Modified degron polypeptide and its use

Abstract

Provided herein are modified degrons exhibiting increased sensitivity to one or more immunomodulatory drugs (IMiDs). Also provided herein are modified cerebrons exhibiting 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 herein are inducibly degradable proteins, inducible cell death systems, and activation condition-controlled polypeptides using modified degrons, modified cerebrons, and methods and / or uses thereof.

Description

[Background technology]

[0001] Cross-reference of related applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 464,394 filed on 5 May 2023, U.S. Provisional Patent Application No. 63 / 511,443 filed on 30 June 2023, U.S. Provisional Patent Application No. 63 / 604,780 filed on 30 November 2023, and U.S. Provisional Patent Application No. 63 / 623,722 filed on 22 January 2024, the disclosures of each thereof being incorporated herein by reference for all purposes.

[0002] Sequence List This application was filed through the Patent Center and includes a sequence listing incorporated herein by reference in its entirety. The XML copy thereof was created in XX / XX / 20XX, named XXXXXUS_sequencelisting.xml, and is X,XXX,XXX bytes in size.

[0003] Currently available cell and gene therapy products can lack control, which can lead to safety concerns in the treated population, such as toxicity. Therefore, additional methods are needed to control and regulate these therapies. [Overview of the project]

[0004] This disclosure provides, in particular, modified degron and celeberon having increased sensitivity to one or more immunomodulatory drugs (IMiDs).

[0005] In some embodiments, this disclosure refers to the amino acid sequence shown in SEQ ID NO: 139, or the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X 10A modified degron is provided, in which X1 is I or R; X2 is F or W; X3 is C or T; X4 is R, D, H, or K; X5 is Q, R, or T; X6 is G or V; X7 is N, T, or A; X8 is L or I; X9 is K or G; and X 10 is L or F, and therein, the modified degron has increased sensitivity to one or more IMiDs compared to degron having the amino acid sequence shown in SEQ ID NO: 40 or 131, and therein, the modified degron has 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 compared to wild-type degron having the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the IMiD is at least one of thalidomide, iverdide, lenalidomide, and pomalidomide, and therein, therein, the IMiD is pomalidomide.

[0006] In some embodiments, the modified deglon contains an amino acid sequence represented by 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 includes SEQ ID NO: 148. In some embodiments, the modified degron functionally associates with a cerebron domain or a variant thereof in an IMiD-responsive manner, and optionally therein, the modified degron functionally associates with a cerebron domain or a variant thereof when exposed to lower concentrations of IMiD compared to unmodified degron. In some embodiments, the cerebron domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 127 or 129. In some embodiments, the cerebron domain or a variant thereof is a modified cerebron that functionally associates with a modified degron when exposed to lower concentrations of IMiD compared to unmodified cerebron. In some embodiments, the cerebron domain or a variant thereof is a modified cerebron described herein.

