Modified degron polypeptides and uses thereof

Abstract

Provided herein are modified degrons with increased sensitivity to one or more immunomodulatory drugs (IMiDs). Also provided herein are modified cereblons with increased sensitivity to one or more IMiDs. Also provided herein are modified degrons exhibiting reduced degradation upon binding to one or more IMiDs. Also provided are inducibly degradable proteins, inducible cell death systems, and activation-conditional control polypeptides employing the modified degrons, modified cereblons, and methods and / or uses thereof.

Description

MODIFIED DEGRON POLYPEPTIDES AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 464,394, filed May 5, 2023, U.S. Provisional Patent Application No. 63 / 511,443, filed June 30, 2023, U.S. Provisional Patent Application No. 63 / 604,780, filed November 30, 2023, and U.S. Provisional Patent Application No. 63 / 623,722, filed January 22, 2024, the disclosures of each of which are hereby incorporated by reference in their entireties for all purposes.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted via Patent Center and is hereby incorporated by reference in its entirety. Said XML file, created on Month XX, 20XX, is named XXXXXUS_sequencelisting.xml, and is X, XXX, XXX bytes in size.BACKGROUND

[0003] Currently available cell and gene therapy products can lack control, which can lead to safety concerns such as toxicity in subjects that receive the therapies. Thus, additional methods of controlling and regulating these therapies are needed.SUMMARY

[0004] The present disclosure provides for, among other things, modified degrons and cerebelons that have increased sensitivity to one or more immunomodulatory drugs (IMiDs).

[0005] In some aspects, the present disclosure provides for a modified degron comprising the amino acid sequence set forth in SEQ ID NO: 139, or the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X10, wherein Xi is I or R; X2is F or W; X3is C or T; X4is R, D, H, or K; X5is Q, R, or T; X6is G or V; X7is N, T, or A; X8is L or I; X9is K or G; and X10 is L or F, and wherein the modified degron has increased sensitivity to one or more IMiDs compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131, optionally wherein the modified degron comprises one or more amino acid substitutions at any one or more positions selected from G30E, Q28T, L33I, R27H, K37G, L38F, I21R AND G30V, R27D, Q28R, N31T, F24W, R27K, I21R, G30V, and N31A, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the IMiD is at least one of thalidomide, iberdomide, lenalidomide, and pomalidomide, optionally wherein the IMiD is pomalidomide.

[0006] In some embodiments, the modified degron comprises an amino acid sequence set forth in any one of SEQ ID NOs: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 226-230, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332,336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, or 788. In some embodiments, the modified degron comprises SEQ ID NO: 148. In some embodiments, the modified degron functionally associates with a cereblon domain or variant thereof in an IMiD responsive manner, optionally wherein the modified degron functionally associates with the cereblon domain or variant thereof when exposed to a lower concentration of IMiD, as compared to an unmodified degron. In some embodiments, the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129. In some embodiments, the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of IMiD, as compared to an unmodified cereblon. In some embodiments, the cereblon domain or variant thereof is a modified cereblon as described in herein.

[0007] In some aspects, the present disclosure also provides for a modified cereblon (CRBN) comprising: (a) one or more amino acid substitutions at positions Q325, V350, H353, Y355, N369, 1371, R373, E377, S379, Q390, A395, S396, H397, or F402 relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to the wildtype CRBN; (b) the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10X11X 12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATKKDMSPQKF WX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is T, F, I, L, V, W, or Y; X9is N, D, or Y; X10 is I, D, E, N, or Q; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is X, D, K, N, Q, or R; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y ; and X25 is G or A, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127 or a truncation thereof comprising the amino acid sequence set forth in SEQ ID NO: 129; (c) the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10X11X12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATKKDMSPQKF WX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is T, F, I, L, V, W, or Y; X9is N, D, or Y; X10 is I, D, E, N, Q, L, V, or M; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is S, D, K, N, Q, R, H, P; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F,H, N, Q, W, or Y; and X25 is G,A, F, I, L, S, or V; (d) one or more amino acid substitutions at any one or more positions selected from: K324, Q325, V350, N351, H353, Y355, H357, T359, N369, 1371, G372, R373, S375, E377, H378, S379, W380, F381, V388, Q390, A395, S396, H397, F402, and G416, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to the wildtype CRBN; (e) the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10X11X 12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATKKDMSPQKF WX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A,I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is V; X9is N, D, or Y; X10 is I, D, E, N, or Q; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is X, D, K, N, Q, or R; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xis is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y; and X25is G or A; or (f) a T359 amino acid substitution. In some embodiments, the T359 amino acid substitution is selected from: a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, optionally wherein the modified CRBN comprises a T359V substitution.

[0008] . In some embodiments, the modified CRBN does not comprise a DDB 1 interacting domain, optionally wherein the modified CRBN further comprises deletion of amino acids 194-247 relative to the wildtype CRBN, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 231 - 249. 256, 258, 260, 262, or 264. In some embodiments, the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, (a) a Q325 amino acid substitution selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution; and one or more amino acid substitutions selected from: a 137 IN substitution, aH397F substitution, and a N369Y substitution; or (b) a Q325H substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 351; or (c) a Q325H substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 355; or (d) a Q325H substitution and a N369Y substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 359; or(e) a Q325H substitution, a N369Y substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 363; or(f) a Q325H substitution, a N369Y substitution, and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 367; or(g) a Q325H substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 371; or (h) a Q325K substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 375; or(i) a Q325K substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 383; or(j) a Q325R substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 403; or (k) a 137 IN substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 331; or (1) a N369Y substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 335; or (m) a N369Y substitution and a I371N substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 339; or (n) a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 343; or (o) a Q325H substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 347; or (p) a Q325K substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 379; or (q) a Q325K substitution and a N369Y substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 387; or(r) a Q325K substitution, a N369Y substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 391; or(s) a Q325K substitution, a N369Y substitution, and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 395; or(t) a Q325K substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 399; or (u) a Q325R substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315; or (v) one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitution, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution; (w) a Q325H substitution, a Q325H substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ IDNO: 544; or (x) a Q325R substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 530; or (y) a Q325R substitution, a N369Y substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 526; or (z) a Q325R substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 524; or (aa) a Q325K substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 532; or (bb) a Q325H substitution, a N369Y substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 528; or (cc) a Q325H substitution, a N369Y substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 534; or (dd) a Q325H substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 522; or (ee) a Q325K substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 542, or (ff) a Q325 amino acid substitution selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution; and (ii) one or more amino acid substitutions selected from: a T359V substitution, a 137 IN substitution, a H397F substitution, and a N369Y substitution.

[0009] In some embodiments, the modified CRBN comprises an amino acid sequence set forth in any one of SEQ ID NOs: 231-249, 256, 258, 260, 262, 264, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363,367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 426, 428, 430,432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, or 544. In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 544, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 530, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 526, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 524, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 532, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 528, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 534, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 522, optionally wherein the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 542.

[0010] The present disclosure also provides an activation-conditional control polypeptide (ACP) complex, comprising: a first polypeptide comprising the modified degron provided herein, and a second polypeptide comprising a ligand binding domain, wherein (i) the first polypeptide further comprises a DNA-binding domain and the second polypeptide further comprises a transcriptional effector domain, or (ii) the first polypeptide further comprises a transcriptional effector domain and the second polypeptide further comprises a DNA-binding domain, and wherein the first and second polypeptides dimerize with each other upon binding of a ligand to the ligand binding domain, thereby forming the ACP complex, and wherein the ACP complex is capable of modulating transcriptional expression of a gene of interest operably linked to an ACP complex-responsive promoter, optionally wherein the DNA- binding domain comprises a DNA zinc finger protein domain, optionally wherein the DNA zinc finger protein domain comprises an amino acid sequence of SEQ ID NO: 57. In some embodiments, the ligand is an IMiD, optionally wherein the IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide, optionally wherein the IMiD is pomalidomide.

[0011] In some embodiments, the transcriptional effector domain is a transcriptional activation domain selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus Rtransactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain); or a transcriptional repressor domain selected from the group consisting of: a Kriippel associated box (KRAB) repression domain; a truncated Kriippel associated box (KRAB) repression domain; a Histone Deacetylase 4 (HDAC4) repressor domain; a Scleraxis (SCX) HLH domain, an Inhibitor of DNA binding 1 (ID1) HLH domain, a HECT domain and RCCl-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a Twist-related protein 1 (TWST1) HLH domain, an Homeobox protein Nkx-2.2 (NKX22) homeodomain, an Inhibitor of DNA binding 1 (ID3) HLH domain, and a Twist-related protein 2 (TWST2) HLH domain, and EED repressor domain; a Repressor Element Silencing Transcription Eactor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

[0012] In some embodiments, the ligand-binding domain is or comprises a component of an E3 ubiquitin ligase complex, a cereblon domain, or a variant thereof. In some embodiments, the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129. In some embodiments, the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of ligand, as compared to an unmodified cereblon. In some embodiments, the cereblon domain or variant thereof is a modified cereblon as described in any one of claims 113-148, 177-187, or 226.

[0013] In some embodiments, the modified degron and the DNA-binding domain or the transcriptional effector domain are linked by a first linker. In some embodiments, the ligandbinding domain and the transcriptional effector domain or the DNA-binding domain are linked by a second linker, optionally wherein the first linker and / or the second linker comprises the amino acid sequence set forth in SEQ ID NO: 169, 171, 173, 265, or 269, 168, 175, or 199. In some embodiments, the first linker and / or the second linker is a glycine- serine linker. In some embodiments, the glycine- serine linker comprises one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, the glycine-serine linker comprises one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). In some embodiments, thelinker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193. In some embodiments, the linker does not have lysine residues. In some embodiments, the first linker and / or the second linker comprises the amino acid sequence set forth in SEQ ID NO: 265 or 269.

[0014] The present disclosure also provides an inducible cell death system comprising: a first polypeptide comprising the modified degron provided herein, operably linked to a first cell-death inducing domain and a second polypeptide comprising a ligand-binding domain operably linked to a second cell death-inducing domain, or a first polypeptide comprising the modified CRBN provided herein, operably linked to a first cell-death inducing domain and a second polypeptide comprising a ligand-binding domain operably linked to a second cell death-inducing domain, wherein the first polypeptide and the second polypeptides are configured to form a complex with each other upon binding of a ligand to the ligand binding domain. In some embodiments, the first and second cell-death inducing domains are identical. In some embodiments, the ligand is an ImiD. In some embodiments, wherein the ImiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide.

[0015] In some embodiments, the first and second cell death inducing domains are derived from a protein selected from the group consisting of: a caspase (e.g., any one of caspases 1-11, such as caspase-3, caspase-6, caspase-7, caspase-8, caspase-9), Diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2-interacting protein 3 (BNIP3), Fas, Fas-associated protein with death domain (FADD), tumor necrosis factor receptor type I-associated death domain protein (TRADD), a TNF receptor (TNF-R), APAF- 1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53-upregulated modulator of apoptosis (PUMA), Noxa, Blk, Hrk, Cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIE), Herpes Simplex Virus thymidine kinase (HSV-TK), Varicella Zoster Virus thymidine kinase (VZV-TK), viral Spike protein, Carboxyl esterase, cytosine deaminase, nitroreductase Fksb, Carboxypeptidase G2, Carboxypeptidase A, Horseradish peroxidase, Einamarase, Hepatic cytochrome P450-2B1, Purine nucleoside phosphorylase, and variants and functional fragments thereof, optionally wherein the first and second cell death inducing domains are derived from caspase-9, optionally wherein the caspase-9 does not comprise a Caspase Activation and Recruitment Domain (CARD) domain sequence.

[0016] In some embodiments, the ligand-binding domain is or comprises a component of an E3 ubiquitin ligase complex, a cereblon domain, or a variant thereof. In someembodiments, the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129. In some embodiments, the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of ligand, as compared to an unmodified cereblon. In some embodiments, the cereblon domain or variant thereof is a modified cereblon as described in any one of claims 5-8. In some embodiments, the ligand-binding domain is or comprises a modified degron, optionally wherein the modified degron or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129. In some embodiments, the modified degron or functionally associates with the modified cereblon when exposed to a lower concentration of ligand, as compared to an unmodified cereblon. In some embodiments, the modified degron or variant thereof is a modified degron as described herein.

[0017] In some embodiments, (i) the modified degron is operably linked to the first celldeath inducing domain by a first linker, (ii) the ligand-binding domain is operably linked to the second cell death-inducing domain by a second linker, or (iii) the modified degron is operably linked to the first cell-death inducing domain by the first linker and the ligandbinding domain operably linked to the second cell death-inducing domain by the second linker. In some embodiments, the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitution, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution. In some embodiments, the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution. In some embodiments, the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution. In some embodiments, the modified degron comprises one or more amino acid substitutions selected from: a Q28T amino acid substitution, a G30E amino acid substitution, and a L33I amino acid substitution, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40, optionally wherein the modified CRBN comprises an 137 IN amino acid substitution, and wherein the modified degron comprises a G30E amino acid substitution. In some embodiments, the modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution. In some embodiments, the modified degron comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution, and the modified degron comprises a Q28T, G30E, and L33I amino acid substitution.

[0018] The present disclosure also provides a cell comprising the inducible cell death system, the modified degron, the modified CRBN, the ACP complex provided herein. In some embodiments, the cell is a human cell. In some embodiments, the cell is a cell therapy modality. In some embodiments, the cell therapy modality is a CAR-T cell or CAR-NK cell.

[0019] The present disclosure also provides a method of inducing cell death, the method comprising expressing the inducible cell death system provided herein in a cell and exposing the cell to the ligand. In some embodiments, the ligand is an IMiD. In some embodiments, the IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, the IMiD is pomalidomide.

[0020] The present disclosure also probides a method of modulating target gene expression, the method comprising expressing the ACP complex provided herein in a cell and exposing the cell to the ligand. In some embodiments, the ligand is an IMiD. In some embodiments, the IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide.

[0021] The present disclosure also provides one or more nucleic acid sequences encoding: the modified degron; the modified CRBN; the inducible cell death system; or the ACP complex provided herein.

[0022] The present disclosure also provides a vector comprising the nucleic acid sequence provided herein. In some embodiments, the vector comprises a promoter. In some embodiments, the promoter is a functional in a mammalian cell.

[0023] The present disclsoure also provides polypeptide complex comprising a first polypeptide comprising a degron and a second polypeptide comprising a cereblon, wherein: (i) the degron is a modified degron provided herein; (ii) the cereblon is a modified cereblon provided herein; or (iii) the degron is a modified degron provided herein, and the cereblon is a modified cereblon provided herein.

[0024] The present disclosure also provides for a modified degron with increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to a reference degron (e.g., an unmodified degron). The present disclosure also provides for, in some embodiments, a modified degron comprising the amino acid sequence set forth in SEQ ID NO: 139, wherein the modified degron has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131.

[0025] The present disclosure further provides for a modified degron comprising the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X10, wherein Xi is I or R; X2 is F orW; X3is C or T; X4is R, D, H, or K; X5is Q, R, or T; X6is G or V; X7is N, T, or A; X8is L or I; X9 is K or G; and X10 is L or F, and wherein the modified degron has increased sensitivity to one or more IMiDs compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131.

[0026] The present disclosure also provides for a modified degron comprising one or more amino acid substitutions at any one or more positions selected from G30E, Q28T, L33I, R27H, K37G, L38F, I21R AND G30V, R27D, Q28R, N31T, F24W, R27K, I21R, G30V, and N31A, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0027] In some embodiments, a modified degron comprises an amino acid sequence set forth in any one of SEQ ID NOs: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 226-230, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, or 424.

[0028] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 140.

[0029] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 142.

[0030] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 144.

[0031] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 146.

[0032] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 148.

[0033] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 150.

[0034] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 152.

[0035] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 154.

[0036] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 156.

[0037] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 158.

[0038] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 160.

[0039] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 162.

[0040] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 164.

[0041] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 166.

[0042] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 226.

[0043] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 227.

[0044] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 228.

[0045] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 229.

[0046] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 230.

[0047] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 276.

[0048] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 280.

[0049] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 284.

[0050] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 288.

[0051] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 292.

[0052] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 296.

[0053] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 300.

[0054] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 304.

[0055] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 308.

[0056] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 312.

[0057] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 316.

[0058] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 320.

[0059] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 324.

[0060] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 328.

[0061] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 332.

[0062] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 336.

[0063] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 340.

[0064] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 344.

[0065] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 348.

[0066] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 352.

[0067] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 356.

[0068] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 360.

[0069] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 364.

[0070] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 368.

[0071] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 372.

[0072] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 376.

[0073] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 380.

[0074] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 384.

[0075] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 388.

[0076] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 392.

[0077] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 396.

[0078] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 400.

[0079] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 404.

[0080] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 408.

[0081] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 412.

[0082] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 416.

[0083] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 420.

[0084] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 424.

[0085] In some embodiments, a modified degron comprises the amino acid sequence set forth in SEQ ID NO: 788.

[0086] In some embodiments, a modified degron functionally associates with a cereblon domain or variant thereof in an IMiD responsive manner. In some embodiments, a modified degron functionally associates with a cereblon domain or variant thereof when exposed to a lower concentration of IMiD, as compared to an unmodified degron. In some embodiments, a cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127. In some embodiments, a cereblon domain or variant thereof comprises an aminoacid sequence as set forth in SEQ ID NO: 129. In some embodiments, a cereblon domain or variant thereof is a modified cereblon that functionally associates with a modified degron when exposed to a lower concentration of IMiD, as compared to an unmodified cereblon. In some embodiments, a cereblon domain or variant thereof is a modified cereblon as described herein. In some embodiments, a cereblon domain or variant thereof is an E3 ubiquitin ligase or a component thereof, and wherein functional association induces degradation of a modified degron, a protein comprising a modified degron, or a polypeptide comprising a modified degron in an IMiD responsive manner.

[0087] In some embodiments, increased sensitivity is or comprises increased degradation of a protein comprising a modified degron and / or a polypeptide comprising a modified degron.

[0088] In some embodiments, an IMiD is at least one of thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide. In some embodiments, an IMiD is iberdomide. In some embodiments, an IMiD is lenalidomide. In some embodiments, an IMiD is thalidomide.

[0089] In some embodiments, the mdoifed degron has increased sensitivity to the one or more IMiDs compared to a degron comprising the amino acid sequence of SEQ ID NO: 131.

[0090] The present disclosure provides for an inducibly degradable protein comprising a target polypeptide operably linked to a modified degron as described herein.

[0091] In some embodiments, a target polypeptide is directly fused to a modified degron or joined by a linker.

[0092] In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 168, 175, or 199. In some embodiments, a linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 169, 171, or 173. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 171. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 199.

[0093] In some embodiments, a linker is a glycine-serine linker. In some embodiments, a linker comprises one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, a linker comprises one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). Insome embodiments, a linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193. In some embodiments, a linker does not have lysine residues.

[0094] The present disclosure provides for a cell comprising a modified degron as described herein, or an inducibly degradable protein as described herein.

[0095] The present disclosure also provides for a method of manufacturing an inducibly degradable protein, the method comprising operably linking a modified degron as described herein to a target polypeptide.

[0096] In some embodiments, a target polypeptide is operably linked to a modified degron by expression of an inducibly degradable protein from a vector comprising a nucleic acid sequence encoding the target polypeptide and modified degron in a single open reading frame.

[0097] In some embodiments, a target polypeptide is directly fused to a modified degron or joined by a linker. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 168, 175, or 199. In some embodiments, a linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 169, 171, or 173. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 171. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a linker comprises the amino acid sequence set forth in SEQ ID NO: 199.

[0098] In some embodiments, a linker is a glycine-serine linker. In some embodiments, a linker comprises one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, a linker comprises one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). In some embodiments, a linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193. In some embodiments, a linker does not have lysine residues.

[0099] In some embodiments, an inducibly degradable protein is degraded upon expression within a mammalian cell and exposure of a cell to an IMiD.

[0100] In some embodiments, an IMiD is at least one of thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide. In someembodiments, an IMiD is lenalidomide. In some embodiments, an IMiD is iberdomide. In some embodiments, an IMiD is thalidomide.

[0101] The present disclosure further provides for a method of promoting target polypeptide degradation, the method comprising expressing an inducibly degradable protein as described herein in a mammalian cell and exposing the cell to an IMiD.

[0102] In some embodiments, an IMiD is at least one of thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide. In some embodiments, an IMiD is iberdomide. In some embodiments, an IMiD is lenalidomide. In some embodiments, an IMiD is thalidomide.

[0103] In some embodiments, a mammalian cell is a human cell.

[0104] In some embodiments, a mammalian cell is a cell therapy modality. In some embodiments, a cell therapy modality is a chimeric antigen receptor (CAR)-T cell or CAR- NK cell.

[0105] The present disclosure provides for an inducible cell death system comprising: a first polypeptide comprising a modified degron as described herein operably linked to a first cell-death inducing domain; a second polypeptide comprising a ligand-binding domain operably linked to a second cell death-inducing domain, wherein the first polypeptide and the second polypeptides are configured to form a complex with each other upon binding of a ligand to the ligand binding domain.

[0106] In some embodiments, first and second cell-death inducing domains are identical.

