs 1 to 171. 173. The chimeric protein of Paragraph 172, wherein the polypeptide of interest comprises a nucleic acid binding domain. 174. The chimeric protein of Paragraph 173, wherein the nucleic acid binding domain comprises a zinc finger domain. 175. The chimeric protein of paragraph 174, wherein the zinc finger domain comprises the sequence MSRPGERPFQCRICMRNFSNMSNLTRHTRTHTGEKPFQCRICMRNFSDRSVLRRHLRTHTGSQKPFQCRICMRNFSDPSNLARHTRTHTGEKPFQCRICMRNFSDRSSLRRHLRTHTGSQKPFQCRICMRNFSQSGTLHRHTRTHTGEKPFQCRICMRNFSQRPNLTRHLRTHLRGS (SEQ ID NO: 62). 176. 176. The chimeric protein of any one of paragraphs 172 to 175, wherein the chimeric protein comprises a chimeric transcription factor and the polypeptide of interest comprises a nucleic acid binding domain and a transcriptional regulator domain. 177. The chimeric protein of paragraph 176, wherein the transcriptional regulator domain is a transcriptional activator. 178. 178. The chimeric protein of paragraph 177, wherein 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; 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). 179. The chimeric protein of paragraph 178, wherein the transcriptional activator is a p65 transcriptional activator comprising the amino acid sequence DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64). 180. An isolated polynucleotide molecule comprising a nucleotide sequence encoding the modified ER-LBD of any one of paragraphs 1-171. 181. 179. An isolated polynucleotide molecule comprising a nucleotide sequence encoding the chimeric protein of any one of paragraphs 172 to 179. 182. A heterologous construct comprising a promoter operably linked to the polynucleotide molecule of Paragraph 180. 183. A heterologous construct comprising a promoter operably linked to the polynucleotide molecule of Paragraph 181. 184. A plasmid comprising the heterologous construct of paragraph 182. 185. A plasmid comprising the heterologous construct of Paragraph 183. 186. A cell comprising the heterologous construct of Paragraph 182 or the plasmid of Paragraph 184. 187. A cell comprising the heterologous construct of Paragraph 183 or the plasmid of Paragraph 185. 188. A gene switch for regulating the transcription of a gene of interest, comprising: (a) the chimeric protein of paragraph 176, wherein the chimeric protein binds to a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to a 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 a gene of interest. 189. 189. The gene switch of paragraph 188, wherein the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. 190. 189. The gene switch of paragraph 188 or 189, wherein 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. 191. 191. The gene switch of paragraph 190, wherein the gene of interest is selected from the group consisting of IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha. 192. 192. The gene switch of paragraph 191, wherein the gene of interest encodes an IL12p70 fusion protein comprising the amino acid sequence of (SEQ ID NO:58). 193. A method for regulating transcription of a gene of interest, comprising: transforming a cell with a target expression cassette comprising (i) a heterologous construct encoding a chimeric protein described in paragraph 176, and (ii) a chimeric transcription factor responsive (CTF responsive) promoter operably linked to a gene of interest; and inducing the chimeric protein to regulate transcription of the gene of interest by contacting the transformed cell with a non-endogenous ligand. 194. 194. The method of paragraph 193, wherein 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. 195. 195. The method of paragraph 193 or 194, wherein modulating transcription comprises activating transcription of the gene of interest. 196. 195. The method of paragraph 193 or 194, wherein modulating transcription comprises suppressing transcription of the gene of interest. 197. The method of any one of paragraphs 193 to 196, wherein the target expression cassette is encoded by a heterologous construct encoding the chimeric protein of paragraph 174. 198. The method of any one of paragraphs 193 to 196, wherein the target expression cassette is encoded by a second heterologous construct. 199. A method for regulating localization of a chimeric protein, the method comprising transforming a cell with a heterologous construct encoding a chimeric protein described in any one of paragraphs 172 to 179, and inducing nuclear localization of the chimeric protein by contacting the transformed cell with a non-endogenous ligand. 200. 200. The method of paragraph 199, wherein the method further comprises culturing the transformed cell under conditions suitable for expression of the chimeric protein prior to inducing nuclear localization. 201. The method of any one of claims 193 or 195-200, wherein 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. 202. The method of any one of paragraphs 193-201, wherein the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. 203. The method of paragraph 201 or 202, wherein the non-endogenous ligand is administered at a concentration at which the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha of SEQ ID NO:1. EXAMPLES

[0318] Below are examples of specific embodiments for carrying out the present disclosure. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but, of course, some experimental error and deviation should be allowed for.

[0319] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology, within the skill of one of ordinary skill in the art. Such techniques are explained fully in the literature, see, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Edition (East, Pennsylvania: Mack Publishing Company, 1992); rd Ed. (Plenum Press) Vols A and B (1992).

[0320] Example 1: ERT2 mutations predicted to modulate ligand binding In silico modeling was performed on 4-OHT, endoxifen, and estradiol binding to a variant of the estrogen receptor alpha known as ERT2, and mutations predicted to have increased sensitivity to 4-OHT compared to ERT2 of SEQ ID NO:2 were identified.