[0007] In some embodiments, the Disclosure also provides modified cerebron (CRBN) comprising: (a) one or more amino acid substitutions at position Q325, V350, H353, Y355, N369, I371, R373, E377, S379, Q390, A395, S396, H397, or F402 compared to wild-type CRBN comprising the amino acid sequence shown in SEQ ID NO: 127, wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to wild-type CRBN; (b) amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX10 X 11 X 12 PX 13 TX 14 X 15 X 16 X 17 X 18 PGYAWTX 19 AX 20 CKICX 21 X 22 [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​(c) Amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10 X 11 X 12 PX 13 TX 14 X 15 X 16 X 17 X 18 PGYAWTX 19 AX 20 CKICX 21 X 22 X 23 IGWKX 24 TATKKDMSPQKFWX 25 In this, X1 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 T, F, I, L, V, W, or Y; X9 is N, D, or Y; X 10 is I, D, E, N; Q, L, V, or M; X 11 is G, N, or Q; X 12 is R, E, N, S, or Y; X 13 The letters are S, D, K, N, Q, R, H, and P; X 14 is E, D, K, Q, S, or Y; X 15 is H, D, Q, or T; X 16 is S, N, or T; X 17 is W, F, or Y; X 18 is F or W; X 19 is V, D, E, N, Q, S, or L; X 20 is Q, D, H, K, or R; X 21 is A, D, E, N, or Q; X22 is S, G, or Q; X 23 is H, D, F, K, Q, Y, I, L, or V; X 24 is F, H, N, Q, W, or Y; and X 25 (d) Compared to wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, K324, Q325, V350, N351, H353, Y355, H357, T359, N369, I371, G372, R373, S375, E377, H378, S379, W380, F381, V388, Q390, A395, S396, H397, F402, (e) Amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10 X 11 X 12 PX 13 TX 14 X 15 X 16 X 17 X 18 PGYAWTX 19 AX 20 CKICX 21 X 22 X 23 IGWKX 24 TATKKDMSPQKFWX 25 In this, X1 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; X 10 is I, D, E, N, or Q; X 11 is G, N, or Q; X 12 is R, E, N, S, or Y; X 13is X, D, K, N, Q, or R; X 14 is E, D, K, Q, S, or Y; X 15 is H, D, Q, or T; X 16 is S, N, or T; X 17 is W, F, or Y; X 18 is F or W; X 19 is V, D, E, N, Q, S, or L; X 20 is Q, D, H, K, or R; X 21 is A, D, E, N, or Q; X 22 is S, G, or Q; X 23 is H, D, F, K, Q, Y, I, L, or V; X 24 is F, H, N, Q, W, or Y; and X 25 is G or A; or (f) a T359 amino acid substitution. In some embodiments, the T359 amino acid substitution is selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, optionally, wherein the modified CRBN comprises the T359V substitution.