[0107] In some embodiments, first and second cell death inducing domains are derived from a protein selected from the group consisting of: a caspase (e.g., any one of caspases 1- 11, such as caspase-3, caspase-6, caspase-7, caspase-8, caspase-9), Diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2-interacting protein 3 (BNIP3), Fas, Fas Associated protein with death domain (FADD), tumor necrosis factor receptor type I- associated death domain protein (TRADD), a TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53- upregulated modulator of apoptosis (PUMA), Noxa, Blk, Hrk, Cytochrome c, Arts, TNF- related cell death-inducing ligand (TRAIL), Herpes Simplex Virus thymidine kinase (HSV- TK), Varicella Zoster Virus thymidine kinase (VZV-TK), viral Spike protein, Carboxyl esterase, cytosine deaminase, nitroreductase Fksb, Carboxypeptidase G2, Carboxypeptidase A, Horseradish peroxidase, Linamarase, Hepatic cytochrome P450-2B1, Purine nucleoside phosphorylase, and variants and functional fragments thereof.

[0108] In some embodiments, first and second cell death inducing domains are derived from caspase-9, optionally wherein the caspase-9 does not comprise a Caspase Activation and Recruitment Domain (CARD) domain sequence.

[0109] In some embodiments, a ligand-binding domain is a component of an E3 ubiquitin ligase complex.

[0110] In some embodiments, a ligand-binding domain is or comprises a cereblon domain or a variant thereof. In some embodiments, a cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127. In some embodiments, a cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 129. In some embodiments, a cereblon domain or variant thereof is a modified cereblon that functionally associates with a modified degron when exposed to a lower concentration of ligand, as compared to an unmodified cereblon. In some embodiments, a cereblon domain or variant thereof is a modified cereblon as described herein. In some embodiments, a modified cereblon (CRBN) comprises, relative to relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitition, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, and wherein the modified degron comprises one or more amino acid substitutions selected from: a Q28T amino acid substitution, a G30E amino acid substitution, and a L33I amino acid substitution, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, modified CRBN comprises an 137 IN amino acid substitution, and wherein a modified degron comprises a G30E amino acid substitution. In some embodiments, a modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution. In some embodiments, a modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution, and wherein the modified degron comprises a Q28T, G30E, and L33I amion acid substitution.

[0111] In some embodiments, a ligand is an IMiD.

[0112] In some embodiments, an IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiDis pomalidomide. In some embodiments, an IMiD is lenalidomide. In some embodiments, an IMiD is iberdomide. In some embodiments, an IMiD is thalidomide.

[0113] The present disclosure, in some embodiments, provides for an inducible cell death system as described herein, wherein (i) a modified degron is operably linked to the first celldeath inducing domain by a first linker, (ii) a ligand-binding domain is operably linked to the second cell death-inducing domain by a second linker, or (iii) a modified degron is operably linked to a first cell-death inducing domain by a first linker and a ligand-binding domain operably linked to a second cell death-inducing domain by a second linker.

[0114] In some embodiments, a first linker and / or a second linker is a glycine- serine linker. In some embodiments, a first linker and / or a second linker comprises one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, a first linker and / or a second linker comprises one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). In some embodiments, a first linker and / or a second linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193. In some embodiments, a first linker and / or a second linker does not have lysine residues.

[0115] The present disclosure provides for a cell comprising an inducible cell death system as described herein.

[0116] In some embodiments, a cell is a human cell.

[0117] In some embodiments, a cell is a cell therapy modality. In some embodiments, a cell therapy modality is a CAR-T cell or CAR-NK cell.

[0118] The present disclosure also provides for a method of inducing cell death, the method comprising expressing an inducible cell death system as described herein in a cell and exposing the cell to a ligand.

[0119] In some embodiments, a ligand is an IMiD.

[0120] In some embodiments, an IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide. In some embodiments, an IMiD is lenalidomide. In some embodiments, an IMiD is iberdomide. In some embodiments, an IMiD is thalidomide.

[0121] The present disclosure provides for an activation-conditional control polypeptide (ACP) complex, comprising a first polypeptide comprising a modified degron as described herein, and a second polypeptide comprising a ligand binding domain, wherein (i) the first polypeptide further comprises a DNA-binding domain and the second polypeptide further comprises a transcriptional effector domain, or (ii) the first polypeptide further comprises atranscriptional effector domain and the second polypeptide further comprises a DNA-binding domain, and wherein the first and second polypeptides dimerize with each other upon binding of a ligand to the ligand binding domain, thereby forming the ACP complex, and wherein the ACP complex is capable of modulating transcriptional expression of a gene of interest operably linked to an ACP complex-responsive promoter.

[0122] In some embodiments, the DNA-binding domain comprises a DNA zinc finger protein domain. In some embodiments, the DNA-binding domain comprises a ZF10-1 domain. In some embodiments, the DNA zinc finger protein domain comprises an amino acid sequence as set forth in SEQ ID NO: 57.

[0123] In some embodiments, a transcriptional effector domain is a transcriptional activation domain selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain), and variants and functional fragments thereof.

[0124] In some embodiments, a transcriptional effector domain is a transcriptional repressor domain selected from the group consisting of: a Kriippel associated box (KRAB) repression domain; a truncated Kriippel associated box (KRAB) repression domain; a Histone Deacetylase 4 (HDAC4) repressor domain; a Scleraxis (SCX) HLH domain, an Inhibitor of DNA binding 1 (ID1) HLH domain, a HECT domain and RCCl-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a Twist-related protein 1 (TWST1) HLH domain, an Homeobox protein Nkx-2.2 (NKX22) homeodomain, an Inhibitor of DNA binding 1 (ID3) HLH domain, and a Twist-related protein 2 (TWST2) HLH domain, and EED repressor domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

[0125] In some embodiments, a ligand-binding domain is a component of an E3 ubiquitin ligase complex.

[0126] In some embodiments, a ligand-binding domain is or comprises a cereblon domain or a variant thereof. In some embodiments, a cereblon domain or variant thereof comprises anamino acid sequence as set forth in SEQ ID NO: 127. In some embodiments, a cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 129. In some embodiments, a cereblon domain or variant thereof is a modified cereblon that functionally associates with a modified degron when exposed to a lower concentration of ligand, as compared to an unmodified cereblon. In some embodiments, a cereblon domain or variant thereof is a modified cereblon as described herein. In some embodiments, a modified cereblon (CRBN) comprises, relative to relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitition, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, and wherein the modified degron comprises one or more amino acid substitutions selected from: a Q28T amino acid substitution, a G30E amino acid substitution, and a L33I amino acid substitution, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, modified CRBN comprises an 137 IN amino acid substitution, and wherein a modified degron comprises a G30E amino acid substitution. In some embodiments, a modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution. In some embodiments, a modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution, and wherein the modified degron comprises a Q28T, G30E, and L33I amino acid substitution.

[0127] In some embodiments, a modified degron and a DNA-binding domain or a transcriptional effector domain are linked by a first linker.

[0128] In some embodiments, a ligand-binding domain and a transcriptional effector domain or a DNA-binding domain are linked by a second linker.

[0129] In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 168, 175, or 199. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in any one of SEQ ID NOs: 169, 171, 173, 265, or 269. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in SEQ ID NO: 169. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in SEQ ID NO: 171. In some embodiments, a first linker and / or a second linkercomprises the amino acid sequence set forth in SEQ ID NO: 173. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in SEQ ID NO: 175. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in SEQ ID NO: 199. In some embodimetns, a first linker and / or a second linker comprises the amino acid sequence set forth in SEQ ID NO: 265. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence set forth in SEQ ID NO: 269.

[0130] In some embodiments, a first linker and / or a second linker is a glycine- serine linker. In some embodiments, a first linker and / or a second linker comprises one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, a first linker and / or a second linker comprises one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a first linker and / or a second linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). In some embodiments, a first linker and / or a second linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193. In some embodiments, a first linker and / or a second linker does not have lysine residues.

[0131] In some embodiments, a ligand is an IMiD.

[0132] In some embodiments, an ImiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an ImiD is pomalidomide. In some embodiments, an ImiD is lenalidomide. In some embodiments, an ImiD is iberdomide. In some embodiments, an ImiD is thalidomide.

[0133] The present discloure provides for cells comprising an ACP complex described herein.

[0134] In some embodiments, a cell is a human cell.

[0135] In some embodiments, a cell is a cell therapy modality. In some embodiments, a cell therapy modality is a CAR-T cell or CAR-NK cell.

[0136] The present disclosure provides for a method of modulating target gene expression, the method comprising expressing an ACP complex as described herein in a cell and exposing the cell to a ligand.

[0137] In some embodiments, a the ligand is an IMiD.

[0138] In some embodiments, an IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide. In some embodiments, an IMiD is lenalidomide. In some embodiments, an IMiD is iberdomide. In some embodiments, an IMiD is thalidomide.

[0139] The present disclosure provides for one or more nucleic acid sequences encoding: a modified degron as described herein; an inducibly degradable protein as described herein; an inducible cell death system as described herein; or an ACP complex as described herein. The present disclosure provides for vectors comprising a nucleic acid as described herein and, optionally, a promoter, preferably a promoter functional in a mammalian cell.

[0140] The present disclosure provides for a modified CRBN comprising one or more amino acid substitutions at positions Q325, V350, H353, Y355, N369, 1371, R373, E377, S379, Q390, A395, S396, H397, or F402 relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to the wildtype CRBN.

[0141] The present disclosure provides for a modified CRBN comprising the amino acid sequenceX1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10X11X 12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATKKDMSPQKF WX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is T, F, I, L, V, W, or Y; X9is N, D, or Y; X10 is I, D, E, N, or Q; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is X, D, K, N, Q, or R; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y ; and X25 is G or A, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127 or a truncation thereof comprising the amino acid sequence set forth in SEQ ID NO: 129.

[0142] The present disclosure provides for a modified cereblon (CRBN) comprising the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10X11X 12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATKKDMSPQKF WX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is T, F, I, L, V, W, or Y; X9is N, D, or Y; X10 is I, D, E, N, Q, L, V, or M; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is S, D, K, N, Q, R, H, P; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K,or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y; and X25is G,A, F, I, L, S, or V.

[0143] The present disclosure provides for a modified cereblon (CRBN) comprising one or more amino acid substitutions at any one or more positions selected from: K324, Q325, V350, N351, H353, Y355, H357, T359, N369, 1371, G372, R373, S375, E377, H378, S379, W380, F381, V388, Q390, A395, S396, H397, F402, and G416, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to the wildtype CRBN.

[0144] In some embodiments, a modified CRBN does not comprise a DDB1 interacting domain.

[0145] In some embodiments, a modification further comprises deletion of amino acids 194-247 relative to the wildtype CRBN.

[0146] In some embodiments, the modified CRBN comprises an amino acid sequence set forth in any one of SEQ ID NOs: 231-249, 256, 258, 260, 262, 264, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363,367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 426, 428, 430,432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466,468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502,504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538,540, 542, or 544.

[0147] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 231.

[0148] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 232.

[0149] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 233.

[0150] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 234.

[0151] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 235.

[0152] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 236.

[0153] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 237.

[0154] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 238.

[0155] In some embodiments a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 239.

[0156] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 240.

[0157] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 241.

[0158] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 242.

[0159] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 243.

[0160] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 244.

[0161] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 245.

[0162] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 246.

[0163] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 247.

[0164] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 248.

[0165] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 249.

[0166] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 256.

[0167] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 258.

[0168] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 260.

[0169] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 262.

[0170] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 264.

[0171] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 275.

[0172] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 279.

[0173] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 283.

[0174] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 287.

[0175] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 291.

[0176] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 295.

[0177] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 299.

[0178] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 303.

[0179] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 307.

[0180] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 311.

[0181] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 315.

[0182] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 319.

[0183] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 323.

[0184] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 327.

[0185] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 331.

[0186] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 335. 1

[0187] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 339.

[0188] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 343.

[0189] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 347.

[0190] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 351.

[0191] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 355.

[0192] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 359.

[0193] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 363.

[0194] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 367.

[0195] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 371.

[0196] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 375.

[0197] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 379.

[0198] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 383.

[0199] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 387.

[0200] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 391.

[0201] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 395.

[0202] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 399.

[0203] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 403.

[0204] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 407.

[0205] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 411.

[0206] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 415.

[0207] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 419.

[0208] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 423.

[0209] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 426.

[0210] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 428.

[0211] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 430.

[0212] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 432.

[0213] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 434.

[0214] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 436.

[0215] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 438.

[0216] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 440.

[0217] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 442.

[0218] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 444.

[0219] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 446.

[0220] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 448.

[0221] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 450.

[0222] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 452.

[0223] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 454.

[0224] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 456.

[0225] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 458.

[0226] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 460.

[0227] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 462.

[0228] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 464.

[0229] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 466.

[0230] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 468.

[0231] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 470.

[0232] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 472.

[0233] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 474.

[0234] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 476.

[0235] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 478.

[0236] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 480.

[0237] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 482.

[0238] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 484.

[0239] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 486.

[0240] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 488.

[0241] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 490.

[0242] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 492.

[0243] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 494.

[0244] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 496.

[0245] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 498.

[0246] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 500.

[0247] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 502.

[0248] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 504.

[0249] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 506.

[0250] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 508.

[0251] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 510.

[0252] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 512.

[0253] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 514.

[0254] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 516.

[0255] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 518.

[0256] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 520.

[0257] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 536.

[0258] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 538.

[0259] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 540.

[0260] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 542.

[0261] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 544.

[0262] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 530.

[0263] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 526.

[0264] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 524.

[0265] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 532.

[0266] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 528.

[0267] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 534.

[0268] In some embodiments, the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 522.

[0269] In some embodiments, one or more amino acid substitutions in a modified CRBN comprises a T359 amino acid substitution, optionally wherein the T359 amino acid substitution is selected from: a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, optionally wherein the modified CRBN comprises a T359V substitution.

[0270] The present disclosure provides for a modified cereblon (CRBN) comprising the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10 X 1 IX 12PX 13TX 14X 15X 16X 17X 18PGYAWTX 19 AX20CKICX21X22X23IGWKX24TAT KKDMSPQKFWX25, wherein XI is K, N, Q, or R; X2 is Q, H, K, or R; X3 is V, E, H, Q, R, or S; X4 is N, A, I, L, or V; X5 is H, P, Q, R, or S; X6 is Y, H, N, R, or S; X7 is H, Q, or W; X8 is V; X9 is N, D, or Y; X10 is I, D, E, N, or Q; XI 1 is G, N, or Q; X12 is R, E, N, S, or Y; X13 is X, D, K, N, Q, or R; X14 is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; X16 is S, N, or T; X17 is W, F, or Y; XI 8 is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24 is F, H, N, Q, W, or Y; and X25 is G or A.

[0271] In some embodiments, a modified CRBN comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 474.

[0272] In some embodiments, a modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, (i) a Q325 amino acid substitution selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution; and (ii) one or more amino acid substitutions selected from: a 137 IN substitution, a H397F substitution, and a N369Y substitution.

[0273] In some embodiments, a modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, (i) a Q325H substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 351; or (ii) a Q325H substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 355; or (iii) a Q325H substitution and a N369Y substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 359; or (iv) a Q325H substitution, a N369Y substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 363; or (v) a Q325H substitution, a N369Y substitution, and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 367; or (vi) a Q325H substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 371; or (vii) a Q325K substitution and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 375; or (viii) a Q325K substitution, a 137 IN substitution, and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 383; or (ix) a Q325R substitution and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 403; or (x) a I371N substitution and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 331; or (xi) a N369Y substitution and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 335; or (xii) a N369Y substitution and a 137 IN substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 339; or (xiii) a N369Y substitution, a 137 IN substitution, and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 343; or (xiv) a Q325H substitution and a H397F substitution , optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 347; or (xv) a Q325K substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 379; or (xvi) a Q325K substitution and a N369Y substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 387; or (xvii) a Q325K substitution, a N369Y substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 391; or (xviii) a Q325K substitution, a N369Y substitution, and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 395; or (xix) a Q325K substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 399; or (xx) a Q325R substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315.

[0274] In some embodiments, a modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitition, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution.

[0275] The present disclosure provides for a polypeptide complex comprising a first polypeptide comprising a degron and a second polypeptide comprising a cereblon, wherein: (i) the degron is a modified degron as described herein; (ii) the cereblon is a modified cereblon as described herein; or (iii) the degron is a modified degron as described herein and the cereblon is a modified cereblon as described herein.

[0276] In some embodiments, a modified cereblon (CRBN) comprises, relative to relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitition, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, and wherein the modified degron comprises one or more amino acid substitutions selected from: a Q28T amino acid substitution, a G30E amino acid substitution,and a L33I amino acid substitution, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, modified CRBN comprises an 137 IN amino acid substitution, and wherein a modified degron comprises a G30E amino acid substitution. In some embodiments, a modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution. In some embodiments, a modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution, and wherein the modified degron comprises a Q28T, G30E, and L33I amion acid substitution.

[0277] In some embodiments, the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, (i) a Q325 amino acid substitution selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution; and (ii) one or more amino acid substitutions selected from: a T359V substitution, a 137 IN substitution, a H397F substitution, and a N369Y substitution.

[0278] In some embodiments, the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, (i) a Q325H substitution, a Q325H substitution, a 137 IN subsitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 544; or (ii) a Q325R substitution, a 137 IN substituion, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 530; or (iii) a Q325R substitution, a N369Y substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 526; or (iv) a Q325R substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 524; or (v) a Q325K substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 532; or (vi) a Q325H substitution, a N369Y substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 528; or (vii) a Q325H substitution, a N369Y substitution, a H397F substitution, and a T359Vsubstitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 534; or (viii) a Q325H substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 522.

[0279] The present disclosure further provides a modified degron comprising the amino acid sequence set forth in SEQ ID NO: 608 , wherein the modified degron exhibits decreased protein degradation upon binding to one or more immunomodulatory drugs (IMiDs) compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131.

[0280] The present disclosure further provides a modified degron comprising the amino acid sequence X1CGFTX2X3X4X5X6NLX7X8HIKX9, wherein Xi is I, H, V, or L; X2is C, T, or S; X3is R, T, N, K, Q, D, or F; X4is Q, C, Y, S, L, T, P, or R; X5is K, H, E, or L; X6is G, E, or L; X7 is L, D, or I; Xs is R, V, or F; and X9 is L or A, and wherein the modified degron exhibits decreased protein degradation upon binding to one or more IMiDs compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131.

[0281] In some embodiments, the modified degron comprises an amino acid seqeunce set forth in any one of SEQ ID NOs: 609-728. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 684. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 688. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 668. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO:704. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 706. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 724. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 690. In some embodiments, the modified degron comprises the amino acid sequence set forth in SEQ ID NO: 702.

[0282] In some embodiments, the modified degron further comprises one or more amino acid substutions selected from the group consisting of: I21R, F24W, C26T, R27D, R27H, R27K, Q28R, Q28T, G30V, G30E, N31T, N31A, L33I, K37G, and L38F, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40. In some embodiments, the one or more amino acid substitutions confer enhanced binding of themodified degron to the IMiD when compared to a degron not comprising the one or more amino acid substitutions.

[0283] In some embodiments, the modified degron functionally associates with a cereblon domain or variant thereof in an IMiD responsive manner. In some embodiments, the modified degron functionally associates with a cereblon domain or variant thereof in an IMiD responsive manner, wherein the modified degron associates with the cereblon domain or variant thereof when exposed to a lower concentration of IMiD, as compared to an unmodified degron. In some embodiments, the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127. In some embodiments, the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 129. In some embodiments, the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of ImiD, as compared to an unmodified cereblon. In some embodiments, the cereblon domain or variant thereof is a modified cereblon as provided herein.

[0284] In some embodiments, the IMiD is at least one of thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, the IMiD is pomalidomide.

[0285] In some embodiments, the modified degron does not comprise the amino acid sequence set forth in SEQ ID NO:40.BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0286] These and other features, aspects, and advantages of the present disclosure will become better understood with regard to the following description, and accompanying drawings.

[0287] FIGs. 1A-1B diagram constructs for assaying libraries of modified degron polepeptides. FIG. 1A details a screening vector comprising EGFP-2a-mCherry operably linked to a degron polypeptide by a linker. FIG. IB diagrams a degron-linker cassette for assaying lysine substitutions in the linker region.

[0288] FIGs. 2A-2B detail an overview of library sorting and isolation of modified degron polypeptides with increased sensitivity to pomalidomide. FIG. 2A diagrams the library sorting method. FIG. 2B shows exemplary flow cytometry plots detailing serial sorting and enrichment of modified degron polypeptides with increased sensitivity to pomalidomide.

[0289] FIG. 3 is a graph showing the identification of modified degron polypeptides enriched in the pool treated with 1 nM pomalidomide.

[0290] FIGs. 4A-4B detail a nanoluciferase degron assay for validation of degrons identified in the library screen. FIG. 4A is a diagram of a dual-luciferase construct comprising a degron-linker cassette. FIG. 4B is a graph showing degradation of luciferase with indicated degron cassettes as measured by reduction of luciferase signal (luminescence).

[0291] FIGs. 5A-5C diagram a dimerization assay of an inducible cell death system comprising modified degron polypeptides. FIG. 5A diagrams the induction of apoptotis by dimerization of caspase-9 by constructs comprising a small molecule-binding domain. FIG. 5B diagrams constructs comprising caspase-9 and a small molecule binding domain that is either cereblon (CRBN) or a variant thereof (SB06097) or a degron (SB06098). FIG. 5C is a diagram of the experimental time course of the caspase-9 dimerization assay.

[0292] FIG. 6 shows the killing efficiency of the indicated modified degron polypeptides compared to the killing efficiency of the wildtype degron.

[0293] FIG. 7 shows an exemplary workflow for a split IMiD ON switch assay for screening mutant CRIMP domains for improved sensitivity to pomalidomide.

[0294] FIG. 8 shows results of a split IMiD ON switch assay for screening mutant CRIMP domains for improved sensitivity to pomalidomide.