[0321] Materials and Methods Available crystal structures of the complexes between estradiol and ERα (PDB:1QKU, 3.2 Å resolution) and between 4-OHT and ERα (PDB:3ERT, 1.9 Å resolution) were used to generate models of the complexes between estradiol and ERT2, 4-OHT and ERT2, and endoxifen and ERT2. The ERT2 sequence differs from ERα in three residues (G400V / M543A / L544A). Only residues 306–551 were used in the structural model because all available structures were resolved in this region alone.

[0322] Using standard protocols (Kannan et al. ACS Omega 2017 Nov 30;2(11):7881-7891.), MD simulations were performed for apo ERT2, ERT2-estradiol, ERT2-4-OHT, and ERT2-endoxifen complexes (each simulation was run for 100 ns in triplicate). Both ERT2 and the bound ligand / drug remained stable during the simulation (using standard procedures). The conformations generated during the second half of the simulation (50 ns) (simulation considered to be equilibrated) were used for subsequent analysis.

[0323] result The first set of mutations was analyzed in silico for improvement of 4-OHT binding. Eighteen mutations to residues in the ligand-binding pocket were selected based on amino acids present in homologous positions in other estrogen receptor proteins. Of the 18 selected mutations, 17 of the mutants bind tighter than wild-type ERT2 by at least 1.8 kcal / mol, with only the M517A mutation appearing to destabilize 4-OHT binding (Figure 1A). Binding energy calculations were then performed to confirm the effect of the mutations on estradiol binding. Compared to 4-OHT (all mutations except M517A favor binding as indicated by negative ΔΔG values), the majority of mutations (Figure 1B) had negligible effects on estradiol binding (ΔΔG values ​​for all mutations are within 2 kcal / mol compared to the ΔΔG values ​​for 4-OHT, which are >2 kcal / mol for the majority of mutations). Only the mutations L409V, M517A, and N407D showed an increase in binding to estradiol of more than 1 kcal / mol, while both L409V and N407D bind 4-OHT tighter by 3 and 4 kcal / mol, respectively. The first set of mutations is shown in Table 6 below.

[0324] (Table 6) TIFF2024513231000014.tif43149

[0325] The second set of mutations was analyzed in silico for improved 4-OHT binding. Molecular docking simulations were performed on 4-OHT and estradiol binding to ERT2 for 19 different mutations at 5 additional sites in the ligand binding pocket (in addition to those shown in Table 6) to identify further mutants with increased sensitivity to 4-OHT compared to wild-type ERT2. Binding energy calculations were performed in agreement with those performed for the first set of mutations. All 19 of the mutations showed improved binding to 4-OHT within the range of 1.8 kcal / mol to 7 kcal / mol (Figure 2). The second set of mutations is shown in Table 7.

[0326] (Table 7) TIFF2024513231000015.tif38148

[0327] The third set of mutations was analyzed in silico for improved 4-OHT binding. A total of 23 mutations at six additional residue positions in the ligand-binding pocket (residues 428, 346, 349, 418, 421, and 424) were selected for molecular docking simulations. Binding energy calculations were performed in agreement with the calculations performed for the first set of mutations. Six of the mutations (L346F, L349M, V418I, V418M, I424M, and M421L) showed improved binding to 4-OHT by at least about 1.5 kcal / mol (Figure 3). These 23 mutations are shown in Table 8.

[0328] (Table 8) TIFF2024513231000016.tif42152

[0329] The fourth set of mutations was analyzed in silico for improved 4-OHT binding. A total of 23 mutations at six additional residue positions in the ligand-binding pocket (residues 528, 343, 388, 522, 414, and 521) were selected for molecular docking simulations. Binding energy calculations were performed in agreement with the calculations performed for the first set of mutations. Eighteen of the 23 mutations showed improved binding to 4-OHT by at least about 1.0 kcal / mol (Figure 4). The fourth set of mutations is shown in Table 9.

[0330] (Table 9) TIFF2024513231000017.tif42152

[0331] The fifth set of mutations was analyzed in silico for improved 4-OHT binding. A total of 38 mutations at five additional residue positions (residues 524, 525, 347, 350, and 351) were selected for molecular docking simulations. Binding energy calculations were performed in agreement with the calculations performed for the first set of mutations. Twenty-eight of the 38 mutations showed improved binding to 4-OHT by at least about 1.0 kcal / mol and up to about 4.5 kcal / mol (Figure 5). The fifth set of mutations is shown in Table 10.

[0332] (Table 10) TIFF2024513231000018.tif90157

[0333] All of the mutations in Sets 1-5 were further analyzed by molecular docking simulations for binding to endoxifen and estradiol to determine the energies of binding to endoxifen and estradiol (calculated as ΔΔG in kcal / mol). Additionally, the difference between the binding energies of endoxifen binding compared to estradiol binding was calculated as ΔΔΔG values. A summary of the binding energies of 4-OHT, endoxifen, and estradiol, respectively, and the binding energy difference of 4-OHT and endoxifen compared to estradiol binding is shown in Table 11.