[0008] In some embodiments, the modified CRBN does not contain a DDB1 interaction domain, and optionally therein the modified CRBN further includes a deletion of amino acids 194-247 compared to the wild-type CRBN, and optionally therein the modified CRBN includes the amino acid sequence shown in SEQ ID NOs. 231-249, 256, 258, 260, 262, or 264. In some embodiments, the modified CRBN contains, compared to a wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, (a) a Q325 amino acid substitution selected from Q325H substitution, Q325K substitution, and Q325R substitution; and one or more amino acid substitutions selected from I371N substitution, H397F substitution, and N369Y substitution; or (b) a Q325H substitution and an I371N substitution, optionally containing an amino acid sequence that is 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, an I371N substitution, and an H397F substitution, optionally containing an amino acid sequence that is 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. (d) containing an acid sequence; or (d) containing an amino acid sequence that is 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) containing an amino acid sequence that is at least 80%, at least 85%, or at least 99% identical to SEQ ID NO: 363; or (e) containing an amino acid sequence that is at least 80%, at least 85%, or at least 99% identical to SEQ ID NO: 363. 0%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence; or (f) Q325H substitution, N369Y substitution, and I371N substitution, which may include 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 amino acid sequence to SEQ ID NO: 367;or (g) Q325H substitution, N369Y substitution, I371N substitution, and H397F substitution, which may contain 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 amino acid sequence to SEQ ID NO: 371; or (h) Q325K substitution and H397F substitution, which may contain at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least and (i) containing the same amino acid sequence as (i) Q325K substitution, I371N substitution, and H397F substitution, which may include the same amino acid sequence as SEQ ID NO: 383 by 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%; or (j) Q325R substitution and H397F substitution, which may include the same amino acid sequence as (i) SEQ ID NO: 403 by at least 80%, at least Contains 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; or (k) I371N substitution and H397F substitution, in cases containing 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 amino acid sequences to SEQ ID NO: 331; or (l) N369Y substitution and H397F substitution, in cases Therefore, it contains an amino acid sequence that is 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 sequence number 335; or (m)N369Y substitution and I371N substitution, in the case thereof, contains an amino acid sequence that is 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 sequence number 339;or (n)N369Y substitution, I371N substitution, and H397F substitution, which may contain 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 amino acid sequences to SEQ ID NO: 343; or (o)Q325H substitution and H397F substitution, which may contain at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% identical amino acid sequences to SEQ ID NO: 347 , containing at least 99% or 100% identical amino acid sequences; or (p)Q325K substitution and I371N substitution, in cases containing 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 amino acid sequences to SEQ ID NO: 379; or (q)Q325K substitution and N369Y substitution, in cases containing at least 80%, at least 85%, at least 90%, at least 95%, at least and It contains 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; or (r) Q325K substitution, N369Y substitution, and H397F substitution, which may include 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 amino acid sequences to SEQ ID NO: 391; or (s) Q325K substitution, N369Y substitution, and I371N substitution, which may include SEQ ID NO: 395 Containing 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 amino acid sequences; or (t)Q325K substitution, N369Y substitution, I371N substitution, and H397F substitution, which may contain 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 amino acid sequences to SEQ ID NO: 399;or (u) Q325R substitution, N369Y substitution, I371N substitution, and H397F substitution, which may include an amino acid sequence that is 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 T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, which may include In this, the Q325 amino acid substitution is selected from Q325H substitution, Q325K substitution, and Q325R substitution, and / or in this case, the T359 amino acid substitution is selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution; (w) Q325H substitution, Q325H substitution, I371N substitution, and T359V substitution, in this case, SEQ ID NO: 544 and at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, less Contains at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; or (x) Q325R substitution, I371N substitution, H397F substitution, and T359V substitution, and possibly contains 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 amino acid sequences to SEQ ID NO: 530; or (y) Q325R substitution, N369Y substitution, H397F substitution, and T359V substitution, and possibly , containing 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 amino acid sequences to SEQ ID NO: 526; or (z)Q325R substitution, I371N substitution, and T359V substitution, which may contain 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 amino acid sequences to SEQ ID NO: 524;or (aa) Q325K substitution, I371N substitution, H397F substitution, and T359V substitution, which may contain 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 amino acid sequences to SEQ ID NO: 532; or (bb) Q325H substitution, N369Y substitution, I371N substitution, H397F substitution, and T359V substitution, which may contain at least identical amino acid sequences to SEQ ID NO: 528. Both contain 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 amino acid sequences; or (cc)Q325H substitution, N369Y substitution, H397F substitution, and T359V substitution, in the case of SEQ ID NO: 534 and at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Contains %, or 100%, the same amino acid sequence as SEQ ID NO: 522; or (dd) Q325H substitution, I371N substitution, H397F substitution, and T359V substitution, which may contain 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% the same amino acid sequence as SEQ ID NO: 522; or (ee) Q325K substitution, I371N substitution, and T359V substitution, which may contain the same amino acid sequence as SEQ ID NO: 522. 542 contains an amino acid sequence that is 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; or (ff) a Q325 amino acid substitution selected from Q325H substitution, Q325K substitution, and Q325R substitution; and (ii) one or more amino acid substitutions selected from T359V substitution, I371N substitution, H397F substitution, and N369Y substitution.

[0009] In some embodiments, the modified CRBNs are sequence numbers 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, The amino acid sequence includes any one of the following: 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 includes the amino acid sequence shown in SEQ ID NO: 544, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 530, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 526, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 524, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 532, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 528, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 534, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 522, and optionally therein, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 542 compared to a wild-type CRBN that includes the amino acid sequence shown in SEQ ID NO: 127.

[0010] The Disclosure also provides an activation-condition-controlled polypeptide (ACP) complex comprising: a first polypeptide comprising a 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 transcription effector domain; or (ii) the first polypeptide further comprises a transcription 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 to form an ACP complex, and wherein the ACP complex is capable of regulating the transcriptional expression of a gene of interest operably linked to an ACP complex-responsive promoter, and optionally, wherein the DNA-binding domain comprises a DNA zinc finger protein domain, and optionally, wherein the DNA zinc finger protein domain comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the ligand is an IMiD, which may be selected from the group consisting of thalidomide, iverdide, lenalidomide, and pomalidomide, and in some embodiments, the IMiD is pomalidomide.