[0295] FIG. 9 shows an exemplary workflow for a split IMiD ON switch assay for screening mutant del.CRBN domains for improved sensitivity to pomalidomide.

[0296] FIG. 10 shows an exemplary heat map of normalized fold activation of reporter for selected mutant del.CRBN domains in response to titration of pomalidomide (100 nM, 10 nM, and 4 nM).

[0297] FIG. 11 shows an exemplary line graph of normalized fold activation of reporter for selected mutant del.CRBN domains in response to titration of pomalidomide (100 nM, 10 nM, and 4 nM).

[0298] FIG. 12 shows an exemplary bar graph of normalized fold activation of reporter for selected mutant del.CRBN domains in response to 4 nM pomalidomide.

[0299] FIGs. 13A-13F show results of an assay to evaluate single vector CRIMP / CRBN combinations and different 2A linkers between switch components.

[0300] FIG. 14 shows results of an assay to evaluate CRBN variants containing combinations of amino acid substitutions.

[0301] FIG. 15 shows results of an assay to evaluate CRBN variants containing combinations of amino acid substitutions.

[0302] FIG. 16 shows an exemplary bar graph of normalized fold activation of reporter for selected mutant del.CRBN domains in response to 10 nM pomalidomide.

[0303] FIG. 17 shows results of an assay to screen for del.CRBN mutants with improved sensitivity to pomalidomide.

[0304] FIG. 18 shows an exemplary bar graph of normalized fold activation of reporter for selected mutant del.CRBN domains in response to 4 nM pomalidomide.

[0305] FIG. 19 shows results of an assay to screen for del.CRBN mutants with improved sensitivity to pomalidomide.

[0306] FIG. 20 shows an exemplary bar graph of normalized fold activation of reporter for selected mutant del.CRBN domains in response to 1 nM pomalidomide.

[0307] FIGs. 21A-21C detail an assay for identifying combination mutants or variants of del.CRBN with improved sensitivity to pomalidomide. FIG. 21A is a diagram of a multicistronic construct including a del.CRBN variant (e.g., a del.CRBN containing combinations of amino acid substitutions), minVPR, Opt2A, CRIMP.mutl, and ZF DNA binding domain (DBD), and a diagram of a reporter construct including a ZF binding site (BS) and mCherry. FIG. 21B is an exemplary workflow for evaluating del.CRBN variants with combinations of amino acid subsitutions. FIG. 21C shows results of an assay to evaluate del.CRBN variants containing combinations of amino acid substitutions.

[0308] FIGs. 22A-22B shows results of an assay to evaluate del.CRBN variants containing combinations of amino acid substitutions.

[0309] FIGs. 23A-23C detail an assay for identifying combination mutants or variants of del.CRBN with improved sensitivity to pomalidomide. FIG. 23A is a diagram of a multicistronic construct including a del.CRBN variant (e.g., a del.CRBN containing combinations of amino acid substitutions), minVPR, Opt2A_2.0, CRIMP.mutl, and ZF DNA binding domain (DBD), and a diagram of a reporter construct including a ZF binding site (BS) and mCherry. FIG. 23B is an exemplary workflow for evaluating del.CRBN variants with combinations of amino acid subsitutions. FIG. 23C shows results of an assay to evaluate del.CRBN variants containing combinations of amino acid substitutions.

[0310] FIGs. 24A-24B shows results of an assay to evaluate del.CRBN variants containing combinations of amino acid substitutions.

[0311] FIG. 25 shows a graph depicting a comparison of performance between constructs containing Opt2A and Opt2A_2.0.

[0312] FIGs. 26A-26B detail an assay for CRIMP variants that are reistant to degradation in the presence of IMiDs (e.g., pomalidomide). FIG. 26A is a schematic detailing the designof constructs encoding CRIMP variants and two fluorescent reporter genes. FIG. 26B depcits an overview of the assay to assess degradation of CRIMP variants in the presence of pomalidomide.

[0313] FIGs. 27A-27D depict graphs showing the result of the degradation assay detailed in FIGs. 26A-26B in three batches of 20 constructs. FIG. 27A depicts a graph of results of the degradation assay for Batch 1. FIG. 27B depicts a graph of results of the degradation assay for Batch 2. FIG. 27C depicts a graph of results of the degradation assay for Batch 3. FIG. 27D depicts a graph of combinede results of the degradation assay for all three batches.

[0314] FIG. 28 is a graph depicting the extent of degradation for indicated CRIMP variants at 0 nM, 0.1 nM, and 4.0 nM of pomalidomide.

[0315] FIG. 29 is a graph showing the activity of the IMiD responsive transcriptional switch in a mouse model at 40 mg / kg pomalidomide.DETAILED DESCRIPTION

[0316] The present disclosure generally relates to modified degron polypeptides with increased sensitivity to pomalidomide. Also provided are inducibly degradable proteins, inducible cell death systems, and activation-conditional control polypeptides employing modified degron polypeptides and methods and / or use thereof as described herein.

[0317] Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0318] The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer disease state, including prophylaxis, lessening in the severity or progression, remission, or cure thereof.

[0319] The term “zzz situ” refers to processes that occur in a living cell growing separate from a living organism, e.g., growing in tissue culture.

[0320] The term “zzz vivo” refers to processes that occur in a living organism.

[0321] The term “mammal” as used herein includes both humans and non-humans, and includes, but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.

[0322] 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 specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm e.g., any of those described herein (e.g., BLASTP and BLASTN or other algorithms availableto persons of skill), or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.

[0323] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0324] Optimal alignment of sequences for comparison can be conducted, e.g., 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).

[0325] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0326] The term “sufficient amount” means an amount sufficient to produce a desired effect, e.g., an amount sufficient to modulate protein aggregation in a cell.

[0327] The term “therapeutically effective amount” is an amount that is effective to ameliorate a symptom of a disease. A therapeutically effective amount can be a “prophylactic ally effective amount” as prophylaxis can be considered therapy.

[0328] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0329] Use of ordinal terms such as “first”, “second”, “third”, etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed, but are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term) to distinguish the claimelements. Similarly, the use of these terms in the specification does not by itself connote any required priority, precedence, or order.Engineered Nucleic Acids and Polypeptides

[0330] The present disclosure provides for, among other things, engineered nucleic acids comprising one or more expression cassettes (e.g., one, two, three, four, five, or more expression cassettes), e.g., any expression cassette described herein. In some embodiments, at least one expression cassette, as provided by the present disclosure, comprises a promoter and an exogenous polynucleotide sequence. In some embodiments, a promoter is operably and / or directly linked to an exogenous polynucleotide sequence. In some embodiments, an exogenous polynucleotide sequence encodes one or more engineered polypeptides provided by the present disclosure, e.g., a modified degron or CRIMP (e.g., fused or operably linked to a polypeptide of interest), a modified CRBN (e.g., fused or operably linked to a polypeptide of interest), an activation-conditional control polypeptide (ACP), an inducibly degradable proteins, an inducible cell death polypeptide monomer, a regulatable cell survival polypeptide, or a chimeric polypeptide, as described herein.

[0331] In some embodiments, an engineered nucleic acid comprises a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence. In some embodiments, a first promoter is operably and / or directly linked to a first exogenous polynucleotide sequence. In some embodiments, a first exogenous polynucleotide sequence encodes one or more engineered polypeptides provided by the present disclosure, e.g., an activation-conditional control polypeptide (ACP), an inducibly degradable proteins, an inducible cell death polypeptide monomer, a regulatable cell survival polypeptide, or a chimeric polypeptide, as described herein. In some embodiments, a single engineered nucleic acid comprises at least one, two, three four, five, or more expression cassettes, e.g., a plurality. In some embodiments, each expression cassette comprises a promoter operably linked to a polynucleotide sequence encoding a protein of interest (e.g., any engineered polypeptide described herein).

[0332] In some embodiments of the present disclosure, an engineered nucleic acid comprises an expression cassette comprising an inducibly degradable protein. An inducibly degradable protein can comprise one or more ligand binding domains (e.g., a ligand binding domain of a degron polypeptide, e.g., a modified degron polypeptide) and at least one target polypeptide. When expressed in a cell, an inducibly degradable protein can be targeted by an E3 ubiquitin ligase via a cognate ligand (e.g., a small molecule) that binds to the ligandbinding domain(s). In some embodiments, an E3 ubiquitin ligase can induce polyubiquitylation upon binding to the inducibly degradable protein, which results in proteasomal degradation of the inducibly degradable protein.

[0333] In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes at least one inducible cell death polypeptide monomer. An inducible cell death polypeptide monomer can comprise one or more ligand binding domains and at least one cell death-inducing domain. When expressed in a cell, the cell death polypeptide monomer is oligomerizable via a cognate ligand (e.g., a small molecule) that binds to the ligand binding domain(s). When the ligand oligomerizes two or more of the cell death polypeptide monomers, a cell death-inducing signal can be generated in the cell. This generally results in cell death. In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes: (a) a first inducible cell death polypeptide monomer comprising a first ligand binding domain and a cell death-inducing domain, and (b) a second inducible cell death polypeptide monomer comprising a second ligand binding domain and a cell-death inducing domain. In some embodiments, an engineered nucleic acid comprises: (a) a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence that encodes a first inducible cell death polypeptide monomer comprising a first ligand binding domain and a cell death-inducing domain; and (b) a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence that encodes a second inducible cell death polypeptide monomer comprising a second ligand binding domain and a cell death-inducing domain. In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes a first inducible cell death polypeptide monomer comprising a first ligand binding domain and a cell death-inducing domain. In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes a second inducible cell death polypeptide monomer comprising a second ligand binding domain and a cell death-inducing domain. In some embodiments, a first inducible cell death polypeptide monomer comprises a degron (e.g., any degron or CRIMP described herein). In some embodiments, a second inducible cell death polypeptide monomer comprises a cereblon domain (e.g., any cereblon domain described herein).

[0334] In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes at least one ACP. The ACP can comprise one or more ligand binding domains and at least one transcription factor comprising at least one nucleic acid-binding domain and at least one transcriptional effector domain. When expressed in a cell, the ACP can undergo nuclear localization upon binding of the ligand binding domain(s) to a cognate ligand. When localized to the cell’s nucleus, the ACP is capable of inducing transcriptional expression of a gene of interest operably linked to an ACP-responsive promoter. In some embodiments, a gene of interest is associated with or causes cell death, e.g., by apoptosis.

[0335] In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes at least one ACP. The ACP can comprise at least one ligand binding domain and at least one transcriptional effector domain. When expressed in a cell and upon binding of the ligand binding domain(s) to a cognate ligand, the ACP is capable of modulating transcriptional expression of a gene of interest operably linked to an ACP-responsive promoter. For example, in some embodiments, when expressed in a cell and upon binding of the ligand binding domain(s) to a cognate ligand, activity of the ACP modulates transcriptional expression of a gene of interest operably linked to an ACP-responsive promoter. Alternatively, in some embodiments, binding of the ligand binding domain(s) to a cognate ligand induces degradation of the ACP, and thus ACP-based modulation of transcriptional expression of a gene of interest is abrogated by the binding to the cognate ligand. In some embodiments, a gene of interest is associated with or causes cell death, e.g., via apoptosis.

[0336] In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence that encodes at least one regulatable cell survival polypeptide comprising at least one ligand binding domain. When expressed in a cell, the at least one cell survival polypeptide is capable of inhibiting at least one cell death-inducing polypeptide and upon binding to a cognate ligand, the cognate ligand inhibits the at least one pro-survival polypeptide. This can result in cell death.

[0337] In some embodiments, an engineered nucleic acid comprises an expression cassette comprising a promoter operably linked to an exogenous polynucleotide sequence having the formula: Cl - L -C2 wherein Cl comprises a polynucleotide sequence encoding at least a first chimeric polypeptide comprising at least a first ligand binding domain and atleast a transcriptional activation domain, L comprises at least a linker polynucleotide sequence, C2 comprises a polynucleotide sequence encoding at least a second chimeric polypeptide comprising a second ligand binding domain and at least a nucleic acid-binding domain. When expressed in a cell, the first chimeric polypeptide and the second chimeric polypeptide can may functionally associate (e.g., directly or indirectly bind, oligomerize, multimerize, etc.) to form an ACP via a cognate ligand that binds to each ligand binding domain. The multimeric ACP can then be capable of inducing transcriptional expression of a gene of interest operably linked to an ACP-responsive promoter. Depending on the gene of interest, this can result in one or more biological responses or events, including but not limited to, cell death.

[0338] In some embodiments, an engineered nucleic acid comprises: (a) a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a first chimeric polypeptide, wherein the first chimeric polypeptide comprises a first ligand binding domain and a transcriptional effector domain, (b) a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a second chimeric polypeptide, wherein the second chimeric polypeptide comprises a second ligand binding domain and a nucleic acid-binding domain. When expressed in a cell, the first chimeric polypeptide and the second chimeric polypeptide may functionally associate (e.g., directly or indirectly bind, oligomerize, multimerize, etc.) to form an ACP via a cognate ligand that binds to each ligand binding domain. The ACP is then capable of inducing or repressing expression of a gene of interest operably linked to an ACP-responsive promoter. In some embodiments, a transcriptional effector domain is a transcriptional activator domain. In some embodiments, a transcriptional effector domain is a transcriptional repressor domain.

[0339] In some embodiments, an engineered nucleic acid comprises: (a) a first expression cassette comprising a first promoter operably linked to a first exogenous polynucleotide sequence encoding a first chimeric polypeptide, wherein the first chimeric polypeptide comprises a first ligand binding domain and a transcriptional activation domain, (b) a second expression cassette comprising a second promoter operably linked to a second exogenous polynucleotide sequence encoding a second chimeric polypeptide, wherein the second chimeric polypeptide comprises a second ligand binding domain and a nucleic acid-binding domain. When expressed in a cell, the first chimeric polypeptide and the second chimeric polypeptide may functionally associate (e.g., directly or indirectly bind, oligomerize, multimerize, etc.) to form an ACP in a ligand dependent manner. The ACP is then capable ofinducing transcriptional expression of a gene of interest operably linked to an ACP- responsive promoter in the cell. Depending on the gene of interest, this can result in one or more biological responses or events, including but not limited to, cell death.

[0340] In some embodiments, a chimeric polypeptide (e.g., a first, second, third, etc. chimeric polypeptide, as described herein) comprises a ligand binding domain and a transcriptional effector domain (e.g., a transcriptional activation domain). In some embodiments, a ligand binding domain and a transcriptional effector domain are linked by a linker sequence (e.g., any linker sequence described herein). In some embodiments, a chimeric polypeptide comprises, from N- to C-terminus, a ligand binding domain, a linker sequence, and a transcriptional effector domain. In some embodiments, a chimeric polypeptide (e.g., a first, second, third, etc. chimeric polypeptide, as described herein) comprises a ligand binding domain and a nucleic acid-binding domain (e.g., a DNA binding domain). In some embodiments, a ligand binding domain and a nucleic acid binding domain are linked by a linker sequence (e.g., any linker sequence described herein). In some embodiments, a chimeric polypeptide comprises, from N- to C-terminus, a ligand binding domain, a linker sequence, and a nucleic acid-binding domain.

[0341] One or more linkers can be used between various domains of the provided engineered polypeptides. Suitable polypeptide linkers can include a glycine-serine linkers comprising one or more serine residues and one or more glycine residues. Exemplary glycine-serine linker sequences can include one or more GGGS (SEQ ID NO: 252) motifs, one or more GGGGS (SEQ ID NO: 253) motifs, or both GGGS (SEQ ID NO: 252) and GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a glycine-serine linker can include 1-20 GGGS (SEQ ID NO: 252) and / or GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a glycine-serine linker includes the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). The present disclosure identifies certain polypeptide linker sequences that may increase or decrease degradation (e.g., via the ubiquitin-protesome pathway) of certain engineered polypeptides comprising said polypeptide linker depending on the presence or absence of certain amino acids within the linker sequence (e.g., one or more lysine residues). In some embodiments, a polypeptide linker sequence comprises one or more lysine residues. In some embodiments, a polypeptide linker sequence comprising one or more lysine residues has increased degradation as compared to a polypeptide linker sequence that does not have said one or more lysine residues (e.g., if said lysine residues were replaced by another amino acid, e.g., alanine). In some embodiments, one or more lysine residues of a polypeptide linker sequence are replaced with another amino acid residue (e.g., alanine). In someembodiments, a polypeptide linker sequence that has one or more lysine residues replaced with another amino acid (e.g., alanine) has decreased degradation as compared to a polypeptide linker sequence that has not had said one or more lysine residues replaced with another amino acid. In some embodiments, a polypeptide linker encoded by an engineered nucleic acid comprises an amino acid sequence such as one or more of: GGGGSGGGGSGGGGSVDGF (SEQ ID NO: 91) and ASGGGGSAS (SEQ ID NO: 92). In some embodiments, a polypeptide linker encoded by the engineered nucleic acid(s) can comprise an amino acid sequence of AEAAAKEAX1AKEAX2AKA (SEQ ID NO: 168), in which XI is A or K; and X2 is A or K. In some embodiments, the linker comprises an amino acid sequence set forth in any one of SEQ ID NOs: 169, 171, 173, or 175. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 169. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 171. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 173. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 175. In some embodiments, the linker comprises the amino acid sequence of SEQ ID NO: 199.

[0342] In some embodiments, one or more expression cassettes can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polynucleotides or effector molecules) can be produced from a single transcript. For example, a multicistronic expression cassette can encode both a first ACP and a second ACP, e.g., both expressed from a single expression cassette driven by a constitutive promoter. In another example, a multicistronic expression cassette can encode both an effector molecule and an antigen recognizing receptor, e.g., both expressed from a single expression cassette driven by an ACP-responsive promoter. Expression cassettes can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first protein of interest (e.g., an engineered polypeptide as described herein) can be linked to a nucleotide sequence encoding a second protein of interest, such as in a first genedinker: second gene 5’ to 3’ orientation. Multicistronic features and options are described herein, e.g., in the section “Multicistronic and Multiple Promoter Systems.”

[0343] In some embodiments, the engineered nucleic acid is selected from: a DNA, a cDNA, an RNA, an mRNA, and a naked plasmid (linear or circular). Also provided herein is an expression vector comprising the engineered nucleic acid.

[0344] In some embodiments, the engineered nucleic acid further comprises an insulator. The insulator can be localized between the first expression cassette and the second expression cassette. An insulator is a cis-regulatory element that has enhancer-blocking or barrierfunction. Enhancer-blocker insulators block enhancers from acting on the promoter of nearby genes. Barrier insulators prevent euchromatin silencing. An example of a suitable insulator of the present disclosure is the A2 insulator as described in Liu M, et al., Nat Biotechnol. 2015 Feb;33(2): 198-203. Additional insulators are described in West et al., Genes & Dev, 002. 16: 271-288, both of which are incorporated by reference in their entirety. Other examples of suitable insulators include, without limitation, an Al insulator, a CTCF insulator, a gypsy insulator, an HS5 insulator, and a P-globin locus insulator, such as cHS4. In some embodiments, the insulator is an A2 insulator, an Al insulator, a CTCF insulator, an HS5 insulator, a gypsy insulator, a P-globin locus insulator, or a cHS4 insulator.Ligand Binding Domains

[0345] In some embodiments of the present disclosure, an engineered polypeptide (e.g., a provided degron, CRIMP, CRBN, etc.) comprises a ligand binding domain. In some embodiments, a ligand binding domain interacts with a ligand such as a cognate ligand. In some embodiments, interaction between a ligand binding domain and a ligand results in oligomerization, e.g., dimerization, of the ligand binding domain and one or more other polypeptides. In some embodiments, interaction between a ligand binding domain and a ligand results in oligomerization, e.g., dimerization, of a plurality of ligand binding domains. In some embodiments, interaction between a ligand binding domain and a ligand results in a functional association between a plurality of ligand binding domains, whereby the functional association results in a biologically relevant output (e.g., increase or decrease in gene expression, increase or decrease in biological signaling, etc.). In some embodiments, a functional association between a plurality of ligand binding domains is direct, in that said plurality of ligand binding domains contact each other directly. In some embodiments, a functional association between a plurality of ligand binding domains is indirect, in that said plurality of ligand binding domains do not contact each other directly (e.g., functional association of said plurality of ligand binding domains may be facilitated by one or more intermediate agents or molecules, e.g., polypeptides, nucleic acids, etc.). In some embodiments, functional association includes oligomerization, multimerization, and / or complex formation between two or more engineered polypeptides of the present disclosure.

[0346] In some embodiments, a ligand binding domain comprises a degron. The terms “degron” and “degron domain,” as used herein, refer to a protein or a portion thereof that is involved in regulation of protein degradation rates. Various degrons including but not limited to short amino acid sequences, structural motifs, and exposed amino acids, can be used invarious embodiments of the present disclosure. Degrons identified from a variety of organisms can be used. See, e.g., Varshazsky A., PNAS 2019 Jan 8; 116(2):358-366, hereby incorporated by reference.