[0334] (Table 11) TIFF2024513231000019.tif224150TIFF2024513231000020.tif233150TIFF2024513231000021.tif251150

[0335] The sixth set of mutations was analyzed in silico to identify variants that destabilize the agonist-bound conformation (i.e., estradiol-bound conformation) and / or stabilize the antagonist-bound conformation (i.e., 4-OHT or endoxifen-bound conformation). The major structural difference between the agonist-bound conformation and the antagonist-bound conformation is in the orientation and docking site of helix 12 (H12, see FIG. 6). A total of 14 mutations at eight residue positions in helix 12 (residues 538, 536, 539, 540, 547, 534, 533, and 537) were selected for analysis. The free energy difference between mutant ERT2 and wild-type ERT2 in the antagonist-bound conformation and the free energy difference between mutant ERT2 and wild-type ERT2 in the agonist-bound conformation were calculated as ΔG values. The ΔΔG values ​​were then calculated, with negative values ​​indicating that the antagonist-bound conformation of the ERT2 mutant was more favorable than the agonist-bound conformation of the mutant, and positive values ​​indicating that the agonist-bound conformation was more favorable than the antagonist-bound conformation. Seven of the 14 mutations (D538K, L536F, L536Y, L536M, L539R, H547A, and V534R) stabilized antagonist confirmation ( FIG. 7 ). The sixth set of mutations is shown in Table 12.

[0336] (Table 12) TIFF2024513231000022.tif37135

[0337] Example 2: ERT2 mutants with increased drug sensitivity identified by transfection screen The ERT2 mutants were analyzed by transfection assay for their ability to induce reporter expression in response to 4-OHT. In the first transfection screen, constructs encoding ERT2 with the mutations described in Example 1 were produced in the background of "wild-type" ERT2 (containing G400V / M543A / L544A / V595A quadruple amino acid substitutions) shown in SEQ ID NO:3. Each ERT2 construct contained the ZF10-1 domain for DNA binding, the p65 transcription activation domain, and the ERT2 mutant. Each construct was tested for sensitivity to 4-OHT. Each mutant was cloned into an expression construct for transfection in the HEK293T+YBTATA_mCherry reporter cell line. In the second transfection screen, constructs encoding the additional ERT2 mutants described in Example 1 were produced and tested for sensitivity to 4-OHT. For the screen, cells were treated with three different concentrations of 4-OHT (0.025, 0.1, and 0.25 uM) and then assayed for mCherry expression by fluorescence-activated cell sorting (FACS) (Figures 8A-8C, 9A-9C, and 10A-10C).

[0338] Materials and Methods HEK293T cells were transduced with a lentivirus encoding a synthetic promoter consisting of four ZF10-1 binding sites linked to the YBTATA minimal promoter, which drives the expression of mCherry. Cells derived from this cell line were referred to as "reporter cells."

[0339] On day 1, reporter cells were seeded at 1.5e5 cells / well in 24-well plates. On day 2, cells were transfected with ERT mutants. A mixture of 0.6ug DNA, 1.8uL Fugene, and 30uL Optimem was made for each well where DNA encoded ZF10-1 fused to p65 and ERT2 mutants. In some screens, a plasmid encoding GFP was included as a control to select transfected cells by flow cytometry. On day 3, cells were split at a ratio of 1:20 and seeded in 96-well plates. Cells were treated with 0, 0.025, 0.1, or 0.25uM 4-OHT. On day 5, media was removed and cells were trypsinized and then resuspended in FACS buffer supplemented with Sytox Red (fluorescent in APC channel) viability dye. Cells were run on a flow cytometer and gated by FSC / SSC for cells, FSC / Sytox Red for live cells, FSC / FSC-Width for single cells, and, when possible, GFP+ for transfected cells (if a transfection control was included). The percentage of mCherry positive cells at each drug concentration was plotted and compared to wild-type ERT2 to identify mutants that were more sensitive to 4-OHT.

[0340] result As shown in Figures 8A-8C, 9A-9C, and 10A-10C, the transfection screen identified mutants with improved induction of mCherry expression compared to ERT2 (SEQ ID NO: 3). In the first transfection screen, improved induction of expression was observed in 17 of the mutants: L354I (SB03498), L391V (SB03505), Q414E (SB03383), L409V (SB03375), S463P (SB03393), L384M (SB03377), L354I+L384M (SB03511), N413D (SB03381), M517A (SB03379), G344M (SB03372), I386V (SB03373), N The greatest improvements in expression were observed for 407D (SB03380), C417S (SB03371), R352K (SB03384), Y537S (SB03389), M388F (SB03579), and G521A (SB03587), with the greatest improvements in expression induced by the following seven mutants: L354I (SB03498), L391V (SB03505), Q414E (SB03383), L409V (SB03375), and S463P (SB03393) (Figures 8A-8C). In a second transfection screen, improved induction of expression was observed in 10 of the mutants: I424L (SB03558), M421L (SB03566), M421F (SB03567), T347E (SB03801), L536M (SB03828), Y537E (SB03839), T347I (SB03802), and T347M (SB03805), V41 8I (SB03550), and V533W (SB03838), and strong improvements in expression were induced by the following six mutants: I424L (SB03558), M421L (SB03566), M421F (SB03567), T347E (SB03801), L536M (SB03828), and Y537E (SB03839) (Figures 9A-9C). In a third transfection screen (Figures 10A-10C), improvements in expression induction were observed for the mutants shown in Table 13.