[0347] The term “degradation sequence” as used herein, refers to a sequence that promotes degradation of an attached protein through either the proteasome or autophagylysosome pathways. Any degradation sequence, e.g., those described herein, can be used for various embodiments of the present disclosure. In some embodiments, a degradation sequence comprises a degron. Degrons from any organism may be used in accordance with the present disclosure, including derivatives and variants thereof. In some embodiments, a degradation sequence is a polypeptide that destabilizes a protein such that half-life of the protein is reduced at least two-fold (e.g., at least two-fold, at least three-fold, at least fourfold, at least five-fold, etc.), when fused to the protein. Any degradation sequence / signal (e.g., of the ubiquitin-proteasome system) may be used in accordance with the present disclosure. A degradation sequence may be operably linked to a cell receptor, but need not be contiguous with it as long as the degradation sequence still functions to direct degradation of the cell receptor. In some embodiments, the degradation sequence induces rapid degradation of the cell receptor. For a discussion of degradation sequences and their function in protein degradation, see, e.g., Kanemaki et al. (2013) Pflugers Arch. 465(3):419-425, Erales et al. (2014) Biochim Biophys Acta 1843(1):216-221 , Schrader et al. (2009) Nat. Chem. Biol. 5(11): 815-822, Ravid et al. (2008) Nat. Rev. Mol. Cell. Biol. 9(9):679-690, Tasaki et al. (2007) Trends Biochem Sci. 32(1 l):520-528, Meinnel et al. (2006) Biol. Chem. 387(7):839- 851, Kim et al. (2013) Autophagy 9(7): 1100-1103, Varshavsky (2012) Methods Mol. Biol. 832: 1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3): 1268-1278; herein incorporated by reference.

[0348] In some embodiments, a degron used in accordance with the present disclosure is a CeReblon IMiD binding Partner (“CRIMP”). As used herein, “CRIMP” may refer to a degron, in particular, a degron that is used in a system that does not result in directed degradation of said degron upon binding of a cognate ligand (e.g., an IMiD). Accordingly, in some embodiments, a degron or modified degron used in accordance with the present disclosure is a CRIMP.

[0349] In some embodiments, a degron is capable of binding, or functionally associating with, CRBN or a variant thereof (e.g., a modified CRBN) in response to an immunomodulatory drug (IMiD). Such functional association may promote ubiquitin pathway-mediated degradation of a target polypeptide (e.g., an engineered polypeptide asdescribed herein, ACP, etc.) In some embodiments, a CRBN polypeptide substrate domain is selected from: IKZF1, IKZF3, Ckla, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or a fragment thereof, that is capable of drug-inducible binding of, or functional association with, CRBN or a variant thereof. In some embodiments, a CRBN polypeptide substrate domain is a chimeric fusion product of native CRBN polypeptide sequences. In some embodiments, a CRBN polypeptide substrate domain is a IKZF3 / ZFP91 / IKZF3 chimeric fusion product having an amino acid sequence of FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRR DAL (SEQ ID NO: 93).

[0350] In some embodiments, a degron (e.g., a modified degron, such as a CRIMP) is capable of binding, or functionally associated with, a CRBN or variant thereof (e.g., a modified CRBN) in response to an IMiD (e.g., IMiD binding at a CRBN ligand binding domain and / or a degron ligand binding domain). In some embodiments, such functional association, e.g., between a modified degron or CRIMP and a modified CRBN, may not lead to targeted degradation of a target polypeptide, but rather may produce a biologically relevant output as a result of the functional association (e.g., increase or decrease in gene expression, increase or decrease in biological signaling, etc.). In some embodiments, the biologically relevant output is produced when a CRIMP and a modified CRBN funtionally associate and bring together an effector domain and a DNA binding domain. For example, in some embedments, a CRIMP may be operably linked to an DNA binding domain, and a modified CRBN may be operably linked to an effector domain, and in the presence of an appropriate ligand (e.g., an IMiD), the CRIMP component and modified CRBN component functionally associate to bring together the effector domain and DNA binding domain to produce an increase or decrease in gene expression at a target gene. In some embedments, a CRIMP may be operably linked to an effector domain, and a modified CRBN may be operably linked to a DNA binding domain, and in the presence of an appropriate ligand (e.g., an IMiD), the CRIMP component and modified CRBN component functionally associate to bring together the effector domain and DNA binding domain to produce an increase or decrease in gene expression at a target gene.

[0351] In some embodiments, a degron comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, a degron comprises the amino acid sequence of SEQ ID NO: 131. In some embodiments, a degron comprises a modified d913 degron. In some embodiments, a degron comprises a modified d913 degron comprising one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 131. In someembodiments, a degron does not comprise the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, a degron does not comprise the amino acid sequence of SEQ ID NO: 40. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 133. In some embodiments, a modified d913 degron comprises one or more modifications / mutations that reduce ubiquitination relative to an unmodified d913 degron having the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, a modified d913 degron comprises one or more modifications / mutations that reduce ubiquitination relative to an unmodified d913 degron having the amino acid sequence of SEQ ID NO: 131. In some embodiments, in a modified d913 degron the modification comprises replacing one or more lysine residues, e.g., relative to an unmodified d913 degron comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, in a modified d913 degron the modification comprises replacing all lysine residues, e.g., relative to an unmodified d913 degron comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, in a modified d913 degron the modification comprises replacing one or more lysine residues with arginine residues, e.g., relative to an unmodified d913 degron comprising the amino acid sequence of SEQ ID NO: 131. In some embodiments, in a modified d913 degron the modification comprises replacing all lysine residues with arginine residues, e.g., relative to an unmodified d913 degron comprising the amino acid sequence of SEQ ID NO: 131, such as a modified degron comprising the amino acid sequence of SEQ ID NO: 133 or 139.

[0352] In some embodiments, a modified degron comprises an amino acid sequence selected from the group consisting of: I21R, F24W, C26T, R27D, R27H, R27K, Q28R, Q28T, G30V, G30E, N31T, N31A, L33I, K37G, and L38F. In some embodiments, the one or more amino acid seqeunces confer increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131.

[0353] In some embodiments, a modified degron comprises the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X10, wherein Xi is I or R; X2is F or W; X3is C or T; X4is R, D, H, or K; X5is Q, R, or T; X6is G or V; X7is N, T, or A; X8is L or I; X9is K or G; and Xio is L or F.

[0354] In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 139. In some embodiments, a modified degron comprises an amino acid sequence set forth in any one of SEQ ID NOs: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 226, 227, 228, 229, 230, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376,380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, or 788. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 140. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 142. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 144. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 146. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 148. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 150. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 152. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 154. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 156. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 158. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 160. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 162. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 164. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 166. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 226. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 227. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 228. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 229. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 230. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 276. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 280. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 284. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 288. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 292. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 296. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 300. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 304. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 308. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 312. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 316. In some embodiments, amodified degron comprises the amino acid sequence of SEQ ID NO: 320. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 324. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 328. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 332. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 336. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 340. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 344. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 348. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 352. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 356. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 360. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 364. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 368. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 372. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 376. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 380. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 384. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 388. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 392. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 396. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 400. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 404. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 408. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 412. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 416. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 420. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 424. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 788.

[0355] In some embodiments, a modified degron comprises an amino acid sequence set forth in any one of SEQ ID NOs: 177, 179, 181, 183, 185, or 187. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 177. In someembodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 179. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 181. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 183. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 185. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 187.

[0356] In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 140. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 142. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 144. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 146. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 148. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 150. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 152. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 154. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 156. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 158. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 160. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity withSEQ ID NO: 162. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 164. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 166. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 226. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 227. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 228. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 229. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 230. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 276. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 280. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 284. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 288. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 292. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 296. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 300. In some embodiments, a modified degron comprises an amino acid sequence having at least 80%(e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 304. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 308. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 312. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 316. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 320. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 324. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 328. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 332. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 336. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 340. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 344. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 348. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 352. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 356. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 360. In someembodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 364. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 368. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 372. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 376. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 380. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 384. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 388. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 392. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 396. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 400. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 404. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 408. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 412. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 416. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 420. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 424. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 788. In some embodiments, a modified degron does not comprise an N-terminal methionine (M) residue.

[0357] In some embodiments, a modified degron (e.g., a CRIMP) has increased sensitivity to a ligand (e.g., an IMiD, e.g., pomalidomide) relative to a degron does not comprise the modification. In some embodiments, a modified degron (e.g., a CRIMP) has increased sensitivity to a ligand (e.g., an IMiD, e.g., pomalidomide) relative to a degron comprising the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, increased sensitivity results in increased degradation of a protein comprising the modified degron. In some embodiments, increased degradation is measured by extinction of a detectable signal produced by a protein. In some embodiments, a detectable signal is fluorescence or luminescence. In some embodiments, a detectable signal is fluorescence. In some embodiments, a detectable signal is luminescence. In some embodiments, increased sensitivity results in increased association with another polypeptide (e.g., a CRBN or modified CRBN described herein). In some embodiments, increased sensitivity results in increased binding of the modified degron (e.g., a CRIMP) to a ligand (e.g., an IMiD, e.g., pomalidomide) relative to a degron comprising the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, increased sensitivity results in increased binding of the modified degron (e.g., a CRIMP) to a ligand (e.g., an IMiD, e.g., pomalidomide) relative to a degron comprising the amino acid sequence of SEQ ID NO: 131.

[0358] In some embodiments, a degron (e.g., a modified degron) used in accordance with the present disclosure comprises an amino acid sequence or nucleic acid sequence as shown in Table C-l, Table C-2, or Table C-4. In some embodiments, a degron (e.g., a modified degron) used in accordance with the present disclosure comprises an amino acid sequence or nucleic acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with an amino acid sequence or nucleic acid sequence as shown in Table C-l, Table C-2, or Table C-4.

[0359] In some embodiments, a modified degron exhibits decreased protein degradation upon binding to a ligand e.g., an IMiD, e.g., pomalidomide). In some embodiments, the decreased protein degradation is decreased degradation of a protein comprising the modifieddegron. In some embodiments, decreased degradation is measured by dereased extinction of a detectable signal produced by a protein relative to a protein that does not comprise the modified degron. In some embodiments, a detectable signal is fluorescence or luminescence. In some embodiments, a detectable signal is fluorescence. In some embodiments, a detectable signal is luminescence.

[0360] In some embodiments, a modified degron polypeptide exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) comprises one or more amino acid substitutions selected from the group consisting of: I21H, I21V, I21L, C26T, C26S, R27T, R27N, R27K, R27Q, R27D, R27F, Q28C, Q28Y, Q28S, Q28L, Q28T, Q28P, Q28R, K29H, K29E, K29L, G30E, G30L, L33D, L33I, R34V, R34F, and L38A.

[0361] In some embodiments, a modified degron exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) comprises the amino acid sequence X1CGFTX2X3X4X5X6NLX7X8HIKX9, wherein Xi is I, H, V, or L; X2 is C, T, or S; X3is R, T, N, K, Q, D, or F; X4is Q, C, Y, S, E, T, P, or R; X5is K, H, E, or L; X6is G, E, or L; X7 is L, D, or I; Xs is R, V, or F; and X9 is L or A.

[0362] In some embodiments, a modified degron exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with an amino acid set forth in any one of SEQ ID NOs: 607-728. In some embodiments, a modified degron exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) comprises and amino acid sequence set forth in any one of SEQ ID NOs: 607-728.

[0363] In some embodiments, a modified degron exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) comprises an amino acid sequence or is encoded by a nucleic acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with a sequence set forth in Table C-6. In some embodiments, a modified degron exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) comprises and amino acid sequence or is encoded by a nucleic acid sequence set forth in Table C-6.

[0364] In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 608. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 608. In some embodiments, a modifieddegron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 610. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 610. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 612. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 612. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 614. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 614. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 616. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 616. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 618. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 618. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 620. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 620. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 622. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 622. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 624. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 624. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 626. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 626. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 628. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 628. In some embodiments, a modified degroncomprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 630. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 630. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 632. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 634. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 634. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 636. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 636. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 638. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 638. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 640. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 640. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 642. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 642. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 644. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 644. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 646. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 646. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 648. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 648. In some embodiments, a modified degroncomprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 650. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 650. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 652. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 652. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 654. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 654. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 656. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 656. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 658. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 658. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 660. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 660. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 662. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 662. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 664. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 664. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 666. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 668. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 668. In some embodiments, a modified degroncomprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 670. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 670. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 672. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 672. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 674. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 674. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 676. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 676. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 678. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 678. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 680. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 680. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 682. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 682. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 684. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 684. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 686. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 686. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 688. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 688. In some embodiments, a modified degroncomprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 690. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 690. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 692. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 692. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 694. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 694. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 696. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 696. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 698. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 698. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 700. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 700. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 702. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 702. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 704. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 704. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 706. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 706. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 708. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 708. In some embodiments, a modified degroncomprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 710. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 710. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 712. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 712. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 714. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 714. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 716. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 716. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 718. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 718. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 720. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 720. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 722. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 722. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 724. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 724. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 726. In some embodiments, a modified degron comprises the amino acid sequence of SEQ ID NO: 726. In some embodiments, a modified degron comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 728. In some embodiments, a modified degron comprises theamino acid sequence of SEQ ID NO: 728. In some embodiments, the modified degron further comprises an N-terminal methionine (M) residue.

[0365] In some embodiments, a modified degron exhibiting decreased protein degradation upon binding to a ligand (e.g., an IMiD, e.g., pomalidomide) further comprises one or more amino acid substitutions that confer enhanced binding of the modified degron to the IMiD when compared to a degron not comprising the one or more amino acid substitutions. In some embodiments, the one or more amino acid substitutions that confer enhanced binding of the modified degron to the IMiD are selected from the group consisting of: I21R, F24W, C26T, R27D, R27H, R27K, Q28R, Q28T, G30V, G30E, N31T, N31A, L33I, K37G, L38F, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40.

[0366] In some embodiments, a cereblon (CRBN), as used in accordance with the present disclosure, is a wild-type CRBN polypeptide, e.g., a CRBN comprising the amino acid sequence of SEQ ID NO: 127. In some embodiments, a CRBN is a modified CRBN. In some embodiments, a modified CRBN comprises mutations that reduce ubiquitination relative to wild-type CRBN. In some embodiments, the DDB1 interacting domain is deleted in a modified CBRN. In some embodiments, amino acids 194-247 are deleted in a modified CBRN, e.g., a modified CRBN comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, a modified CRBN comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 127. In some embodiments, a modified CRBN comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 129.

[0367] In some embodiments, a modified CRBN comprises the amino acid sequence set forth in any one of SEQ ID NOs: 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 256, 258, 260, 262, 264, 275, 279, 283, 287, 291, 295,299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367,371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 426, 428, 430, 432,434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468,470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504,506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540,542, or 544. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 231. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 232. In some embodiments, a modified CRBN comprises the aminoacid sequence of SEQ ID NO: 233. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 234. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 235. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 236. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 237. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 238. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 239. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 240. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 241. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 242. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 243. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 244. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 245. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 246. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 247. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 248. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 249. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 256. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 258. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 260. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 262. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 264. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 275. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 279. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 283. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 287. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 291. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 295. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 299. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 303. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 307. In some embodiments, a modified CRBN comprises theamino acid sequence of SEQ ID NO: 311. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 315. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 319. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 323. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 327. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 331. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 335. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 339. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 343. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 347. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 351. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 355. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 359. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 363. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 367. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 371. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 375. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 379. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 383. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 387. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 391. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 395. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 399. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 403. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 407. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 411. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 415. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 419. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 423. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 426. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 428. In some embodiments, a modified CRBNcomprises the amino acid sequence of SEQ ID NO: 430. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 43. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 434. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 436. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 438. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 440. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 442. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 444. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 446. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 448. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 450. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 452. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 454. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 456. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 458. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 460. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 462. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 464. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 466. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 468. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 470. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 472. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 474. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 476. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 478. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 480. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 482. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 484. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 486. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 490. In some embodiments, a modifiedCRBN comprises the amino acid sequence of SEQ ID NO: 492. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 494. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 496. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 498. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 500. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 502. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 504. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 506. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 508. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 510. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 512. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 514. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 516. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 518. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 520. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 522. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 524. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 528. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 530. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 532. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 534. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 536. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 538. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 540. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 542. In some embodiments, a modified CRBN comprises the amino acid sequence of SEQ ID NO: 544.

[0368] In some embodiments, a modified CRBN comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with any one of SEQ ID NOs: 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 256, 258, 260, 262, 264, 275, 279, 283,287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355,359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411, 415, 419, 423, 426,428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462,464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498,500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534,536, 538, 540, 542, or 544. In some embodiments, a modified CRBN does not comprise an N-terminal methionine (M) residue.

[0369] In some embodiments, a CRBN (e.g., a modified CRBN) used in accordance with the present disclosure comprises an amino acid sequence or nucleic acid sequence as shown in Table C-l, Table C-3, Table C-4, or Table C-5. In some embodiments, a CRBN (e.g., a modified CRBN) used in accordance with the present disclosure comprises an amino acid sequence or nucleic acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with an amino acid sequence or nucleic acid sequence as shown in Table C-l, Table C-3, Table C-4, or Table C-5.Ligands and Cognate Ligand Pairs

[0370] The present disclosure identifies certain ligands that are useful for binding to ligand binding domains, as discussed herein. In many embodiments of the present disclosure, a ligand may bind to a particular ligand binding domain (e.g., a ligand domain in a provided degron or CRIMP, or a provided CRBN). A given ligand that consistently binds to a given ligand binding domain can be referred to as a cognate ligand pair.

[0371] In some embodiments, a ligand used in accordance with the present disclosure is an immunomodulatory imide drug (IMiD). In some embodiments, an IMiD is an FDA- approved drug. In some embodiments, an IMiD is thalidomide. In some embodiments, an IMiD is a variant or derivative of thalidomide. In some embodiments, an IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide. In some embodiments, an IMiD is pomalidomide. In some embodiments, an IMiD is thalidmide. In some embodiments, and IMiD is iberdomide. In some embodiments, an IMiD is lenalidomide.Inducibly Degradable Proteins

[0372] In some embodiments, an engineered polypeptide of the present disclosure comprises an inducibly degradable polypeptide or protein. In some embodiments, an inducibly degradable protein comprises at least one ligand binding domain and at least onetarget polypeptide that is targeted for degradation. When expressed in a cell, an inducibly degradable protein can be targeted by an E3 ubiquitin ligase via a cognate ligand (e.g., a small molecule) that binds to the ligand binding domain(s). In some embodiments, an E3 ubiquitin ligase can induce polyubiquitylation upon binding to the inducibly degradable protein, which results in proteasomal degradation of the inducibly degradable protein, and subsequent degradation of the target polypeptide.

[0373] In some embodiments, an inducibly degradable protein comprises a degron (e.g., any degron described herein, e.g., any of those in Table C-l, Table C-2, or Table C-4) operably linked to a target polypeptide. In some embodiments, a provided degron or modified degron is directly fused to a target polypeptide, e.g., as a fusion protein. In some embodiments, a provided degron or modified degron is operably linked to a target polypeptide with a linker (e.g., any linker described herein).Cell Death-Inducing Domains

[0374] In some embodiments, an engineered polypeptide of the present disclosure comprises an inducible cell death polypeptide. Inducible cell death polypeptides can include one or more ligand binding domains and at least one cell death-inducing domain. When expressed in a cell, a provided inducible cell death polypeptide can be used as a component of an inducible cell death system, where a cognate ligand that binds a ligand binding domain of one inducible cell death polypeptide may be provided resulting in that ligand binding domain functionally associating with another inducible cell death polypeptide thereby forming a complex that causes or promotes cell death. For example, in some embodiments, a first polypeptide comprising a modified degron (e.g., any modified degron provided herein, such as a CRIMP) comprising a first ligand binding domain is operably linked to a first celldeath inducing domain, and a second polypeptide comprising a second ligand binding domain is operably linked to a second cell death inducing domain, and the first polypeptide and the second polypeptide form a complex when at least one ligand binding domain is bound by an appropriate cognate ligand (e.g., an IMiD), and the formed complex causes or promotes cell death. In some embodiments, a CRBN (e.g., any modified CRBN as described herein) comprises the second ligand binding domain. In some embodiments, the first cell-death inducing domain and the second cell-death inducing domain are the same (e.g., form a homodimer complex when the inducible cell death system is in the presence of an appropriate cognate ligand).

[0375] Exemplary cell death-inducing domains can be derived from a protein such as one or more of: a caspase (e.g., any one of caspases 1-11, such as caspase 3, caspase 6, caspase 7, caspase 8, caspase 9), Diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2-interacting protein 3 (BNIP3), Fas, Fas -associated protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), a TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53-upregulated modulator of apoptosis (PUMA), Noxa, Blk, Hrk, Cytochrome c, Arts, TNF-related apoptosis-inducing ligand (TRAIE), Herpes Simplex Virus thymidine kinase (HSV-TK), Varicella Zoster Virus thymidine kinase (VZV-TK), viral Spike protein, Carboxyl esterase, cytosine deaminase, nitroreductase Fksb, Carboxypeptidase G2, Carboxypeptidase A, Horseradish peroxidase, Einamarase, Hepatic cytochrome P450-2B1, Purine nucleoside phosphorylase, or variants or functional fragments thereof. A“variant” as used herein may refer to mutants, splice variants, conformations, isoforms, allelic variants, species variants and species homologs of a biomolecule, e.g., a polynucleotide or a polypeptide. In some embodiments, a variant biomolecule is a natural variant. In some embodiments, a variant biomolecule is an engineered variant. In some embodiments, a variant is a sequence optimized variant, e.g., a polynucleotide sequence optimized variant. In some embodiments, the term “variant” refers to a polynucleotide variant, e.g., a gene or regulatory element, comprising one or more different nucleotides as compared to a reference (or “parent”) sequence. Accordingly, variant polynucleotide sequences may comprise at least one mutation, substitution, insertion, or deletion as compared to their respective reference sequence. “Variants” may have a sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence (e.g., a naturally occurring sequence or fragment or derivative thereof, or an engineered sequence). In some embodiments, based on context, a variant may refer to a “functional fragment.” A “functional fragment” of a biomolecule, e.g., a polynucleotide or polypeptide, may refer to a fragment of a reference biomolecule (i.e., shorter and / or smaller) with the same or similar functional activity of the reference biomolecule. It is contemplated that a similar functional activity could be greater, about equal, or less than the functional activity of the reference biomolecule, as long as the functional fragment achieves at least a portion of the activity of the reference biomolecule. When the reference biomolecule is a polypeptide, it is contemplated that the polypeptide fragment retains at least 1%, 5%, 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the reference polypeptide in a suitable assay. For example, when the reference biomolecule is a polypeptide, it is contemplated that the polypeptide fragment may be a polypeptide that has been cleaved or otherwise modified to be shorter and / or smaller than the reference polypeptide, but still retains functional activity of the reference polypeptide, such as binding to a particular receptor; when the reference biomolecule is a polynucleotide, it is contemplated that the polynucleotide fragment retains some of the same activity of the reference polynucleotide. For example, in the case of a polynucleotide encoding a protein, it is contemplated that the polynucleotide fragment encodes a protein having least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the protein encoded by the reference polynucleotide in a suitable assay. In the case of a polynucleotide that acts a regulatory element (e.g., a promoter or enhancer), it is contemplated that the polynucleotide fragment has at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the activity as reference polynucleotide in a suitable assay. Exemplary cell death-inducing domain sequences can be found in Table C-l.