[0341] (Table 13) TIFF2024513231000023.tif135128

[0342] Example 3: Mutants with increased drug sensitivity identified by transduction screening ERT2 mutants were analyzed by three transduction screens for their ability to induce reporter expression in response to 4-OHT. Mutant L354I+L384M (identified in the first transfection screen) was included in all three transduction screens. The lentivirus vectors encoding ERT2 mutants that showed improved response to 4-OHT in the transfection screen from Example 2 were cloned. The reporter cell line described in Example 2 was transduced with lentiviruses encoding ERT2 mutants, and the mutants' ability to induce mCherry expression in response to various 4-OHT concentrations was evaluated.

[0343] Materials and Methods For the transduction screen, on day 1, reporter cells were seeded at 2e5 cells / well in 12-well plates. On day 2, cells were transduced with lentivirus encoding the lead ERT mutant from the transfection screen. On days 3 and 4, cells were passaged to maintain <90% confluency on the plate. On day 5, cells were seeded in 96-well plates and treated with 0, 0.001, 0.0025, 0.004, 0.025, 0.05, 0.1, or 0.25 uM 4-OHT. On day 8, media was removed and cells were trypsinized and then resuspended in FACS buffer supplemented with Sytox Red (fluorescence in APC channel) viability dye. Cells were run on a flow cytometer, gated by FSC / SSC for live cells and FSC / Sytox Red for live cells, and the percentage of mCherry positive cells at each drug concentration was plotted to find the more sensitive mutant compared to wild type ERT2 (Figure 11A). The percentage of mCherry positive cells at two of the drug concentrations, 0.004 and 0.025uM 4-OHT, is also shown in the bar graph (Figure 11B).

[0344] result As shown in Figures 11A and 11B, the first transduction screen confirmed improved 4-OHT response for several mutants identified in the transfection screen from Example 2 as having improved 4-OHT binding. In particular, mutants L354I, L391V, Q414E, L409V, S463P, L384M, and L354I+L384M all showed improved 4-OHT response compared to wild-type ERT2 (construct 3422, SEQ ID NO: 3). ERT2 mutants showing improved 4-OHT binding in the first transduction screen are shown in Table 14.

[0345] (Table 14) TIFF2024513231000024.tif93128

[0346] As shown in FIG. 12, the second transduction screen confirmed the improved 4-OHT response for the mutants identified in the transfection screen from Example 2. In particular, mutants M517A, and N413D, as well as L354I+L384M, showed improved 4-OHT response compared to wild-type ERT2 (construct 3422). In particular, the improved 4-OHT response of the L354I+L384M mutant was confirmed in both the first and second transduction screens. ERT2 mutants showing improved 4-OHT binding in the second transduction screen are shown in Table 15.

[0347] (Table 15) TIFF2024513231000025.tif48128

[0348] As shown in Figure 13, the third transduction screen confirmed the improved 4-OHT response for the mutants identified in the transfection screen from Example 2. In particular, mutants I524L, M421L, and L354I+L384M showed improved 4-OHT response compared to wild type ERT2 (construct 3422). ERT2 mutants showing improved 4-OHT binding in the third transduction screen are shown in Table 16.

[0349] (Table 16) TIFF2024513231000026.tif48128

[0350] Example 4: ERT2 mutant library screen to develop an endoxifen ON switch Materials and Methods ERT2 mutant combinatorial library screen HEK293T cells were transduced with SB04401, a combinatorial ERT2 mutant library (Figure 14A) composed of approximately 800 unique ER-LBD variants, each of which is a unique combination of the mutants listed in Table 18 along with the rationale for its inclusion. To generate homogenous cell lines, each with a unique ER-LBD variant per cell, viral integration of transduced HEK293T cells was quantified by copy number assay. Cell lines with an average viral integration of <1 copy of the ERT2 mutant library per cell were identified and subjected to puromycin selection. The selected cell lines were then transduced with the SB01066 mCherry reporter (Figure 14B). The transduced cells were then tested for sensitivity to endoxifen and 4-OHT via the mCherry reporter, which would be expressed if the ER-LBD variant was sensitive to test concentrations as low as approximately 0.1 nM up to approximately 1 uM. Cells that express mCherry and thus respond to endoxifen treatment are selected, followed by isolating genomic DNA from the selected cells.Variants are identified from isolated genomic DNA by PCR amplification of ERT2 coding sequence, followed by insertion of the PCR product into pcr4 TOPO vector (Life Technologies).Colonies obtained are then submitted for Sanger sequencing.Identified mutants are cloned into constructs such as SB06136-SB06153 (ZF10-1_p65_ERT2 mutants).

[0351] (Table 18) TIFF2024513231000027.tif130153

[0352] Validation of ERT2 mutants in U87MG using mCherry reporter Approximately 16 hours prior to transduction, 100k U87MG:1066 cells were seeded into 16 wells in a 12-well plate format. During transduction, cells in each well were transduced with 100k pg of virus of the ERT2 variant construct. After transduction, cells were split into 50k cells per well in a 24-well plate format and treated with drug-free media or a range of endoxifen or 4-OHT drug conditions. Approximately 2 days after treatment of cells with endoxifen or 4-OHT, cells were harvested and mCherry reporter expression was quantified by flow cytometry.

[0353] result Screening of the ERT2 mutant library identified a subset of ERT2 mutants that were sensitive to endoxifen at 1 nM. Of this subset, 15 variants (Table 19) were tested in U87MG cells for their ability to activate the mCherry reporter (SB01066, FIG. 14B) at a range of endoxifen (FIG. 15A) and 4-OHT (FIG. 15B) concentrations of 0 pM, 10 pM, 50 pM, 100 pM, and 1 nM. Expression of mCherry was quantified approximately 24 hours after treatment with endoxifen or 4-OHT. As demonstrated, all 15 variants were sensitive to endoxifen and 4-OHT at concentrations of approximately 1 nM or less, while the negative controls U87MG and U87MG:1066 negative control showed no sensitivity.

[0354] (Table 19) TIFF2024513231000028.tif82153

[0355] Selected ERT2 variants from the previous screen (Table 20) were further tested for sensitivity to endoxifen and 4-OHT compared to wild-type ERT2. Testing showed that while activation of wild-type ERT2 begins at about 25 nM endoxifen and about 25 nM 4-OHT, the three ERT2 variants tested activate mCherry expression at 1 nM and 0.1 nM endoxifen, and 1 nM and 0.1 nM 4-OHT (Figures 16A-16D). An exemplary heat map shows the fold activation of mCherry expression of the constructs tested at various concentrations, including 2.2 pM estradiol (Figure 16B). It was confirmed that ERT2 is ER-alpha mutated to be insensitive to estradiol, and the lead ERT2 variant remains insensitive to biologically observed concentrations of estradiol. Fold activation was calculated by dividing the gMFI mCherry levels for each test condition by the gMFI mCherry levels in U87MG cells transduced with the reporter (SB01066).

[0356] (Table 20) TIFF2024513231000029.tif32153

[0357] Example 5: Validation of ERT2 mutants in NK cells Materials and Methods Validation of ERT2 mutants using mCherry reporter in NK cells After 10 days of feeder cell activation, NK cells were co-transduced with ERT2 mutant virus and reporter SB01066 virus (experimental set-up 1). Two days after transduction, transduced NK cells were treated with endoxifen or 4-OHT at a range of concentrations: 0 nM, 0.01 nM, 0.1 nM, 1 nM, and 10 nM. On day 4, cells were checked for mCherry expression by flow cytometry.

[0358] Experimental setup 1 TIFF2024513231000030.tif38128

[0359] Validation of ERT2 mutants using an IL12 reporter in NK cells After 10 days of feeder cell activation, NK cells were transduced with ERT2 / IL12 vector (Table 22, experimental set-up 2). Two days after transduction, transduced NK cells were treated with endoxifen at a range of concentrations: 0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, and 1000 nM. On day 4, cells were checked for IL-12 expression via Luminex.

[0360] Experimental setup 2 TIFF2024513231000031.tif39128

[0361] result Selected ERT2 variants from the previous screen (Table 21, Figures 17A-17B) were further tested for sensitivity to endoxifen and 4-OHT in primary NK cells. Among the variants tested, SB06142 (variant 77, L354I / L391V / N413D / M421L / S463P / M517A / H524L), SB06136 (variant 81, L384M / L391V / N413D / M421L / S463P / H524L), and SB06145 (variant 62, L409V / N413D / Q414E / M421L / S463P / H524L) were sensitive to both endoxifen and 4-OHT at a concentration of 0.1 nM (Figures 17A-17B).

[0362] (Table 21) TIFF2024513231000032.tif82152

[0363] To demonstrate delivery of therapeutic polypeptides, ERT2 / IL-12 vectors were constructed as shown in Table 22 and tested for sensitivity to endoxifen in primary NK cells. Testing of the ERT2 / IL-12 vectors in NK cells showed that TL10009 showed the best induction and fold change in IL-12, along with SB06142 and ERT2-L354I / L391V / N413D / S463P / H524L from crIL12 CD16 CS (Figures 18A-18B).

[0364] (Table 22) TIFF2024513231000033.tif133151

[0365] Table 17: Sequence TIFF2024513231000034.tif177151TIFF2024513231000035.tif232152TIFF2024513231000036.tif232152TIFF2024513231000037.tif232152TIFF2024513231000038.tif232152TIFF2024513231000039.tif232150TIFF2024513231000040.tif232152TIFF2024513231000041.tif232152TIFF2024513231000042.tif232152TIFF2024513231000043.tif232152TIFF2024513231000044.tif232152TIFF2024513231000045.tif232151TIFF2024513231000046.tif232151TIFF2024513231000047.tif232152TIFF2024513231000048.tif227154TIFF2024513231000049.tif232152TIFF2024513231000050.tif232150TIFF2024513231000051.tif232150TIFF2024513231000052.tif232150TIFF2024513231000053.tif232150TIFF2024513231000054.tif232152TIFF2024513231000055.tif232150TIFF2024513231000056.tif232150TIFF2024513231000057.tif232150TIFF2024513231000058.tif232152TIFF2024513231000059.tif232150TIFF2024513231000060.tif232150TIFF2024513231000061.tif232150TIFF2024513231000062.tif232150TIFF2024513231000063.tif232151TIFF2024513231000064.tif232150TIFF2024513231000065.tif232150TIFF2024513231000066.tif232150TIFF2024513231000067.tif232150TIFF2024513231000068.tif232150TIFF2024 513231000069.tif232150TIFF2024513231000070.tif232150TIFF2024513231000071.tif23 2150TIFF2024513231000072.tif232150TIFF2024513231000073.tif232150TIFF202451323 1000074.tif232150TIFF2024513231000075.tif232150TIFF2024513231000076.tif218151.