[0376] In some embodiments, a cell death-inducing domain is derived from Caspase 9. In some embodiments, a cell death-inducing domain comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, a cell death-inducing domain comprises the amino acid sequence of SEQ ID NO: 123. A cell death-inducing domain can include or be derived from Caspase 9, e.g., the amino acid sequence shown in SEQ ID NO: 39 or 123. In some embodiments, a derivative of Caspase-9 comprises an inducible Caspase-9 (“iCasp-9”), which is capable of inducing apoptosis due to drug-based dimerization, e.g., the amino acid sequence shown in SEQ ID NO: 48 or 125. In some aspects, the caspase domain or derivative or functional fragment thereof thereof, e.g., inducible Casp-9, does not comprise a Caspase Activation and Recruitment Domain (CARD) domain sequence.

[0377] In some embodiments, an inducible cell death system comprises a first polypeptide comprising a degron or modified degron (e.g., any degron or modified degron described herein) operably linked to a first Caspase 9 polypeptide monomer and a second polypeptide operably linked to a second Caspase 9 polypeptide monomer, where the first Caspase 9 polypeptide monomer and the second Caspase 9 polypeptide monomer form a complex (e.g., dimerize) in the presence of an appropriate cognate ligand (e.g., when a ligand domain is bound by an IMiD).

[0378] A cell death-inducing domain can include BAX, e.g., the amino acid sequence shown in SEQ ID NO: 32.Regulatable Cell Survival Polypeptides

[0379] In some embodiments, an engineered polypeptide of the present disclosure comprises a regulatable cell survival polypeptide. A regulatable cell survival polypeptide can comprise at least one ligand binding domain. When expressed in a cell, a provided regulatable cell surivival polypeptide can be used as a component of an regulatable cell survival system, where a cognate ligand that binds a ligand binding domain of one regulatable cell survival polypeptide may be provided resulting in a that ligand binding domain functionally associating with another regulatable cell survival polypeptide, thereby forming a complex that promotes cell survival.

[0380] For example, in some embodiments, a first polypeptide comprising a modified degron (e.g., any modified degron provided herein, such as a CRIMP) which comprises a first ligand binding domain is operably linked to a first cell survival polypeptide, and a second polypeptide comprising a second ligand binding domain is operably linked to a second cell survival polypeptide, and the first polypeptide and the second polypeptide form a complex when at least one ligand binding domain is bound by an appropriate cognate ligand (e.g., an IMiD), and the formed complex promotes cell survival. In some embodiments, a CRBN (e.g., any modified CRBN as described herein) comprises the second ligand binding domain.

[0381] Exemplary cell survival polypeptides include one or more of XIAP, Bcl-2, Bcl- xL, Bcl-w, Bcl-2-related protein Al (BCL2A1), Mcl-1, FLICE-like inhibitory protein (c- FLIP), and an adenoviral E1B-19K protein. A cell survival polypeptide can include XIAP. A cell survival polypeptide can include wild-type XIAP, e.g., having the amino acid sequence SEQ ID NO: 107. A cell survival polypeptide can include modified XIAP. A modified XIAP can include one or more amino acid substitutions with reference to SEQ ID NO: 107.Activation-Conditional Control Polypeptides (ACPs)

[0382] In some embodiments, an engineered polypeptide of the present disclosure comprises an ACP. In some embodiments, an engineered polypeptide of the present disclosure may form an ACP complex in the presence of a ligand. For example, in some embodiments, a first ACP comprising a first polypeptide comprising a first ligand binding domain that is operably linked to a transcriptional effector domain (e.g., a transciptional repressor or transcriptional activator), and a second ACP comprising a second polypeptidecomprising a second ligand binding domain that is operably linked to a DNA binding domain, form an ACP complex when at least one ligand binding domain is bound by an appropriate cognate ligand, and the formed ACP complex facilitates a biologically relevant output (e.g., increase or decrease in gene expression, increase or decrease in biological signaling, etc.).

[0383] In some embodiments, the ACP is or comprises a transcriptional modulator. In some embodiments, the ACP is or comprises a transcriptional repressor. In some embodiments, the ACP is or comprises a transcriptional activator. In some embodiments, the ACP is or comprises a transcription factor. In some embodiments, an ACP comprises a DNA- binding domain. In some embodiments, an ACP comprises a transcriptional effector domain. In some embodiments, the ACP comprises a DNA-binding domain and a transcriptional effector domain. In some embodiments, the transcription factor includes a zinc-finger- containing transcription factor. In some embodiments, the zinc-finger-containing transcription factor may be a synthetic transcription factor. In some embodiments, the ACP DNA-binding domain comprises a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the DNA-binding domain comprises a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, an ACP can include one or more ligand binding domains.

[0384] In some embodiments, an ACP used in accordance with the present disclosure comprises a polypeptide comprising a ligand binding domain operably linked to a transcriptional effector domain. In some embodiments, an ACP comprises a degron or modified degron (e.g., any degron or modified degron provided herein, such as a CRIMP) operably linked to a transcriptional effector domain (e.g., any transcriptional effector domain described herein). In some embodiments, an ACP comprises a degron or modified degron comprising a sequence as shown in Table C-l, Table C-2, or Table C-4 operably linked to a transcriptional effector domain comprising a sequence as shown in Table 1, Table 2, Table C- 1, or Table C-2. In some embodiments, an ACP comprises a CRBN (e.g., any CRBN or modified CRBN provided herein) operably linked to a transcriptional effector domain (e.g., any transcriptional effector domain described herein). In some embodiments, an ACP comprises a CRBN comprising a sequence as shown in Table C-l, Table C-3, Table C-4, or Table C-5 operably linked to a a transcriptional effector domain comprising a sequence as shown in Table 1, Table 2, Table C-l, or Table C-2.Nucleic Acid Binding Domains

[0385] The present disclosure, in some embodiments, provides for engineered polypeptides comprising at least one nucleic acid-binding domain (e.g., a DNA binding domain). In some embodiments, an engineered polypeptide comprises at least one ligand binding domain and at least one nucleic acid-binding domain.

[0386] In some embodiments, an engineered polypeptide comprises at least one transcription factor. In some embodiments, a transcription factor comprises at least one nucleic acid-binding domain. In some embodiments, a transcription factor comprises at least one nucleic acid-binding domain and at least one transcriptional effector domain.

[0387] In some aspects, the nucleic acid-binding domain comprises a DNA-binding zinc finger protein domain (ZF protein domain). In some aspects, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). In some aspects, the transcriptional effector domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein- Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); a histone acetyltransferase (HAT) core domain of the human El A- associated protein p300 (p300 HAT core activation domain); a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; an HP1 alpha chromoshadow repression domain, and variants or functional fragments thereof. In some embodiments, a transcriptional effector domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain), or a variant or functional fragments thereof. In some embodiments, a transcriptional effector domain comprises a p65 domain, or a variant or functional fragments thereof.

[0388] In some embodiments, the ZF protein domain is modular in design and is composed of zinc finger arrays (ZFA). A zinc finger array comprises multiple zinc finger protein motifs that are linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity to any desired nucleic acid sequence. The ZFmotifs can be directly adjacent to each other, or separated by a flexible linker sequence. In some embodiments, a ZFA is an array, string, or chain of ZF motifs arranged in tandem. A ZFA can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs. The ZFA can have from 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.

[0389] In some embodiments, a ZF protein domain comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more ZFAs. In some embodiments, a ZF domain comprises from 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, the ZF protein domain comprises one to ten ZFA(s). In some embodiments, the ZF protein domain comprises at least one ZFA. In some embodiments, the ZF protein domain comprises at least two ZFAs. In some embodiments, the ZF protein domain comprises at least three ZFAs. In some embodiments, the ZF protein domain comprises at least four ZFAs. In some embodiments, the ZF protein domain comprises at least five ZFAs. In some embodiments, the ZF protein domain comprises at least ten ZFAs.

[0390] An exemplary ZF protein domain is shown in the sequence SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTH TGSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTH TGSQKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHL RGS (SEQ ID NO: 57). In some embodiments, a ZF protein domain comprises the amino acid sequence of SEQ ID NO: 57.Transcriptional Effector Domains

[0391] The present disclosure provides for, in some embodiments, engineered polypeptides comprising at least one transcriptional effector domain. In some embodiments, an engineered polypeptide may comprise at least one ligand binding domain and at least one transcriptional effector domain.

[0392] In some embodiments, an inducible cell death polypeptide as provided herein can include at least one transcriptional effector domain. In some embodiments, an ACP as provided herein can include at least one transcriptional effector domain. In some embodiments, an inducible cell death polypeptide comprises at least one ligand binding domain and at least one transcriptional effector domain. In some embodiments, an ACP comprises at least one ligand binding domain and at least one transcriptional effector domain.

[0393] In someembodiments, a transcriptional effector domain comprises one or more of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); a histone acetyltransferase (HAT) core domain of the human E1A- associated protein p300 (p300 HAT core activation domain); a Kriippel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)- methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain. In some embodiments, a transcriptional effector domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain). In some embodiments, a transcriptional effector domain comprises a p65 domain.

[0394] In some embodiments, the transcriptional effector domain comprises a transcriptional repressor domain. In some embodiments, the transcriptional repressor domain is selected from the group consisting of: a Kriippel associated box (KRAB) repression domain; a truncated Kriippel associated box (KRAB) repression domain; a Histone Deacetylase 4 (HDAC4) repressor domain; a Scleraxis (SCX) HLH domain, an Inhibitor of DNA binding 1 (ID1) HLH domain, a HECT domain and RCCl-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a Twist-related protein 1 (TWST1) HLH domain, an Homeobox protein Nkx-2.2 (NKX22) homeodomain, an Inhibitor of DNA binding 1 (ID3) HLH domain, and a Twist-related protein 2 (TWST2) HLH domain, and EED repressor domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain. In some embodiments, the transcriptional effector domain comprises a transcriptional activation domain. In some aspects, the transcriptional activation domain is selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activationdomain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain). In some embodiments, a transcriptional activator domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain). In some embodiments, a transcriptional activator domain comprises a p65 domain. Transcriptional activation domains can also be referred to as transcriptional activator domains.

[0395] In some embodiments, an engineered nucleic acid of the present disclosure comprises a polynucleotide encoding an inducible cell death polypeptide. In some embodiments, an engineered nucleic acid comprises a polynucleotide encoding an ACP. In some embodiments, an inducible cell death polypeptide as provided herein comprises at least one transcription factor comprising at least one transcriptional effector domain. In some embodiments, an ACP as provided herein comprises at least one transcription factor comprising at least one transcriptional effector domain. In some embodiments, an inducible cell death polypeptide as provided herein comprises at least one transcription factor comprising at least one nucleic acid-binding domain and at least one transcriptional effector domain. In some embodiments, an ACP as provided herein comprises at least one transcription factor comprising at least one nucleic acid-binding domain and at least one transcriptional effector domain.. In addition, an ACP may comprise at least one ligand binding domain and at least one transcription factor comprising at least one nucleic acidbinding domain and at least one transcriptional effector domain.

[0396] An engineered nucleic acid provided by the present disclosure can encode an effector domain, such as a transcriptional effector domain. In some embodiments a transcriptional effector domain comprises an effector domain (e.g., activator domain or repressor domain) of a transcription factor. Transcription factor effector domains are also known as transactivation domains, and act as scaffold domains for proteins such as transcription coregulators that act to activate or repress transcription of genes. Any suitable transcriptional effector domain can be used including, but not limited to, a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain consisting of four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein- Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains, the tripartite activator is known as a VPR activation domain; a histone acetyltransferase (HAT) core domain of the human ElA- associated protein p300, known as a p300 HAT core activation domain; a Kruppel associatedbox (KRAB) repression domain; a truncated Kruppel associated box (KRAB) repression domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain, or any combination thereof. In some embodiments, a transcriptional effector domain is a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain). In some embodiments, a transcriptional effector domain comprises a p65 domain.

[0397] In some embodiments, a transcriptional effector domain used in accordance with the present disclosure is a VPR domain. In some embodiments, a transcriptional effector domain used in accordance with the present disclosure is a min VPR domain. In some embodiments, the min VPR domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 197. In some embodiments, the min VPR domain comprises an amino acid sequence as set forth in SEQ ID NO: 197. In some embodiments, the min VPR domain comprises an amino acid sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with SEQ ID NO: 545. In some embodiments, the min VPR domain comprises an amino acid sequence as set forth in SEQ ID NO: 545.

[0398] Exemplary transcriptional effector domain protein sequences are shown in Table 1. Exemplary transcriptional effector domain nucleotide sequences are shown in Table 2. In some embodiments, a transcriptional effector domain used in accordance with the present disclosure comprises a sequence shown in Table 1 or Table 2. In some embodiments, a transcriptional effector domain used in accordance with the present disclosure comprises a sequence having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with a sequence shown in Table 1 or Table 2.Table 1. Transcriptional Effector Domain (Protein)Table 2. Transcriptional Effector Domain (Nucleotide)Promoters

[0399] In some embodiments, an engineered nucleic acid of the present disclosure comprises one or more promoters, e.g., in one or more expression cassettes. In some embodiments, an engineered nucleic acid of the present disclosure comprises a first expression cassette comprising a first promoter operably linked to an exogenous polynucleotide sequence. In some embodiments, an engineered nucleic acid of the present disclosure comprises a second expression cassette comprising a promoter operably linked to a second exogenous polynucleotide sequence encoding one or more effector molecules. In some embodiments, the first expression cassette and second expression cassette are each encoded by a separate engineered nucleic acid of the present disclosure. In some embodiments, the first expression cassette and the second expression cassette are encoded by the same engineered nucleic acid of the present disclosure.

[0400] In some embodiments, a promoter is an ACP-responsive promoter. In some embodiments, an ACP-responsive promoter of the present disclosure comprises an ACP- binding domain and a promoter sequence. In some embodiments, the ACP-responsive promoter is operable linked to a nucleotide sequence encoding an effector molecule (e.g., a protein of interest, e.g., any engineered polypeptide described herein).

[0401] A “promoter” refers to a control region of a nucleic acid sequence at which initiation and rate of transcription of the remainder of a nucleic acid sequence are controlled. In some embodiments, a promoter comprises sub-regions at which regulatory proteins or other molecules may bind, such as RNA polymerase, an ACP, and / or transcription factors. Promoters may be constitutive, inducible, repressible, tissue-specific or any combination thereof. In some embodiments, a promoter is a constitutive promoter. In some embodiments, a promoter is an inducible promoter. A promoter drives expression or drives transcription of the nucleic acid sequence that it regulates. Herein, a promoter is considered to be “operably linked” when it is in a correct functional location and orientation in relation to a nucleic acid sequence it regulates to control (“drive”) transcriptional initiation and / or expression of that sequence.

[0402] In some embodiments, a promoter may be one naturally associated with a particular gene or sequence, as may be obtained by isolating the 5’ non-coding sequences located upstream of the coding segment of said gene or sequence. Such a promoter can be referred to as “endogenous.” In some embodiments, a coding nucleic acid sequence may be positioned under the control of a recombinant or heterologous promoter, which refers to apromoter that is not normally associated with the encoded sequence in its natural environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that are not “naturally occurring” such as, for example, those that contain different elements of different transcriptional regulatory regions and / or mutations that alter expression through methods of genetic engineering. In addition to producing nucleic acid sequences of promoters and enhancers synthetically, sequences may be produced using recombinant cloning and / or nucleic acid amplification technology, including polymerase chain reaction (PCR) (see, e.g., U.S. Pat. No. 4,683,202 and U.S. Pat. No. 5,928,906).

[0403] Promoters of an engineered nucleic acid of the present disclosure may be “inducible promoters,” which refer to promoters that are characterized by regulating (e.g., initiating or activating) transcriptional activity when in the presence of, influenced by or contacted by a signal. The signal may be endogenous or a normally exogenous condition (e.g., light), compound (e.g., chemical or non-chemical compound) or protein (e.g., an engineered polypeptide described herein, e.g., an ACP) that contacts an inducible promoter in such a way as to be active in regulating transcriptional activity from the inducible promoter. Activation of transcription may involve directly acting on a promoter to drive transcription or indirectly acting on a promoter by inactivation a repressor (e.g., an engineered polypeptide described herein) that is preventing the promoter from driving transcription. Conversely, deactivation of transcription may involve directly acting on a promoter to prevent transcription or indirectly acting on a promoter by activating a repressor that then acts on the promoter.

[0404] In some embodiments, a promoter is “responsive to” or “modulated by” a local tumor state (e.g., inflammation or hypoxia) or signal if in the presence of that state or signal, transcription from the promoter is activated, deactivated, increased, or decreased. In some embodiments, the promoter comprises a response element. A “response element” is a short sequence of DNA within a promoter region that binds specific molecules (e.g., transcription factors, or an engineered polypeptide as described herein, such as an ACP) that modulate (regulate) gene expression from the promoter. Response elements that may be used in accordance with the present disclosure include, without limitation, a phloretin-adjustable control element (PEACE), a zinc-finger DNA-binding domain (DBD), an interferon-gamma- activated sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121- 34, incorporated herein by reference), an interferon-stimulated response element (ISRE) (Han, K. J. et al. J Biol Chem. 2004 Apr 9;279(15): 15652-61, incorporated herein byreference), a NF-kappaB response element (Wang, V. et al. Cell Reports 2012; 2(4): 824-839, incorporated herein by reference), and a 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 sensitivity of the response element to its cognate binding molecule. Tandem repeats can be labeled 2X, 3X, 4X, 5X, etc. to denote the number of repeats present.

[0405] Non-limiting examples of responsive promoters (also referred to as “inducible promoters”) (e.g., TGF-beta responsive promoters) are listed in Table 3. Additionally, Table 3 provides exemplary promoter and transcription factor combinations that can be used in accordance with the present disclosure, as well as the effect of a select inducer molecule on said promoter and transcription factor combinations. For example, transcription factor response to an inducer is shown in the TF column (where A represents activation; DA represents deactivation; and DR represents derepression) and transgene transcription response to an inducer is shown in the T column (where B represents binding; D represents dissociation; and n.d. represents not determined) (see Homer, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m, and references cited therein). Non-limiting examples of components that may be included in an inducible promoter (e.g., minimal promoters and responsive elements) are shown in Table 4.Table 3. Exemplary Inducible PromotersTable 4. Exemplary Components of Inducible Promoters

[0406] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EFla) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the spleen focus-forming virus (SFFV) promoter, the simian virus 40 (SV40) promoter, and the ubiquitin C (UbC) promoter. In some embodiments, the promoter is a constitutive promoter. Exemplary constitutive promoters are shown in Table 5.Table 5. Exemplary Constitutive Promoters

[0407] In some embodiments, the promoter sequence is derived from a promoter selected from: minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, API response element, TCF-EEF response element promoter fusion, Hypoxia responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule responsive promoters, and tandem repeats thereof.

[0408] In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, the constitutive promoter is selected from: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEFlaVl, hCAGG, hEFlaV2, hACTb, heIF4Al, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0409] In some embodiments, an ACP-responsive promoter is a synthetic promoter. In some embodiments, the ACP-responsive promoter comprises a minimal promoter. In some embodiments, the ACP-binding domain comprises one or more zinc finger binding sites. In some embodiments, an ACP-binding domain comprises 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, or more zinc finger binding sites. In some embodiments, the ACP-binding domain comprises one zinc finger binding site. In some embodiments, the ACP-binding domain comprises two zinc finger binding sites. In some embodiments, the ACP-binding domain comprises three zinc finger binding sites. In some embodiments, the ACP-binding domain comprises four zinc finger binding sites. In some embodiments, an ACP-binding domain comprises the polynucleotide sequence of: cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtc GGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcg cctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTT CTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 100).