[0366] While the present disclosure has been specifically shown and described with reference to preferred and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the disclosure and the appended claims.

[0367] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A modified estrogen receptor ligand binding domain (ER-LBD) comprising an amino acid sequence corresponding to amino acids 282-595 of SEQ ID NO:1, the modified ER-LBD comprises a G400V amino acid substitution, a M543A amino acid substitution, and a L544A amino acid substitution, and one or more additional amino acid substitutions; the one or more additional amino acid substitutions are within a region of SEQ ID NO:1 selected from the group consisting of positions 343-354, 380-392, 404-463, and 517-540, and position 547; the modified ER-LBD has, as a result of the one or more additional amino acid substitutions, greater sensitivity and / or selectivity for 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; Optionally, the modified ER-LBD further comprises a V595A amino acid substitution; and / or Optionally, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. Modified ER-LBD.

2. 2. The modified ER-LBD of claim 1, wherein the one or more additional amino acid substitutions are at one or more positions of SEQ ID NO:1 selected from the group consisting of 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.

3. a. the one or more positions include position 343 of SEQ ID NO:1; 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; b. the one or more positions include position 344 of SEQ ID NO:1; Optionally, the amino acid substitution at position 344 of SEQ ID NO:1 is G344M; c. the one or more positions include position 345 of SEQ ID NO:1; Optionally, the amino acid substitution at position 345 of SEQ ID NO:1 is L345S; d. the one or more positions include position 346 of SEQ ID NO:1; 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; e. the one or more positions include position 347 of SEQ ID NO:1; Optionally, 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; f. the one or more positions include position 348 of SEQ ID NO:1; Optionally, the amino acid substitution at position 348 of SEQ ID NO:1 is N348K; g. the one or more positions include position 349 of SEQ ID NO:1; 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; h. the one or more positions include position 350 of SEQ ID NO:1; 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; i. the one or more positions include position 351 of SEQ ID NO:1; 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; j. the one or more positions include position 352 of SEQ ID NO:1; Optionally, the amino acid substitution at position 352 of SEQ ID NO:1 is R352K; k. the one or more positions include position 354 of SEQ ID NO:1; 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; l. the one or more positions include position 380 of SEQ ID NO:1; Optionally, the amino acid substitution at position 380 of SEQ ID NO:1 is E380Q; m. the one or more positions include position 384 of SEQ ID NO:1; 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; n. the one or more positions include position 386 of SEQ ID NO:1; Optionally, the amino acid substitution at position 386 of SEQ ID NO:1 is I386V; o. the one or more positions include position 387 of SEQ ID NO:1; 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; p. the one or more positions include position 388 of SEQ ID NO:1; Optionally, the amino acid substitution at position 388 of SEQ ID NO:1 is selected from the group consisting of M388I, M388L, and M388F; q. the one or more positions include position 389 of SEQ ID NO:1; Optionally, the amino acid substitution at position 389 of SEQ ID NO:1 is I389M; r. the one or more positions include position 391 of SEQ ID NO:1; 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; s. the one or more positions include position 392 of SEQ ID NO:1; Optionally, the amino acid substitution at position 392 of SEQ ID NO:1 is V392M; t. the one or more positions include position 404 of SEQ ID NO:1; 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; u. the one or more positions include position 407 of SEQ ID NO:1; Optionally, the amino acid substitution at position 407 of SEQ ID NO:1 is N407D; v. the one or more positions include position 409 of SEQ ID NO:1; Optionally, the amino acid substitution at position 409 of SEQ ID NO:1 is L409V; w. the one or more positions include position 413 of SEQ ID NO:1; Optionally, the amino acid substitution at position 413 of SEQ ID NO:1 is N413D; x. the one or more positions include position 414 of SEQ ID NO:1; Optionally, the amino acid substitution at position 414 of SEQ ID NO:1 is Q414E; y. the one or more positions include position 417 of SEQ ID NO:1; Optionally, the amino acid substitution at position 417 of SEQ ID NO:1 is C417S; z. the one or more positions include position 418 of SEQ ID NO:1; 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; aa. the one or more positions include position 420 of SEQ ID NO:1; 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; bb. the one or more positions include position 421 of SEQ ID NO:1; 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; cc. the one or more positions include position 422 of SEQ ID NO:1; Optionally, the amino acid substitution at position 422 of SEQ ID NO:1 is V422I; dd. the one or more positions include position 424 of SEQ ID NO:1; 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; ee. the one or more positions include position 428 of SEQ ID NO:1; 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; ff. the one or more positions include position 463 of SEQ ID NO:1; Optionally, the amino acid substitution at position 463 of SEQ ID NO:1 is S463P; gg. the one or more positions include position 517 of SEQ ID NO:1; Optionally, the amino acid substitution at position 517 of SEQ ID NO:1 is M517A; hh. the one or more positions include position 521 of SEQ ID NO:1; 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; ii. the one or more positions include position 522 of SEQ ID NO:1; Optionally, the amino acid substitution at position 522 of SEQ ID NO:1 is selected from the group consisting of M522I, M522L, and M522V; jj. the one or more positions include position 524 of SEQ ID NO:1; 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; kk. the one or more positions include position 525 of SEQ ID NO:1; 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; ll. the one or more positions include position 526 of SEQ ID NO:1; Optionally, the amino acid substitution at position 526 of SEQ ID NO:1 is Y526L; mm, wherein the one or more positions include position 527 of SEQ ID NO:1; Optionally, the amino acid substitution at position 527 of SEQ ID NO:1 is S527N; nn. the one or more positions include position 528 of SEQ ID NO:1; Optionally, the amino acid substitution at position 528 of SEQ ID NO:1 is selected from the group consisting of M528F, M528I, and M528V; oo. the one or more positions include position 533 of SEQ ID NO:1; Optionally, the amino acid substitution at position 533 of SEQ ID NO:1 is selected from the group consisting of V533F and V533W; pp. the one or more positions include position 534 of SEQ ID NO:1; Optionally, the amino acid substitution at position 534 of SEQ ID NO:1 is selected from the group consisting of V534Q and V534R; qq. the one or more positions include position 536 of SEQ ID NO:1; 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; rr. the one or more positions include position 537 of SEQ ID NO:1; Optionally, the amino acid substitution at position 537 of SEQ ID NO:1 is selected from the group consisting of Y537E and Y537S; ss. the one or more positions include position 538 of SEQ ID NO:1; Optionally, the amino acid substitution at position 538 of SEQ ID NO:1 is selected from the group consisting of D538G and D538K; tt. the one or more positions include position 539 of SEQ ID NO:1; Optionally, the amino acid substitution at position 539 of SEQ ID NO:1 is selected from the group consisting of L539A and L539R; The one or more positions include position 540 of SEQ ID NO:1; Optionally, the amino acid substitution at position 540 of SEQ ID NO:1 is selected from the group consisting of L540A and L540F; or vv. the one or more positions include position 547 of SEQ ID NO:1; Optionally, the amino acid substitution at position 547 of SEQ ID NO:1 is H547A. The modified ER-LBD of claim 2.