[0410] In some embodiments, an ACP-responsive promoter comprises an enhancer that promotes transcription when an antigen recognizing receptor engages a cognate antigen, e.g., an antigen expressed on a target cell. Enhancers can include, but are not limited to, enhancers enriched in the ATAC-seq of activated T cells (Gate et al. Nat Genet. Author manuscript; available in PMC 2019 Jan 9; herein incorporated by reference for all purposes) or enhancers associated with upregulated genes in single-cell RNA seq data (Xhangolli et al. Genomics Proteomics Bioinformatics. 2019 Apr; 17(2): 129- 139. Doi: 10.1016 / j.gpb.2019.03.002; herein incorporated by reference for all purposes). In some embodiments, an enhancer is or comprises a synthetic enhancer. Synthetic enhancers can include multiple iterations of transcription factor binding sites. In some embodiments, a synethetic enhancer comprises oneor more (e.g., one, two, three, four, five, or more) iterations of one or more distinct transcription factor binding sites. In some embodiments, a synthetic enhancer comprises four iterations of two distinct transcription factor binding sites in an aaaabbbb or abababab organization. Illustrative non-limiting examples of genes from which enhancers can be derived include, but are not limited to, ATF2, ATF7, BACH1, BATF, Bcl-6, Blimp-1, BMI1, CBFB, CREB1, CREM, CTCF, E2F1, EBF1, EGR1, ETV6, FOS, F0XA1, F0XA2, GATA3, HIF1A, IKZF1, IKZF2, IRF4, JUN, JUNB, JUND, Lefl, NFAT, NFIA, NFIB, NFKB, NR2F1, Nur77, PU.l, RELA, RUNX3, SCRT1, SCRT2, SP1, STAT4, STAT5A, T- Bet, Tcf7, ZBED1, ZNF143, or ZNF217.Multicistronic and Multiple Promoter Systems

[0411] In some embodiments, engineered nucleic acids are configured to produce multiple polypeptides. For example, nucleic acids may be configured to produce 2-20 different polypeptides. In some embodiments, engineered nucleic acids are configured to produce 2-20, 2-19, 2-18, 2-17, 2-16, 2-15, 2-14, 2-13, 2-12, 2-11, 2-10, 2-9, 2-8, 2-7, 2-6, 2- 5, 2-4, 2-3, 3-20, 3-19, 3-18, 3-17, 3-16, 3-15, 3-14, 3-13, 3-12, 3-11, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12, 4-11, 4-10, 4-9, 4-8, 4-7, 4-6, 4- 5, 5-20, 5-19, 5-18, 5-17, 5-16, 5-15, 5-14, 5-13, 5-12, 5-11, 5-10, 5-9, 5-8, 5-7, 5-6, 6-20, 6-19, 6-18, 6-17, 6-16, 6-15, 6-14, 6-13, 6-12, 6-11, 6-10, 6-9, 6-8, 6-7, 7-20, 7-19, 7-18, 7-17, 7-16, 7-15, 7-14, 7-13, 7-12, 7-11, 7-10, 7-9, 7-8, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8- 13, 8-12, 8-11, 8-10, 8-9, 9-20, 9-19, 9-18, 9-17, 9-16, 9-15, 9-14, 9-13, 9-12, 9-11, 9-10, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12- 13, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-20, 14-19, 14-18, 14-17, 14-16, 14- 15, 15-20, 15-19, 15-18, 15-17, 15-16, 16-20, 16-19, 16-18, 16-17, 17-20, 17-19, 17-18, 18- 20, 18-19, or 19-20 polypeptides. In some embodiments, engineered nucleic acids are configured to produce 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 polypeptides. In some embodiments, engineered nucleic acids are configured to produce two polypeptides. In some embodiments, engineered nucleic acids are configured to produce three polypeptides. In some embodiments, engineered nucleic acids are configured to produce four polypeptides. In some embodiments, engineered nucleic acids are configured to produce five polypeptides.

[0412] In some embodiments, a provided engineered nucleic acid can be multicistronic, i.e., more than one separate polypeptide (e.g., multiple exogenous polynucleotides or effectormolecules, or engineered polypeptides as described herein) can be produced from a single transcript. In some embodiments, engineered nucleic acids can be multicistronic through the use of various linkers, e.g., a polynucleotide sequence encoding a first exogenous polynucleotide or effector molecule can be linked to a nucleotide sequence encoding a second exogenous polynucleotide or effector molecule, such as in a first gene: I inker: second gene 5’ to 3’ orientation. In some embodiments, a linker polynucleotide sequence comprises a polynucleotide sequence encoding a 2A ribosome skipping element, such as T2A. In some embodiments, a 2A ribosome skipping element comprises E2A, P2A, or F2A ribosome skipping elements. In some embodiments, a 2A ribosome skipping element comprises a P2A ribosome skipping element. In some embodiments, a P2A ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 195. In some embodiments, a P2A ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 271. In some embodiments, a 2A ribosome skipping element comprises a T2A ribosome skipping element. In some embodiments, a T2A ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 267. In some embodiments, a 2A ribosome skipping element comprises an E2A G4S T2A (Opt2A) ribosome skipping element. In some embodiments, an E2A G4S T2A (Opt2A) ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 265. In some embodiments, a 2A ribosome skipping element comprises a P2A 3-T2A 2 (Opt2A 2.0) ribosome skipping element. In some embodiments, a P2A 3-T2A 2 (Opt2A 2.0) ribosome skipping element comprises an amino acid sequence as set forth in SEQ ID NO: 269. 2A ribosome skipping elements allow production of separate polypeptides encoded by the first and second genes are produced during translation. In some embodiments, a linker is or comprises a cleavable linker polypeptide sequence, such as a Furin cleavage site or a TEV cleavage site, wherein following expression the cleavable linker polypeptide is cleaved such that separate polypeptides encoded by the first and second genes are produced. In some embodiments, a cleavable linker sequence comprises a flexible linker sequence that further promotes cleavage. In some embodiments, a flexible linker comprises a Gly-Ser-Gly sequence).

[0413] In some embodiments, a linker polynucleotide sequence comprises a polynucleotide sequence encoding an Internal Ribosome Entry Site (IRES), such that separate polypeptides encoded by the first and second genes are produced during translation. In some embodiments, a linker polynucleotide sequence comprises a polynucleotide sequence encoding a splice acceptor, such as a viral splice acceptor.

[0414] In some embodiments, a linker comprises combination of linker sequences, such as a Furin-2A linker that can produce separate polypeptides through 2A ribosome skipping followed by further cleavage of the Furin site to allow for complete removal of 2A residues. In some embodiments, a combination of linkers can include a Furin sequence, a flexible linker, and 2A linker. Accordingly, in some embodiments, the linker is a Furin-Gly-Ser-Gly- 2A fusion polypeptide. In some embodiments, a linker is a Furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0415] In general, a multicistronic system used in accordance with the present disclosure may comprise any number or combination of linkers, to express any number of genes or portions thereof (e.g., an engineered nucleic acid can encode a first, a second, and a third effector molecule or engineered polypeptide as described herein, each separated by linkers such that separate polypeptides encoded by the first, second, and third effector molecules are produced).

[0416] ‘ ‘Linkers,” as used herein can refer to polypeptides that link a first polypeptide sequence and a second polypeptide sequence or the multicistronic linkers as described herein.Post-Transcriptional Regulatory Elements

[0417] In some embodiments, an engineered nucleic acid of the present disclosure comprises a post-transcriptional regulatory element (PRE). In some embodiments, a PRE can enhance gene expression via enabling tertiary RNA structure stability and 3’ end formation. Non-limiting examples of PREs include the Hepatitis B virus PRE (HPRE) and the Woodchuck Hepatitis Virus PRE (WPRE). In some embodiments, the post-transcriptional regulatory element is a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE). In some embodiments, the WPRE comprises the alpha, beta, and gamma components of the WPRE element. In some embodiments, the WPRE comprises the alpha component of the WPRE element.Payload molecules

[0418] In some embodiments, an engineered nucleic acid of the present invention (e.g., and engineered nucleic acid encoding an ACP) comprises one or more nucleic acid seqeunces encoding a payload molecule. In some embodiments, an ACP controls expression of the one or more pay load molecules.

[0419] In some embodiments, a payload molecule is an effector molecule. Any suitable effector molecule known in the art can be encoded by the engineered nucleic acid orexpressed by the engineered cell. Suitable effector molecules can be grouped into therapeutic classes based on structure similarity, sequence similarity, or function. Effector molecule therapeutic classes include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, co-activation molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0420] In some embodiments, each effector molecule is independently selected from a therapeutic class, wherein the therapeutic class is selected from: a cytokine, a chemokine, a homing molecule, a growth factor, a co-activation molecule, a tumor microenvironment modifier a, a receptor, a ligand, an antibody, a polynucleotide, a peptide, and an enzyme.

[0421] In some embodiments, a effector molecule is a chemokine. Chemokines are small cytokines or signaling proteins secreted by cells that can induce directed chemotaxis in cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C and XC, all of which exert biological effects by binding selectively to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that may be encoded by the engineered nucleic acids of the present disclosure include: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1, or any combination thereof. In some embodiments, the chemokine is selected from: CCL21a, CXCL10, CXCL11, CXCL13, a CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0422] In some embodiments, a effector molecule is a cytokine. Non-limiting examples of cytokines that may be encoded by the engineered nucleic acids of the present disclosure include: ILl-beta, IL2, IL4, IL6, IL7, IL10, IL12, an IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, Type I interferons, Interferon-gamma, and TNF-alpha, or any combination thereof. In some embodiments, the cytokine is selected from: ILl-beta, IL2, IL4, IL6, IL7, IL 10, IL 12, an IL12p70 fusion protein, IL15, IL 17 A, IL18, IL21, IL22, Type I interferons, Interferon-gamma, and TNF-alpha.

[0423] In some embodiments, a payload molecule is a homing molecule. “Homing,” refers to active navigation (migration) of a cell to a target site (e.g., a cell, tissue (e.g., tumor), or organ). A “homing molecule” refers to a molecule that directs cells to a target site. In some embodiments, a homing molecule functions to recognize and / or initiate interaction of an engineered cell to a target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, PDGFR, anti-integrin alpha4,beta7; anti-MAdCAM; CCR9; CXCR4; SDF1; MMP-2;CXCR1; CXCR7; CCR2; CCR4; and GPR15, or any combination thereof. In someembodiments, the homing molecule is selected from: anti-integrin alpha4,beta7 ; anti- MAdCAM; CCR9; CXCR4; SDF1; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

[0424] In some embodiments, a payload molecule is a growth factor. Suitable growth factors for use as an effector molecule include, but are not limited to, FLT3L and GM-CSF, or any combination thereof. In some embodiments, the growth factor is selected from: FLT3L and GM-CSF.

[0425] In some embodiments, a payload molecule is a co-activation molecule. Suitable co-activation molecules for use as an effector molecule include, but are not limited to, c-Jun, 4-1BBL and CD40L, or any combination thereof. In some embodiments, the co-activation molecule is selected from: c-Jun, 4-1BBL and CD40L.

[0426] A “tumor microenvironment” is the cellular environment in which a tumor exists, including surrounding blood vessels, immune cells, fibroblasts, bone marrow-derived inflammatory cells, lymphocytes, signaling molecules and the extracellular matrix (ECM) (see, e.g., Pattabiraman, D.R. & Weinberg, R.A. Nature Reviews Drug Discovery 13, 497- 512 (2014); Balkwill, F.R. et al. J Cell Sci 125, 5591-5596, 2012; and Li, H. et al. J Cell Biochem 101(4), 805-15, 2007). Suitable tumor microenvironment modifiers for use as an effector molecule include, but are not limited to, adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof. In some embodiments, the tumor microenvironment modifier is selected from: adenosine deaminase, TGFbeta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

[0427] In some embodiments, a payload molecule is a TGFbeta inhibitor. Suitable TGFbeta inhibitors for use as an effector molecule include, but are not limited to, an anti- TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, or combinations thereof. In some embodiments, the TGFbeta inhibitors are selected from: an anti-TGFbeta peptide, an anti-TGFbeta antibody, a TGFb-TRAP, and combinations thereof.

[0428] In some embodiments, a payload molecule is an immune checkpoint inhibitor. Suitable immune checkpoint inhibitors for use as an effector molecule include, but are not limited to, anti-PD-1 antibodies, anti-PD-Ll antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti-TIM-3 antibodies, anti-TIGIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti-B7-H3 antibodies, anti-B7-H4 antibodies, anti- HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, antiphosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREMl antibodies, and anti-TREM2 antibodies, or any combination thereof. In some embodiments, the immune checkpoint inhibitors are selected from: anti-PD-1 antibodies, anti-PD-Llantibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-LAG-3 antibodies, anti- TIM-3 antibodies, anti-TIGIT antibodies, anti- VISTA antibodies, anti-KIR antibodies, anti- B7-H3 antibodies, anti-B7-H4 antibodies, anti-HVEM antibodies, anti-BTLA antibodies, anti-GAL9 antibodies, anti-A2AR antibodies, anti-phosphatidylserine antibodies, anti-CD27 antibodies, anti-TNFa antibodies, anti-TREMl antibodies, and anti-TREM2 antibodies.

[0429] Illustrative immune checkpoint inhibitors include pembrolizumab (anti-PD- 1 ; MK-3475 / Keytruda® - Merck), nivolumamb (anti-PD- 1; Opdivo® - BMS), pidilizumab (anti-PD-1 antibody; CT-011 - Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti- PD-L1; Bavencio® - Pfizer), durvalumab (anti-PD-Ll; MEDI4736 / Imfinzi® - Medimmune / AstraZeneca), atezolizumab (anti-PD-Ll; Tecentriq® - Roche / Genentech), BMS-936559 (anti-PD-Ll - BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy ® - BMS), lirilumab (anti-KIR; BMS), monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0430] In some embodiments, a payload molecule is a VEGL inhibitor. Suitable VEGE inhibitors for use as an effector molecule include, but are not limited to, anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof. In some embodiments, the VEGF inhibitors comprise anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

[0431] In some embodiments, each payload molecule is a human-derived payload molecule.Secrection Signals

[0432] In general, the one or more payload molecules comprise a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the payload molecule’s N-terminus that direct newly synthesized proteins destined for secretion or membrane insertion to the proper protein processing pathways. In embodiments with two or more payload molecules, each pay load molecule can comprise a secretion signal (S). In embodiments with two or more payload molecules, each payload molecule can comprise a secretion signal such that each payload molecule is secreted from an engineered cell. In embodiments, the second expression cassette comprising one or more units of (L - E)x further comprises a polynucleotide sequence encoding a secretion signal peptide (S). In embodiments, for each X the corresponding secretion signal peptide is operably associated with the payload molecule. In embodiments, the second expression cassette comprising an ACP-responsive promoter and a second exogenous polynucleotide sequence having the formula: (L -S- E)x.

[0433] The secretion signal peptide operably associated with a payload molecule can be a native secretion signal peptide native secretion signal peptide (e.g., the secretion signal peptide generally endogenously associated with the given pay load molecule). The secretion signal peptide operably associated with a pay load molecule can be a non-native secretion signal peptide native secretion signal peptide. Non-native secretion signal peptides can promote improved expression and function, such as maintained secretion, in particular environments, such as tumor microenvironments. Non-limiting examples of non-native secretion signal peptide are shown in Table 14.Table 14. Exemplary Signal Secretion PeptidesEngineered Cells

[0434] Also provided herein are engineered cells, and methods of producing engineered cells, that comprise one or more engineered nucleic acids of the present disclosure.Accordingly, provided engineered cells, which typically contain one or more engineered nucleic acids as described herein, do not occur in nature. In some embodiments, engineered cells are isolated (e.g., an engineered cell may be substantially isolated from other cell types including non-engineered cells and / or other distinct engineered cells). In some embodiments, an engineered cell is isolated for a particular use (e.g., any use described herein).. In some embodiments, an engineered cell comprises one or more vectors that comprises one or more engineered nucleic acids as described. In some embodiments, an engineered cell comprises one or more loci within its genome that comprise one or more engineered nucleic acids as described herein. In some embodiments, engineered cells comprise at least one promoter operably linked to an exogenous polynucleotide sequence (e.g., a polynucleotide sequence encoding any engineered polypeptide as described herein).

[0435] An engineered cell of the present disclosure, in some embodiments, comprises an engineered nucleic acid integrated into the cell’s genome. In some embodiments, an engineered cell comprises an engineered nucleic acid capable of expression withoutintegrating into the cell’s genome. In some embodiments, an engineered cell comprises a vector that comprises an engineered nucleic acid. In some embodiments, an engineered cell is contacted with an engineered nucleic acid (e.g., in the form of an mRNA) so that said engineered nucleic acid is expressed in the engineered cell.Engineered Cell Types

[0436] In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a human cell. In some embodiments, an engineered cell or isolated engineered cell is a human primary cell. In some embodiments, an primary cell is a somatic cell. In some embodiments, a primary cell is a stem cell. In some embodiments, a primary cell is an induced pluripotent stem cell (iPSC). In some embodiments, an engineered cell is derived from a subject. In some embodiments, an engineered cell is allogeneic with reference to a subject.

[0437] An engineered cell of the present disclosure, in some embodiments, can be made using a cell isolated from a subject. In some embodiments, a subject is known to have, or is suspected of having, cancer. In some embodiments, an engineered cell is made using a cancer cell isolated from a subject. Cell isolation methods 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. An engineered cell can be allogenic with reference to a subject being administered a treatment. Allogenic modified cells can be HLA-matched to a subject being administered a treatment. An engineered cell can be a cultured cell, such as an ex vivo cultured cell. An engineered cell can be an ex vivo cultured cell, such as a primary cell isolated from a subject. Cultured cell can be cultured with one or more cytokines.

[0438] In some embodiments, an engineered or isolated engineered cell of the present disclosure is selected from: a T cell (e.g., a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell), a cytotoxic T lymphocyte (CTL), a regulatory T cell, a Natural Killer T (NKT) cell, a Natural Killer (NK) cell, a B cell, a tumor-infiltrating lymphocyte (TIL), an innate lymphoid cell, a mast cell, an eosinophil, a basophil, a neutrophil, a myeloid cell, a macrophage (e.g., an Ml macrophage or an M2 macrophage), a monocyte, a dendritic cell, an erythrocyte, a platelet cell, a neuron, an oligodendrocyte, an astrocyte, a placode-derived cell, a Schwann cell, a cardiomyocyte, an endothelial cell, a nodal cell, a microglial cell, a hepatocyte, a cholangiocyte, a beta cell, a human embryonic stem cell (ESC), an ESC-derived cell, apluripotent stem cell, a mesenchymal stromal cell (MSC), an induced pluripotent stem cell (iPSC), and an iPSC-derived cell.

[0439] In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a T cell (e.g., a , a CD8+ T cell, a CD4+ T cell, or a gamma-delta T cell). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a cytotoxic T lymphocyte (CTL). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a regulatory T cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a Natural Killer T (NKT) cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a Natural Killer (NK) cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a B cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a tumor-infiltrating lymphocyte (TIL). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an innate lymphoid cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a mast cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an eosinophil. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a basophil. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a neutrophil. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a myeloid cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a macrophage e.g., an Ml macrophage or an M2 macrophage). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a monocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a dendritic cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an erythrocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a platelet cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a neuron. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an oligodendrocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an astrocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a placode-derived cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a Schwann cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a cardiomyocyte. In some embodiments, anengineered cell or isolated engineered cell of the present disclosure is an endothelial cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a nodal cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a microglial cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a hepatocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a cholangiocyte. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a beta cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a human embryonic stem cell (ESC). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an ESC-derived cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a pluripotent stem cell. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a mesenchymal stromal cell (MSC). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an induced pluripotent stem cell (iPSC). In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is an iPSC-derived cell. In some embodiments, an engineered cell is autologous. In some embodiments, an engineered cell is allogeneic. In some embodiments, an engineered cell or isolated engineered cell of the present disclosure is a CD34+ cell, a CD3+ cell, a CD8+ cell, a CD16+ cell, and / or a CD4+ cell.

[0440] In some embodiments, an engineered cell of the present disclosure is a cell therapy modality. In some embodiments, a cell therapy modality can express one or more therapeutic proteins. In some embodiments, a therapeutic protein is a chimeric antigen receptor (CAR) or a modified T cell receptor (TCR). In some embodiments, a cell therapy modality is a CAR-T cell or a CAR-NK cell. In some embodiments, a cell therapy modality is a CAR-T cell. In some embodiments, a cell therapy modality is a CAR-NK cell.

[0441] In some embodiments, an engineered cell of the present disclosure is a tumor cell. In some embodiments, an engineered cell of the present disclosure is selected from: an adenocarcinoma cell, a bladder tumor cell, a brain tumor cell, a breast tumor cell, a cervical tumor cell, a colorectal 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.

[0442] In some embodiments, an engineered cell of the present disclosure is a bacterial cell. In some embodiments, an engineered cell of the present disclosure is selected from:Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

[0443] Also provided herein are methods of culturing the engineered cells of the present disclosure. Provided engineered cells may be cultured using any suitable method. One skilled in the art will recognize that culturing conditions will depend on the particular engineered cell of interest. One skilled in the art will recognize that culturing conditions will depend on the specific downstream use of an engineered cell, for example, specific culturing conditions for subsequent administration of an engineered cell to a subject.Methods of Engineering Cells

[0444] Also provided herein are compositions and methods for engineering cells with any nucleic acid as described herein.

[0445] Provided engineered cells may be engineered through introduction (z.e., delivery) of one or more polynucleotides (e.g., engineered nucleic acids) 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. One skilled in the art will appreciate the choice of delivery method can depend on the specific cell type to be engineered.

[0446] In some embodiments, an engineered cell is transduced using an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. In some embodiments, an oncolytic virus is a recombinant oncolytic virus. In some embodiments, a recombinant oncolytic virus comprises an engineered nucleic acid as provided herein. In some embodiments, an oncolytic virus is a recombinant oncolytic virus comprising a firstexpression cassette and a second expression cassette. In some embodiments, an oncolytic virus further comprises a third expression cassette.