4. a. the one or more additional amino acid substitutions are two amino acid substitutions, optionally each of the two amino acid substitutions being at a position in SEQ ID NO: 1 selected from the group consisting of 343, 345, 347, 348, 351, 354, 384, 387, 388, 389, 391, 392, 404, 418, 421, 521, 524, and 525; Optionally, 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; Optionally, 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; Optionally, the two amino acid substitutions are at positions 421 and 392 of SEQ ID NO:1, 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; Optionally, the two amino acid substitutions are at positions 354 and 391 of SEQ ID NO:1, 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; Optionally, the two amino acid substitutions are at positions 354 and 384 of SEQ ID NO:1, 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; Optionally, the two amino acid substitutions are at positions 354 and 387 of SEQ ID NO:1, 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; Optionally, the two amino acid substitutions are at positions 387 and 391, 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; Optionally, the two amino acid substitutions are at positions 384 and 387 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 387 of SEQ ID NO:1 is L387M; Optionally, the two amino acid substitutions are at positions 384 and 391 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 391 of SEQ ID NO:1 is L391F; and / or b. the one or more additional amino acid substitutions are three amino acid substitutions, optionally each of the three amino acid substitutions being at a position in SEQ ID NO: 1 selected from the group consisting of 343, 347, 351, 354, 388, 391, 404, 414, 418, 463, 521, 524, and 525; Optionally, the three amino acid substitutions are at positions 354, 384, and 391 of SEQ ID NO:1, 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; Optionally, the three amino acid substitutions are at positions 414, 463, and 524 of SEQ ID NO:1, 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; and / or c. the one or more additional amino acid substitutions are four amino acid substitutions, optionally each of the four amino acid substitutions being at a position in SEQ ID NO: 1 selected from the group consisting of 343, 347, 351, 354, 384, 388, 391, 404, 413, 418, 463, 521, 524, and 525; Optionally, the four amino acid substitutions are at positions 354, 384, 391, and 418 of SEQ ID NO:1, 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; Optionally, the four amino acid substitutions are at positions 343, 388, 521, and 404 of SEQ ID NO:1, 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; Optionally, the four amino acid substitutions are at positions 524, 347, 351, and 525 of SEQ ID NO:1, 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; Optionally, the four amino acid substitutions are at positions 354, 384, 391, and 463 of SEQ ID NO:1, 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; Optionally, the four amino acid substitutions are at positions 384, 391, 413, and 524 of SEQ ID NO:1, 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; and / or d. the one or more additional amino acid substitutions are five amino acid substitutions, optionally each of the five amino acid substitutions at positions in SEQ ID NO: 1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, and 524; Optionally, the five amino acid substitutions are at positions 384, 409, 413, 463, and 524 of SEQ ID NO:1, 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; Optionally, the five amino acid substitutions are at positions 391, 413, 414, 463, and 524 of SEQ ID NO:1, 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; Optionally, the five amino acid substitutions are at positions 391, 414, 421, 463, and 524 of SEQ ID NO:1, 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; Optionally, the five amino acid substitutions are at positions 354, 409, 413, 421, and 524 of SEQ ID NO:1, 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; Optionally, the five amino acid substitutions are at positions 354, 409, 421, 463, and 524 of SEQ ID NO:1, 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; and / or e. the one or more additional amino acid substitutions are six amino acid substitutions, optionally each of the six amino acid substitutions at a position in SEQ ID NO: 1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, and 524; Optionally, the six amino acid substitutions are at positions 384, 391, 413, 421, 463, and 524 of SEQ ID NO:1, 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; Optionally, the six amino acid substitutions are at positions 409, 413, 414, 421, 463, and 524 of SEQ ID NO:1, 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; Optionally, the six amino acid substitutions are at positions 354, 391, 409, 413, 414, and 524 of SEQ ID NO:1, 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; and / or f. the one or more additional amino acid substitutions are seven amino acid substitutions, optionally each of the seven amino acid substitutions at a position in SEQ ID NO: 1 selected from the group consisting of 354, 384, 391, 409, 413, 414, 421, 463, 517, and 524; Optionally, the seven amino acid substitutions are at positions 354, 384, 409, 413, 421, 463, and 524 of SEQ ID NO:1, 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; Optionally, the seven amino acid substitutions are at positions 354, 391, 413, 421, 463, 517, and 524 of SEQ ID NO:1, 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; Optionally, the seven amino acid substitutions are at positions 354, 391, 413, 414, 421, 517, and 524 of SEQ ID NO:1, 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; and / or g. the one or more additional amino acid substitutions 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, 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; The modified ER-LBD of claim 1.