[0447] In some embodiments, a virus as provided in accordance with the present disclosure is an oncolytic virus. In some embodiments, avirus is a recombinant virus that encodes one more transgenes encoding one or more pay load molecules, such as any of the engineered nucleic acids described herein. In some embodiments, an engineered cell is engineered via transduction with an oncolytic virus.Viral-Mediated Delivery

[0448] Any suitable viral vector-based delivery platform can be used to make engineered cells of the present disclosure. In general, a viral vector-based delivery platform engineers a cell through introducing (i.e., delivering) a nucleic acid payload (e.g., any engineered nucleic acid as described herein) into a host cell. A viral vector-based delivery platform may comprise engineered or recombinant viruses to deliver engineered nucleic acids of the present disclosure to a cell.

[0449] A viral vector-based delivery platform can deliver one or more nucleic acid payloads, e.g., an engineered nucleic acid, gene, or transgene as described herein, within the same nucleic acid. In some embodiments, a viral vector-based delivery platform (e.g., a platform using a recombinant virus or an engineered virus) can deliver one or more transgenes, including, but not limited to, any engineered nucleic acid described herein that encodes one or more pay load molecules. In some embodiments, one or more transgenes encoding one or more pay load molecules can be configured to express the one or more pay load molecules. A viral vector-based delivery platform can encode one or more genes in addition to the one or more transgenes (e.g., transgenes encoding the one or more pay load molecules), such as viral genes needed for viral infectivity and / or viral production (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.), referred to as cis-acting elements or genes.

[0450] A viral vector-based delivery platform can comprise more than one viral vector, such as separate viral vectors encoding the engineered nucleic acids, genes, or transgenes described herein, and referred to as trans-acting elements or genes. For example, a helperdependent viral vector-based delivery platform can provide additional genes needed for viral infectivity and / or viral production on one or more additional separate vectors in addition to the vector encoding the one or more effector molecules. One viral vector can deliver more than one engineered nucleic acids, such as one vector that delivers engineered nucleic acidsthat are configured to produce two or more effector molecules. More than one viral vector can deliver more than one engineered nucleic acids, such as more than one vector that delivers one or more engineered nucleic acid configured to produce one or more effector molecules. The number of viral vectors used can depend on the packaging capacity of the above mentioned viral vector-based vaccine platforms, and one skilled in the art can select the appropriate number of viral vectors.

[0451] In general, any of the viral vector-based systems can be used for the in vitro production of molecules, such as effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules. The selection of an appropriate viral vector-based system will depend on a variety of factors, such as cargo / payload size, immunogenicity of the viral system, target cell of interest, gene expression strength and timing, and other factors appreciated by one skilled in the art.

[0452] Viral vector-based delivery platforms can be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, a herpes simplex virus, a adenovirus, a measles virus, an influenza virus, a Indiana vesiculovirus, a Newcastle disease virus, a vaccinia virus, a poliovirus, a myxoma virus, a reovirus, a mumps virus, a Maraba virus, a rabies virus, a rotavirus, a hepatitis virus, a rubella virus, a dengue virus, a chikungunya virus, a respiratory syncytial virus, a lymphocytic choriomeningitis virus, a morbillivirus, a lentivirus, a replicating retrovirus, a rhabdovirus, a Seneca Valley virus, a sindbis virus, and any variant or derivative thereof. Other exemplary viral vector-based delivery platforms are described in the art, such as vaccinia, fowlpox, selfreplicating alphavirus, marabavirus, 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 lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev. (2011) 239(1): 45-61, Sakuma et al., Lentiviral vectors: basic to translational, Biochem J. (2012) 443(3):603-18, Cooper et al., Rescue of splicing-mediated intron loss maximizes expression in lentiviral vectors containing the human ubiquitin C promoter, Nucl. Acids Res. (2015) 43 (1): 682-690, Zufferey et al., Self-Inactivating Lentivirus Vector for Safe and Efficient In vivo Gene Delivery, J. Virol. (1998) 72 (12): 9873-9880).

[0453] The sequences may be preceded with one or more sequences targeting a subcellular compartment. Upon introduction (i.e. delivery) into a host cell, infected cells (i.e.,an engineered cell) can express, and in some case secrete, the one or more effector molecules. Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, e.g., Salmonella typhi vectors, and the like will be apparent to those skilled in the art from the description herein.

[0454] The viral vector-based delivery platforms can be a virus that targets a tumor cell, herein referred to as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, an oncolytic herpes simplex virus, an oncolytic adenovirus, an oncolytic measles virus, an oncolytic influenza virus, an oncolytic Indiana vesiculovirus, an oncolytic Newcastle disease virus, an oncolytic vaccinia virus, an oncolytic poliovirus, an oncolytic myxoma virus, an oncolytic reovirus, an oncolytic mumps virus, an oncolytic Maraba virus, an oncolytic rabies virus, an oncolytic rotavirus, an oncolytic hepatitis virus, an oncolytic rubella virus, an oncolytic dengue virus, an oncolytic chikungunya virus, an oncolytic respiratory syncytial virus, an oncolytic lymphocytic choriomeningitis virus, an oncolytic morbillivirus, an oncolytic lentivirus, an oncolytic replicating retrovirus, an oncolytic rhabdovirus, an oncolytic Seneca Valley virus, an oncolytic sindbis virus, and any variant or derivative thereof. Any of the oncolytic viruses described herein can be a recombinant oncolytic virus comprising one more transgenes (e.g., an engineered nucleic acid) encoding one or more effector molecules. The transgenes encoding the one or more effector molecules can be configured to express the one or more effector molecules.

[0455] 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).

[0456] The viral vector-based delivery platform can be retrovirus-based. In general, retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the one or more engineered nucleic acids (e.g., transgenes encoding the one or more effector molecules) into the target cell to provide permanent transgene expression. Retroviral-based delivery systems include, but are not limited to, those based upon murine leukemia, virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency vims (SIV), human immunodeficiency vims (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et ah, J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59(1990); Wilson et ah, J. Virol. 63:2374-2378 (1989); Miller et al, J, Virol. 65:2220-2224 (1991); PCT / US 94 / 05700). Other retroviral systems include the Phoenix retrovirus system.

[0457] The viral vector-based delivery platform can be lentivirus-based. In general, lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as ViraPower systems (ThermoFisher) or pLenti systems (Cell Biolabs). . Lentiviral- based delivery platforms can be SIV, or FIV-based. Other exemplary lentivirus-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; 6,955,919, each herein incorporated by reference for all purposes.

[0458] The viral vector-based delivery platform can be adenovirus-based. In general, adenoviral based vectors are capable of very high transduction efficiency in many cell types, do not require cell division, achieve high titer and levels of expression, and can be produced in large quantities in a relatively simple system. In general, adenoviruses can be used for transient expression of a transgene within an infected cell since adenoviruses do not typically integrate into a host’s genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655, each herein incorporated by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Pat. Nos. 5585362; 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 WO96 / 13597, each herein incorporated by reference for all purposes.

[0459] The viral vector-based delivery platform can be adeno-associated virus (AAV)- based. Adeno-associated virus (“AAV”) vectors may be used to transduce cells with engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). AAV systems can be used for the in vitro production of effector molecules, or used in vivo and ex vivo gene therapy procedures, e.g., for in vivo delivery of the engineered nucleic acids encoding one or more effector molecules (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; US patent publications US 2003-0138772, US 2007 / 0036760, and US 2009 / 0197338; 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 applicationsI l lWO 2010 / 138263 and WO 93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each herein incorporated by reference for all purposes). Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Pat. No, 5,173,414; Tratschin et ah, Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et ah, Mol. Cell, Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:64666470 (1984); and Samuiski et ah, J. Virol. 63:03822-3828 (1989), each herein incorporated by reference for all purposes. In general, 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.RhlO, AAV11 and variants thereof.

[0460] The viral vector-based delivery platform can be a virus-like particle (VLP) platform. In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo / payload (e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids 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 to produce VLPs using any suitable method. Production of VLPs are described in more detail in Seow et al. (Mol Ther. 2009 May; 17(5): 767-777), herein incorporated by reference for all purposes.

[0461] The viral vector-based delivery platform can be engineered to target (i.e., infect) a range of cells, target a narrow subset of cells, or target a specific cell. In general, the envelope protein chosen for the viral vector-based delivery platform will determine the viral tropism. The virus used in the viral vector-based delivery platform can be pseudotyped to target a specific cell of interest. The viral vector-based delivery platform can be pantropic and infect a range of cells. Lor example, pantropic viral vector-based delivery platforms can include the VSV-G envelope. The viral vector-based delivery platform can be amphotropic and infect mammalian cells. Accordingly, one skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein for targeting a desired cell type.Lipid Structure Delivery Systems

[0462] Engineered nucleic acids of the present disclosure (e.g., any of the engineered nucleic acids described herein) can be introduced into a cell using a lipid-mediated delivery system. In general, a lipid-mediated delivery system uses a structure composed of an outer lipid membrane enveloping an internal compartment. Examples of lipid-based structures include, but are not limited to, a lipid-based nanoparticle, a liposome, a micelle, an exosome, a vesicle, an extracellular vesicle, a cell, or a tissue. Lipid structure delivery systems can deliver a cargo / payload (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0463] A lipid-based nanoparticle can include, but is not limited to, a unilamellar liposome, a multilamellar liposome, and a lipid preparation. As used herein, a “liposome” is a generic term encompassing in vitro preparations of lipid vehicles formed by enclosing a desired cargo, e.g., an engineered nucleic acid, such as any of the engineered nucleic acids described herein, within a lipid shell or a lipid aggregate. Liposomes may be characterized as having vesicular structures with a bilayer membrane, generally comprising a phospholipid, and an inner 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, the liposomes are neutral in charge. Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of a desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys.Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each herein incorporated by reference for all purposes.

[0464] A multilamellar liposome is generated spontaneously when lipids comprising phospholipids are suspended in an excess of aqueous solution such that multiple lipid layers are separated by an aqueous medium. Water and dissolved solutes are entrapped in closed structures between the lipid bilayers following the lipid components undergoing selfrearrangement. A desired cargo (e.g., a polypeptide, a nucleic acid, a small molecule drug, anengineered nucleic acid, such as any of the engineered nucleic acids described herein, a viral vector, a viral-based delivery system, etc.) can be encapsulated in the aqueous interior of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the polypeptide / nucleic acid, interspersed within the lipid bilayer of a liposome, entrapped in a liposome, complexed with a liposome, or otherwise associated with the liposome such that it can be delivered to a target entity. Lipophilic molecules or molecules with lipophilic regions may also dissolve in or associate with the lipid bilayer.

[0465] A liposome used according to the present embodiments can be made using any suitable method. Preparations of liposomes are described in further detail in WO 2016 / 201323, International Applications PCT / US85 / 01161 and PCT / US89 / 05040, and U.S. Patents 4,728,578, 4,728,575, 4,737,323, 4,533,254, 4,162,282, 4,310,505, and 4,921,706; each herein incorporated by reference for all purposes.

[0466] Liposomes can be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patent No. 5,962,016; 5,030,453; 6,680,068, U.S. Application 2004 / 0208921, and International Patent Applications W003 / 015757A1, WO04029213A2, and W002 / 100435A1, each hereby incorporated by reference in their entirety.

[0467] Lipid- mediated gene delivery methods are described, for instance, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; W091 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987), each herein incorporated by reference for all purposes.

[0468] Exosomes are small membrane vesicles of endocytic origin that are released into the extracellular environment following fusion of multivesicular bodies with the plasma membrane. The size of exosomes ranges between 30 and 100 nm in diameter. Their surface consists of a lipid bilayer from the donor cell’s cell membrane, and they contain cytosol from the cell that produced the exosome, and exhibit membrane proteins from the parental cell on the surface. Any exosomes useful for the delivery of nucleic acids can be used in accordance with the present disclosure, e.g., the exosomes described in more detail in U.S. Pat. No. 9,889,210, herein incorporated by reference for all purposes.

[0469] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle comprising a membrane that encloses an internal space. In general, extracellular vesicles comprise all membrane-bound vesicles that have a smaller diameter than the cell from which they are derived. Generally extracellular vesicles range in diameter from 20 nmto 1000 nm, and can comprise various macromolecular cargo either within the internal space, displayed on the external surface of the extracellular vesicle, and / or spanning the membrane. The cargo can comprise 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, fragments of cells, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesiculated organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of the late endosome with the plasma membrane). Extracellular vesicles can be derived from a living or dead organism, explanted tissues or organs, and / or cultured cells.

[0470] As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The exosome comprises lipid or fatty acid and polypeptide and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The exosome can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. An exosome is a species of extracellular vesicle. Generally, exosome production / biogenesis does not result in the destruction of the producer cell. Exosomes and preparation of exosomes are described in further detail in WO 2016 / 201323, which is hereby incorporated by reference in its entirety.

[0471] As used herein, the term “nanovesicle” (also referred to as a “microvesicle”) refers to a cell-derived small (between 20-250 nm in diameter, more preferably 30-150 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from the cell by direct or indirect manipulation such that said nanovesicle would not be produced by said producer cell without said manipulation. In general, a nanovesicle is a sub-species of an extracellular vesicle. Appropriate manipulations of the producer cell include but are not limited to serial extrusion, treatment with alkaline solutions, sonication, or combinations thereof. The production of nanovesicles may, in some instances, result in the destruction of said producer cell. Preferably, populations of nanovesicles are substantially free of vesicles that are derived from producer cells by way of direct budding from the plasma membrane or fusion of the late endosome with the plasma membrane. Thenanovesicle comprises lipid or fatty acid and polypeptide, and optionally comprises a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting moiety), a polynucleotide (e.g., a nucleic acid, RNA, or DNA, such as any of the engineered nucleic acids described herein), a sugar (e.g., a simple sugar, polysaccharide, or glycan) or other molecules. The nanovesicle, once it is derived from a producer cell according to said manipulation, may be isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof.

[0472] Lipid nanoparticles (LNPs), in general, are synthetic lipid structures that rely on the amphiphilic nature of lipids to form membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver cargo / pay loads, such as any of the engineered nucleic acids or viral systems described herein, by absorbing into the membrane of target cells and releasing the cargo into the cytosol. Lipids used in LNP formation can be cationic, anionic, or neutral. The lipids can be synthetic or naturally derived, and in some instances biodegradable. Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as the cationic, neutral, anionic, and amphipathic lipids. In some instances, 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 influence overall LNP size and stability. In an example, the lipid composition comprises 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 a PEG or PEG-conjugated lipid, a sterol, 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.

[0473] Micelles, in general, are spherical synthetic lipid structures that are formed using single-chain lipids, where the single-chain lipid’s hydrophilic head forms an outer layer or membrane and the single-chain lipid’ s hydrophobic tails form the micelle center. Micelles typically refer to lipid structures only containing a lipid mono-layer. Micelles are described in more detail in Quader et al. (Mol Ther. 2017 Jul 5; 25(7): 1501-1513), herein incorporated by reference for all purposes.

[0474] Nucleic-acid vectors, such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids. Similarly, viral delivery systems exposed directly to serum can trigger an undesired immune responseand / or neutralization of the viral delivery system. Therefore, encapsulation of an engineered nucleic acid and / or viral delivery system can be used to avoid degradation, while also avoiding potential off-target effects. In certain examples, an engineered nucleic acid and / or viral delivery system is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP. Encapsulation of an engineered nucleic acid and / or viral delivery system within an LNP can be carried out using any suitable method, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device. Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices. In an example, the desired lipid formulation, such as MC3 or MC3-like containing compositions, is provided to the droplet generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired agents, such that the delivery vector and desired agents are fully encapsulated within the interior of the MC3 or MC3-like based LNP. In an example, the droplet generating device can control the size range and size distribution of the LNPs produced. Lor example, the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. Pollowing droplet generation, the delivery vehicles encapsulating the cargo / payload (e.g., an engineered nucleic acid and / or viral delivery system) can be further treated or engineered to prepare them for administration.Nanoparticle Delivery

[0475] Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Nanomaterial vehicles, importantly, can be made of non-immunogenic materials and generally avoid eliciting immunity to the delivery vector itself. These materials can include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymeric materials. Nanomaterial particles are described in more detail in Riley et al. (Recent Advances in Nanomaterials for Gene Delivery — A Review. Nanomaterials 2017, 7(5), 94), herein incorporated by reference for all purposes.Genomic Editing Systems

[0476] A genomic editing systems can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure. In general, a “genomic editing system” refers to any system for integrating an exogenous gene into a host cell’s genome. Genomic editing systems include, but are not limited to, atransposon system, a nuclease genomic editing system, and a viral vector-based delivery platform.

[0477] A transposon system can be used to integrate an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure, into a host genome. Transposons generally comprise terminal inverted repeats (TIR) that flank a cargo / payload nucleic acid and a transposase. The transposon system can provide the transposon in cis or in trans with the TIR-flanked cargo. A transposon system can be a retrotransposon system or a DNA transposon system. In general, transposon systems integrate a cargo / payload (e.g., an engineered nucleic acid) randomly into a host genome. Examples of transposon systems include systems using a transposon of the Tcl / mariner transposon superfamily, such as a Sleeping Beauty transposon system, 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 herein incorporated by reference for all purposes. Another example of a transposon system includes a PiggyBac transposon system, described in more detail in U.S. Patent Nos. 6,218,185 and 6,962,810, each of which is herein incorporated by reference for all purposes.

[0478] A nuclease genomic editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid of the present disclosure. Without wishing to be bound by theory, in general, the nuclease-mediated gene editing systems used to introduce an exogenous gene take advantage of a cell’s natural DNA repair mechanisms, particularly homologous recombination (HR) repair pathways. Briefly, following an insult to genomic DNA (typically a double-stranded break), a cell can resolve the insult by using another DNA source that has identical, or substantially identical, sequences at both its 5’ and 3’ ends as a template during DNA synthesis to repair the lesion. In a natural context, HDR can use the other chromosome present in a cell as a template. In gene editing systems, exogenous polynucleotides are introduced into the cell to be used as a homologous recombination template (HRT or HR template). In general, any additional exogenous sequence not originally found in the chromosome with the lesion that is included between the 5’ and 3’ complimentary ends within the HRT (e.g., a gene or a portion of a gene) can be incorporated (i.e., “integrated”) into the 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 an 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 acidsdescribed herein, such as any of the engineered nucleic acids encoding one or more effector molecules).

[0479] In some examples, a HR template can be linear. Examples of linear HR templates include, but are not limited to, a linearized plasmid vector, a ssDNA, a synthesized DNA, and a PCR amplified DNA. In particular examples, a HR template can be circular, such as a plasmid. A circular template can include a supercoiled template.

[0480] The identical, or substantially identical, sequences found at the 5’ and 3’ ends of the HR template, with respect to the exogenous sequence to be introduced, are generally referred to as arms (HR arms). HR arms can be identical to regions of the endogenous genomic target locus (i.e., 100% identical). HR arms in some examples can be substantially identical to regions of the endogenous genomic target locus. While substantially identical HR arms can be used, it can be advantageous for HR arms to be identical as the efficiency of the HDR pathway may be impacted by HR arms having less than 100% identity.

[0481] Each HR arm, i.e., the 5’ and 3’ HR arms, can be the same size or different sizes. Each HR arm can each be greater than or equal to 50, 100, 200, 300, 400, or 500 bases in length. Although HR arms can, in general, be of any length, practical considerations, such as the impact of HR arm length and overall template size on overall editing efficiency, can also be taken into account. An HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical to, or substantially identical to, regions of an endogenous genomic target locus immediately adjacent to a cleavage site. Each HR arms can be identical, or substantially identical to, regions of an endogenous genomic target locus within a certain distance of a 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 of each other.

[0482] A nuclease genomic editing system can use a variety of nucleases to cut a target genomic locus, including, but not limited to, a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) family nuclease or derivative thereof, a Transcription activator-like effector nuclease (TALEN) or derivative thereof, a zinc-finger nuclease (ZFN) or derivative thereof, and a homing endonuclease (HE) or derivative thereof.

[0483] A CRISPR-mediated gene editing system can be used to engineer a host genome to encode an engineered nucleic acid, such as an engineered nucleic acid encoding one or more of the effector molecules described herein. CRISPR systems are described in more detail in M. Adli (“The CRISPR tool kit for genome editing and beyond” Nature Communications; volume 9 (2018), Article number: 1911), herein incorporated by referencefor all that it teaches. In general, a CRISPR-mediated gene editing system comprises a CRISPR-associated (Cas) nuclease and a RNA(s) that directs cleavage to a particular target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 systems comprised of a Cas9 nuclease and a RNA(s) that has a CRISPR RNA (crRNA) domain and a trans-activating CRISPR (tracrRNA) domain. The crRNA typically has two RNA domains: a guide RNA sequence (gRNA) that directs specificity through base-pair hybridization to a target sequence (“a defined nucleotide sequence”), e.g., a genomic sequence; and an RNA domain that hybridizes to a tracrRNA. A tracrRNA can interact with and thereby promote recruitment of a nuclease (e.g., Cas9) to a genomic locus. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can be a single polynucleotide, also referred to as a single guide RNA (sgRNA). While the Cas9 system is illustrated here, other CRISPR systems can be used, such as the Cpf 1 system. Nucleases can include derivatives thereof, such as Cas9 functional mutants, e.g., a Cas9 “nickase” mutant that in general mediates cleavage of only a single strand of a defined nucleotide sequence as opposed to a complete double- stranded break typically produced by Cas9 enzymes.

[0484] In general, the components of a 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 separately produced and used to form the RNP complex. In some CRISPR systems, each component can be separately produced in vitro and contacted (i.e., “complexed”) with each other in vitro to form the RNP complex. The in vitro produced RNP can then be introduced (i.e., “delivered”) into a cell’s cytosol and / or nucleus, e.g., a T cell’s cytosol and / or nucleus. The in vitro produced RNP complexes 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 (LNP), virus like particles (VLP), and sonication. In a particular example, in vitro produced RNP complexes can be delivered to a cell using a Nucleofactor / Nucleofection® electroporationbased delivery system (Lonza®). Other electroporation systems include, but are not limited to, MaxCyte electroporation systems, Miltenyi CliniMACS electroporation systems, Neon electroporation systems, and BTX electroporation systems. CRISPR nucleases, e.g., Cas9, can be produced in vitro (i.e., synthesized and purified) using any suitable method. CRISPR system RNAs, e.g., an sgRNA, can be produced in vitro (i.e., synthesized and purified) using a variety of RNA production techniques, such as in vitro transcription or chemical synthesis.

[0485] An in vitro produced RNP complex can be complexed at different ratios of nuclease to gRNA. An in vitro produced RNP complex can be also be used at different amounts in a CRISPR-mediated editing system. For example, depending on the number of cells desired to be edited, the total RNP amount added can be adjusted, such as a reduction in the amount of RNP complex added when editing a large number of cells in a reaction.

[0486] In some CRISPR systems, each component (e.g., Cas9 and an sgRNA) can be separately encoded by a polynucleotide with each polynucleotide 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 multicistronic vector, see description of exemplary multicistronic systems below) and introduced into a cell. Following expression of each polynucleotide encoded CRISPR component within a cell (e.g., translation of a nuclease and transcription of CRISPR RNAs), an RNP complex can form within the cell and can then direct site-specific cleavage.

[0487] Some RNPs can be engineered to have moieties that promote delivery of the RNP into the nucleus. For example, a Cas9 nuclease can have a nuclear localization signal (NLS) domain such that if a Cas9 RNP complex is delivered into a cell’s cytosol or following translation of Cas9 and subsequent RNP formation, the NLS can promote further trafficking of a Cas9 RNP into the nucleus.

[0488] The engineered cells described herein can be engineered using non-viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using non-viral methods. The engineered cells described herein can be engineered using viral methods, e.g., the nuclease and / or CRISPR mediated gene editing systems described herein can be delivered to a cell using viral methods such as adenoviral, retroviral, lentiviral, or any of the other viral-based delivery methods described herein.

[0489] In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target the same gene or general genomic locus at more than target nucleotide sequence. For example, two separate CRISPR compositions can be provided to direct cleavage at two different target nucleotide sequences within a certain distance of each other. In some CRISPR systems, more than one CRISPR composition can be provided such that each separately target opposite strands of the same gene or general genomic locus. For example, two separate CRISPR “nickase” compositions can be provided to direct cleavage at the same gene or general genomic locus at opposite strands.

[0490] In general, the features of a CRISPR-mediated editing system described herein can apply to other nuclease-based genomic editing systems. TALEN is an engineered site-specific nuclease, which is composed of the DNA- binding domain of TALE (transcription activator-like effectors) and the catalytic domain of restriction endonuclease Fokl. By changing the amino acids present in the highly variable residue region of the monomers of the DNA binding domain, different artificial TALENs can be created to target various nucleotides sequences. The DNA binding domain subsequently directs the nuclease to the target sequences and creates a double-stranded break. TALEN-based systems are described in more detail in U.S. Ser. No. 12 / 965,590; U.S. Pat. No. 8,450,471; U.S. Pat. No. 8,440,431; U.S. Pat. No. 8,440,432; U.S. Pat. No. 10,172,880; and U.S. Ser. No. 13 / 738,381, all of which are incorporated by reference herein in their entirety. ZFN-based editing systems are described in more detail in U.S. Patent Nos. 6,453,242; 6,534,261; 6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317; 7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent Publication Nos. 2005 / 0064474; 2007 / 0218528; 2005 / 0267061, all incorporated herein by reference in their entireties for all purposes.Other Engineering Delivery Systems

[0491] Various additional means are available to a skilled artisan for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target recipient entity, such as any of the lipid structures described herein. A skilled artisan will understand which methods are suitable for introducing engineered nucleic acids into a cell or other biological system.

[0492] In some embodiments, electroporation is used to deliver polynucleotides (e.g., any engineered nucleic acid described herein) to recipient entities. Electroporation is a method of internalizing a cargo / payload into a target cell or entity’s interior compartment through applying an electrical field to transiently permeabilize the outer membrane or shell of the target cell or entity. In general, the method involves placing cells or target entities between two electrodes in a solution containing a cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cells is then disrupted, i.e., permeabilized, by applying a transient set voltage that allows the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the example of cells, at least some, if not a majority, 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, electroporation solution composition, 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 efficiency ofinternalization desired, and the viability desired. Optimization of such criteria are within the scope of those skilled in the art. A variety devices and protocols can be used for electroporation. Examples include, but are not limited to, Neon® Transfection System, MaxCyte® Flow Electroporation™, Lonza® Nucleofector™ systems, and Bio-Rad® electroporation systems.

[0493] Other means for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into a cell or other target re...

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A modified degron comprising the amino acid sequence set forth in SEQ ID NO: 139, or the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X10, wherein Xi is I or R; X2 is F or W; X3is C or T; X4is R, D, H, or K; X5is Q, R, or T; X6is G or V; X7is N, T, or A; Xs is L or I; X9 is K or G; and X10 is L or F, and wherein the modified degron has increased sensitivity to one or more IMiDs compared to a degron comprising the amino acid sequence set forth in SEQ ID NO: 40 or 131, optionally wherein the modified degron comprises one or more amino acid substitutions at any one or more positions selected from G30E, Q28T, L33I, R27H, K37G, L38F, I21R AND G30V, R27D, Q28R, N31T, F24W, R27K, I21R, G30V, and N31A, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40, optionally wherein the IMiD is at least one of thalidomide, iberdomide, lenalidomide, and pomalidomide, optionally wherein the IMiD is pomalidomide.

2. The modified degron of claim 1, wherein the modified degron comprises an amino acid sequence set forth in any one of SEQ ID NOs: 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 226-230, 276, 280, 284, 288, 292, 296, 300, 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, or 788, optionally wherein the modified degron comprises SEQ ID NO: 148.

3. The modified degron of any one of claims 1 or 2, wherein the modified degron functionally associates with a cereblon domain or variant thereof in an IMiD responsive manner, optionally wherein the modified degron functionally associates with the cereblon domain or variant thereof when exposed to a lower concentration of IMiD, as compared to an unmodified degron.

4. The modified degron of any one of claims 1-3, wherein the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129, optionally wherein the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of IMiD, as compared to an unmodified cereblon, optionally wherein the cereblon domain or variant thereof is a modified cereblon as described in any one of claims 5-8.A modified cereblon (CRBN) comprising: a. one or more amino acid substitutions at positions Q325, V350, H353, Y355, N369, 1371, R373, E377, S379, Q390, A395, S396, H397, or F402 relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to the wildtype CRBN; b. the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10X11X12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATK KDMSPQKFWX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is T, F, I, L, V, W, or Y; X9is N, D, or Y; X10 is I, D, E, N, or Q; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is X, D, K, N, Q, or R; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y; and X25 is G or A, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127 or a truncation thereof comprising the amino acid sequence set forth in SEQ ID NO: 129; c. the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10X11X12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATK KDMSPQKFWX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H, Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is T, F, I, L, V, W, or Y; X9is N, D, or Y; X10 is I, D, E, N, Q, L, V, or M; Xu is G, N, or Q; X12 is R, E, N, S, or Y; X13 is S, D, K, N, Q, R, H, P; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y; and X25 is G,A, F, I, L, S, or V;d. one or more amino acid substitutions at any one or more positions selected from: K324, Q325, V350, N351, H353, Y355, H357, T359, N369, 1371, G372, R373, S375, E377, H378, S379, W380, F381, V388, Q390, A395, S396, H397, F402, and G416, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, and wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) as compared to the wildtype CRBN; e. the amino acid sequenceX1X2CQETEITTKNEIFSESECGPMAAYX3X4PX5GX6VX7EX8ETVYKACNEX9EX 10X11X12PX13TX14X15X16X17X18PGYAWTX19AX20CKICX21X22X23IGWKX24TATK KDMSPQKFWX25, wherein Xi is K, N, Q, or R; X2is Q, H, K, or R; X3is V, E, H,Q, R, or S; X4is N, A, I, L, or V; X5is H, P, Q, R, or S; X6is Y, H, N, R, or S; X7is H, Q, or W; X8is V; X9is N, D, or Y; X10 is I, D, E, N, or Q; Xu is G, N, or Q; X12 isR, E, N, S, or Y; X13 is X, D, K, N, Q, or R; Xi4is E, D, K, Q, S, or Y; X15 is H, D, Q, or T; Xi6 is S, N, or T; X17 is W, F, or Y; Xi8is F or W; X19 is V, D, E, N, Q, S, or L; X20 is Q, D, H, K, or R; X21 is A, D, E, N, or Q; X22 is S, G, or Q; X23 is H, D, F, K, Q, Y, I, L, or V; X24is F, H, N, Q, W, or Y; and X25 is G or A; or f. a T359 amino acid substitution, optionally wherein the T359 amino acid substitution is selected from: a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, optionally wherein the modified CRBN comprises a T359V substitution.

6. The modified CRBN of claim 5, wherein the modified CRBN does not comprise a DDB 1 interacting domain, optionally wherein the modified CRBN further comprises deletion of amino acids 194-247 relative to the wildtype CRBN, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 231 - 249. 256, 258, 260, 262, or 264.

7. The modified CRBN of claim 5 or 6, wherein the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, a) a Q325 amino acid substitution selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution; and one or more amino acid substitutions selected from: a 137 IN substitution, a H397F substitution, and a N369Y substitution; orb) a Q325H substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 351; or c) a Q325H substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 355; or d) a Q325H substitution and a N369Y substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 359; or e) a Q325H substitution, a N369Y substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 363; or f) a Q325H substitution, a N369Y substitution, and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 367; or g) a Q325H substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 371; or h) a Q325K substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 375; or i) a Q325K substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 383; or j) a Q325R substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%,at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 403; or k) a 137 IN substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 331; or l) a N369Y substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 335; or m) a N369Y substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 339; or n) a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 343; or o) a Q325H substitution and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 347; or p) a Q325K substitution and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 379; or q) a Q325K substitution and a N369Y substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 387; or r) a Q325K substitution, a N369Y substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 391; ors) a Q325K substitution, a N369Y substitution, and a 137 IN substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 395; or t) a Q325K substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 399; or u) a Q325R substitution, a N369Y substitution, a 137 IN substitution, and a H397F substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 315; or v) one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitution, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution; w) a Q325H substitution, a Q325H substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 544; or x) a Q325R substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 530; or y) a Q325R substitution, a N369Y substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 526; or z) a Q325R substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 524; or aa) a Q325K substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 532; or bb) a Q325H substitution, a N369Y substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 528; or cc) a Q325H substitution, a N369Y substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 534; or dd) a Q325H substitution, a 137 IN substitution, a H397F substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 522; or ee) a Q325K substitution, a 137 IN substitution, and a T359V substitution, optionally comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 542, or ff) a Q325 amino acid substitution selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution; and (ii) one or more amino acid substitutions selected from: a T359V substitution, a 137 IN substitution, a H397F substitution, and a N369Y substitution.

8. The modified CRBN of any one of claims 5-7, wherein the modified CRBN comprises an amino acid sequence set forth in any one of SEQ ID NOs: 231-249, 256, 258, 260, 262, 264, 275, 279, 283, 287, 291, 295, 299, 303, 307, 311, 315, 319, 323, 327, 331, 335, 339,343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, 399, 403, 407, 411,415, 419, 423, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454,456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490,492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526,528, 530, 532, 534, 536, 538, 540, 542, or 544, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 544, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 530, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 526, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 524, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 532, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 528, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 534, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 522, optionally wherein the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, optionally wherein the modified CRBN comprises the amino acid sequence set forth in SEQ ID NO: 542.

9. An activation-conditional control polypeptide (ACP) complex, comprising: a first polypeptide comprising the modified degron of any one of claims 1-4, and a second polypeptide comprising a ligand binding domain, wherein (i) the first polypeptide further comprises a DNA-binding domain and the second polypeptide further comprises a transcriptional effector domain, or (ii) the first polypeptide further comprises a transcriptional effector domain and the second polypeptide further comprises a DNA-binding domain, and wherein the first and second polypeptides dimerize with each other upon binding of a ligand to the ligand binding domain, thereby forming the ACP complex, and wherein the ACP complex is capable of modulating transcriptional expression of a gene of interest operably linked to an ACP complex-responsive promoter, optionally wherein the DNA-binding domain comprises a DNA zinc finger protein domain, optionally wherein the DNA zinc finger protein domain comprises an amino acid sequence of SEQ ID NO: 57;optionally wherein the ligand is an IMiD, optionally wherein the IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide, optionally wherein the IMiD is pomalidomide.

10. The ACP complex of claim 9, wherein the transcriptional effector domain is a transcriptional activation domain selected from the group consisting of: a Herpes Simplex Virus Protein 16 (VP 16) activation domain; an activation domain comprising four tandem copies of VP 16, a VP64 activation domain; a p65 activation domain of NFKB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tripartite activator comprising the VP64, the p65, and the Rta activation domains (VPR activation domain); a tripartite activator comprising the VP64, the p65, and the HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of the human ElA-associated protein p300 (p300 HAT core activation domain); or a transcriptional repressor domain selected from the group consisting of: a Kruppel associated box (KRAB) repression domain; a truncated Kruppel associated box (KRAB) repression domain; a Histone Deacetylase 4 (HDAC4) repressor domain; a Scleraxis (SCX) HLH domain, an Inhibitor of DNA binding 1 (ID1) HLH domain, a HECT domain and RCCl-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a Twist-related protein 1 (TWST1) HLH domain, an Homeobox protein Nkx-2.2 (NKX22) homeodomain, an Inhibitor of DNA binding 1 (ID3) HLH domain, and a Twist-related protein 2 (TWST2) HLH domain, and EED repressor domain; a Repressor Element Silencing Transcription Factor (REST) repression domain; a WRPW motif of the hairy-related basic helix-loop-helix repressor proteins, the motif is known as a WRPW repression domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and an HP1 alpha chromoshadow repression domain.

11. The ACP complex of claim 9 or 10, wherein the ligand-binding domain is or comprises a component of an E3 ubiquitin ligase complex, a cereblon domain, or a variant thereof, optionally wherein the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129, optionally wherein the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of ligand, as compared to an unmodifiedcereblon, optionally wherein the cereblon domain or variant thereof is a modified cereblon as described in any one of claims 113-148, 177-187, or 226.

12. The ACP complex of any one of claims 9-11, wherein the modified degron and the DNA- binding domain or the transcriptional effector domain are linked by a first linker, optionally wherein the ligand-binding domain and the transcriptional effector domain or the DNA-binding domain are linked by a second linker, optionally wherein the first linker and / or the second linker comprises the amino acid sequence set forth in SEQ ID NO: 169, 171, 173, 265, or 269, 168, 175, or 199, optionally wherein the first linker and / or the second linker is a glycine-serine linker, optionally wherein the glycine- serine linker comprises one or more GGGS (SEQ ID NO: 252) motifs, optionally wherein the glycineserine linker comprises one or more GGGGS (SEQ ID NO: 253) motifs, optionally wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254), optionally wherein the linker comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193, optionally wherein the linker does not have lysine residues, optionally wherein the first linker and / or the second linker comprises the amino acid sequence set forth in SEQ ID NO: 265 or 269.

13. An inducible cell death system comprising: a first polypeptide comprising the modified degron of any one of claims 1-4, operably linked to a first cell-death inducing domain and a second polypeptide comprising a ligand-binding domain operably linked to a second cell death-inducing domain, or a first polypeptide comprising the modified CRBN of any one of claims 5-8, operably linked to a first cell-death inducing domain and a second polypeptide comprising a ligand-binding domain operably linked to a second cell death-inducing domain, wherein the first polypeptide and the second polypeptides are configured to form a complex with each other upon binding of a ligand to the ligand binding domain, optionally wherein the first and second cell-death inducing domains are identical, optionally wherein the ligand is an ImiD, optionally wherein the ImiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide.

14. The inducible cell death system of claim 13, wherein the first and second cell death inducing domains are derived from a protein selected from the group consisting of: a caspase (e.g., any one of caspases 1-11, such as caspase-3, caspase-6, caspase-7, caspase- 8, caspase-9), Diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2-interacting protein 3 (BNIP3), Fas, Fas -associated protein with death domain (FADD), tumor necrosis factor receptor type I-associated death domain protein (TRADD), a TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondria-derived activator of caspases (SMAC), Omi, Bmf, Bid, Bim, p53-upregulated modulator of apoptosis (PUMA), Noxa, Blk, Hrk, Cytochrome c, Arts, TNF-related cell death-inducing ligand (TRAIL), Herpes Simplex Virus thymidine kinase (HSV-TK), Varicella Zoster Virus thymidine kinase (VZV-TK), viral Spike protein, Carboxyl esterase, cytosine deaminase, nitroreductase Fksb, Carboxypeptidase G2, Carboxypeptidase A, Horseradish peroxidase, Linamarase, Hepatic cytochrome P450-2B1, Purine nucleoside phosphorylase, and variants and functional fragments thereof, optionally wherein the first and second cell death inducing domains are derived from caspase-9, optionally wherein the caspase-9 does not comprise a Caspase Activation and Recruitment Domain (CARD) domain sequence.

15. The inducible cell death system of claim 13 or 14, wherein (a) the ligand-binding domain is or comprises a component of an E3 ubiquitin ligase complex, a cereblon domain, or a variant thereof, optionally wherein the cereblon domain or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129, optionally wherein the cereblon domain or variant thereof is a modified cereblon that functionally associates with the modified degron when exposed to a lower concentration of ligand, as compared to an unmodified cereblon, optionally wherein the cereblon domain or variant thereof is a modified cereblon as described in any one of claims 5-8; or (b) the ligandbinding domain is or comprises a modified degron, optionally wherein the modified degron or variant thereof comprises an amino acid sequence as set forth in SEQ ID NO: 127 or 129, optionally wherein the modified degron or functionally associates with the modified cereblon when exposed to a lower concentration of ligand, as compared to an unmodified cereblon, optionally wherein the modified degron or variant thereof is a modified degron as described in any one of claims 1-4.

16. The inducible cell death system of any one of claims 13 - 15, wherein (i) the modified degron is operably linked to the first cell-death inducing domain by a first linker, (ii) the ligand-binding domain is operably linked to the second cell death-inducing domain by a second linker, or (iii) the modified degron is operably linked to the first cell-death inducing domain by the first linker and the ligand-binding domain operably linked to the second cell death-inducing domain by the second linker.

17. The inducible cell death system of any one of claims 13-16, the ACP complex of any one of claims 89-102, or 163-168, or the polypeptide complex of claim 149, wherein the modified CRBN comprises, relative to a wildtype CRBN comprising the amino acid sequence set forth in SEQ ID NO: 127, one or more amino acid substitutions selected from: a T359 amino acid substitution, a Q325 amino acid substitution, a N369Y amino acid substitution, a 137 IN amino acid substitution, and a H397F amino acid substitution, optionally wherein the Q325 amino acid substitution is selected from a Q325H substitution, a Q325K substitution, and a Q325R substitution, and / or optionally wherein the T359 amino acid substitution is selected from a T359V substitution, a T359F substitution, a T359I substitution, a T359L substitution, a T359W substitution, and a T359Y substitution, and wherein the modified degron comprises one or more amino acid substitutions selected from: a Q28T amino acid substitution, a G30E amino acid substitution, and a L33I amino acid substitution, relative to a wildtype degron comprising the amino acid sequence set forth in SEQ ID NO: 40, optionally wherein the modified CRBN comprises an 137 IN amino acid substitution, and wherein the modified degron comprises a G30E amino acid substitution, optionally wherein the modified CRBN comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution; or optionally wherein the modified degron comprises a Q325R, N369Y, 137 IN, and H397F amino acid substitution, and wherein the modified degron comprises a Q28T, G30E, and L33I amino acid substitution.

18. A cell comprising the inducible cell death system of any one of claims 13-17, the modified degron of any one of claims 1-4, the modified CRBN of any one of claims 5-8, or the ACP complex of any one of claims 9-12, optionally wherein the cell is a human cell, optionally wherein the cell is a cell therapy modality, optionally wherein the cell therapy modality is a CAR-T cell or CAR-NK cell.

19. A method of inducing cell death, the method comprising expressing the inducible cell death system of any one of claims 13-17 in a cell and exposing the cell to the ligand, optionally wherein the ligand is an IMiD, optionally wherein the IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide, optionally wherein the IMiD is pomalidomide.

20. A method of modulating target gene expression, the method comprising expressing the ACP complex of any one of claims 9-12 in a cell and exposing the cell to the ligand, optionally wherein the ligand is an IMiD, optionally wherein the IMiD is selected from the group consisting of: thalidomide, iberdomide, lenalidomide, and pomalidomide.

21. One or more nucleic acid sequences encoding: the modified degron of any one of claims 1-4; the modified CRBN of any one of claims 5-8; the inducible cell death system of any one of claims 13-17; or the ACP complex of any one of claims 9-12.

22. A vector comprising the nucleic acid sequence of claim 21 and, optionally, a promoter, preferably a promoter functional in a mammalian cell.

23. A polypeptide complex comprising a first polypeptide comprising a degron and a second polypeptide comprising a cereblon, wherein: (i) the degron is a modified degron of any one of claims 1-4; (ii) the cereblon is a modified cereblon of any one of claims 5-8; or (iii) the degron is a modified degron of any one of claims 1-4, and the cereblon is a modified cereblon of any one of claims 5-8.

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