5. A chimeric protein comprising a polypeptide of interest fused to the modified ER-LBD of claim 1, Optionally, the polypeptide of interest comprises a nucleic acid binding domain, optionally the nucleic acid binding domain comprises a zinc finger domain, optionally the nucleic acid binding domain comprises a zinc finger domain, optionally the zinc finger domain has the sequence: MSRPGERPFQCRICMRNFSNMSNLTRHTRTHTGEKPFQCRICMRNFSDRSVLRRHLRTHTG SQKPFQCRICMRNFSDPSNLARHTRTHTGEKPFQCRICMRNFSDRSSLRRHLRTHTGS QKPFQCRICMRNFSQSGTLHRHTRTHTGEKPFQCRICMRNFSQRPNLTRHLRTHLRGS (SEQ ID NO:62) Including, Optionally, the chimeric protein comprises a chimeric transcription factor, and the polypeptide of interest comprises a nucleic acid binding domain and a transcription regulator domain, and optionally the transcription regulator domain is a transcription activator, and optionally the transcription activator is selected from the group consisting of: 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 domain (VPH activation domain); and the histone acetyltransferase core domain of human E1A-associated protein p300 (p300 HAT core activation domain); or Optionally, the transcription regulator domain comprises: DEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPMLMEYPEAITRLVTGAQRPPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISS (SEQ ID NO: 64) A p65 transcription activator comprising the amino acid sequence Chimeric proteins.

6. 2. An isolated polynucleotide molecule comprising a nucleotide sequence encoding the modified ER-LBD of claim 1.

7. A heterologous construct comprising a promoter operably linked to the polynucleotide molecule of claim 6.

8. A plasmid comprising the heterologous construct of claim 7.

9. A cell comprising the heterologous construct of claim 7.

10. 1. A gene switch for regulating transcription of a gene of interest, comprising: (a) the chimeric protein of claim 5, which binds to a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to the gene of interest; (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. Including, gene switches.

11. a. the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen; and / or 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 coactivator molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme; and / or c. the gene of interest encodes a cytokine selected from the group consisting of IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha; and / or d. The gene of interest is: MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEV LGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYS GRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEES LPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFC VQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGSGGGSGGGSGG GSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLE LTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS (SEQ ID NO:58) encoding an IL12p70 fusion protein comprising the amino acid sequence The gene switch of claim 10.

12. 1. A method for modulating transcription of a gene of interest, comprising: Transforming a cell with (i) a heterologous construct encoding the chimeric protein of claim 5 and (ii) a target expression cassette comprising a chimeric transcription factor-responsive (CTF-responsive) promoter operably linked to the gene of interest; contacting the transformed cell with a non-endogenous ligand, thereby inducing the chimeric protein to regulate transcription of the gene of interest; Including, Optionally, the method further comprises culturing the transformed cell under conditions suitable for expression of the chimeric protein prior to inducing the chimeric protein to regulate transcription; Optionally, regulating transcription comprises activating transcription of the gene of interest; Optionally, the target expression cassette is encoded by the heterologous construct encoding the chimeric protein of claim 5, or the target expression cassette is encoded by a second heterologous construct, Optionally, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. method.

13. 1. A method for modulating localization of a chimeric protein, comprising: Transforming a cell with a heterologous construct encoding the chimeric protein of claim 5; contacting the transformed cell with a non-endogenous ligand to induce nuclear localization of the chimeric protein; Including, Optionally, the method further comprises culturing the transformed cells under conditions suitable for expression of the chimeric protein prior to inducing the nuclear localization; Optionally, the non-endogenous ligand is selected from the group consisting of 4-hydroxytamoxifen, N-desmethyltamoxifen, tamoxifen-N-oxide, and endoxifen. method.

14. the transformed cell is in a human or animal; contacting the transformed cell with the non-endogenous ligand comprises administering a pharmacological dose of the ligand to the human or animal. The method of claim 12.

15. 15. The method of claim 14, wherein the non-endogenous ligand is administered at a concentration such that the non-endogenous ligand is substantially inactive on the wild-type estrogen receptor alpha of SEQ ID NO:1.

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