[0011] In some embodiments, the transcription effector domains include: herpes simplex virus protein 16 (VP16) activation domain; activation domains containing four tandem copies of VP16; VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; a tri-component activator containing VP64, p65, and Rta activation domains (VPR activation domain); a tri-component activator containing VP64, p65, and HSF1 activation domains (VPH activation domain); and the histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 A transcriptional activation domain selected from the group consisting of (HAT core activation domain); or a Kruppel-associated box (KRAB) repressor domain; a cleavage-type Kruppel-associated box (KRAB) repressor domain; a histone deacetylase 4 (HDAC4) repressor domain; a scleracsis (SCX) HLH domain, a DNA binding inhibitor 1 (ID1) HLH domain, a HECT domain and an RCC1-like domain-containing protein 2 (HERC2) Cyt-b5 domain, a twist-associated protein 1 (TWST1) HLH domain, or a homeobox protein Nkx-2.2 (NKX22) homeodomain. The transcriptional repressor domain is selected from the group consisting of the DNA binding inhibitor 1 (ID3) HLH domain, the twist-related protein 2 (TWST2) HLH domain, and the EED repressor domain; the repressor element silencing transcription factor (REST) ​​repressor domain; the WRPW motif of the hairy-associated basic helix-loop helix repressor protein, the motif of which is known as a WRPW repressor domain; the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and the HP1 alpha-chromoshadow repressor domain.

[0012] In some embodiments, the ligand-binding domain is a component of, or comprises, an E3 ubiquitin ligase complex, a cereblon domain, or a variant thereof. In some embodiments, the cereblon domain or a variant thereof comprises the amino acid sequence shown in SEQ ID NO: 127 or 129. In some embodiments, the cereblon domain or a variant thereof is a modified cereblon that functionally associates with modified degron when exposed to lower concentrations of ligand compared to unmodified cereblon. In some embodiments, the cereblon domain or a 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 transcription effector domain are linked by a first linker. In some embodiments, the ligand-binding domain and the transcription effector domain or DNA-binding domain are linked by a second linker, which may include the amino acid sequence shown in SEQ ID NOs: 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 includes one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, the glycine-serine linker includes one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, the linker includes the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). In some embodiments, the linker includes the amino acid sequence shown in any one of SEQ ID NOs. 45, 46, 91, 92, 191, or 193. In some embodiments, the linker does not have a lysine residue. In some embodiments, the first linker and / or the second linker includes the amino acid sequence shown in SEQ ID NOs. 265 or 269.

[0014] The present invention also provides an inducible cell death system comprising: a first polypeptide comprising a modified degron provided herein, operably linked to a first cell death induction domain, and a second polypeptide comprising a ligand-binding domain operably linked to a second cell death induction domain; or a first polypeptide comprising a modified CRBN provided herein, operably linked to a first cell death induction domain, and a second polypeptide comprising a ligand-binding domain operably linked to a second cell death induction domain, wherein the first and 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 induction domains are identical. In some embodiments, the ligand is ImiD. In some embodiments, ImiD is selected from the group consisting of thalidomide, iverdide, lenalidomide, and pomalidomide.

[0015] In some embodiments, the first and second cell death induction domains include caspases (e.g., any one of caspases 1-11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, etc.), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-related protein with death domain (FADD), tumor necrosis factor receptor 1-associated death Domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspase (SMAC), Omi, Bmf, Bid, Bim, p53 upregulator of apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-associated cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK) is derived from proteins selected from the group consisting of viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamorase, liver cytochrome P450-2B1, purine nucleoside phosphorylase, and their variants and functional fragments, wherein the first and second cell death induction domains are derived from caspase 9, wherein caspase 9 does not contain the caspase activation and recruitment domain (CARD) domain sequence.

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

[0017] In some embodiments, (i) the modified degron is operably linked to a first cell death induction domain by a first linker, (ii) the ligand-binding domain is operably linked to a second cell death induction domain by a second linker, or (iii) the modified degron is operably linked to a first cell death induction domain by a first linker, and the ligand-binding domain is operably linked to a second cell death induction domain by a second linker. In some embodiments, the modified CRBN includes one or more amino acid substitutions selected from the T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, compared to a wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127. In some embodiments, the Q325 amino acid substitution is selected from the Q325H substitution, Q325K substitution, and Q325R substitution. In some embodiments, the T359 amino acid substitution is selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution. In some embodiments, the modified deglon includes one or more amino acid substitutions selected from Q28T amino acid substitution, G30E amino acid substitution, and L33I amino acid substitution, compared to wild-type deglon having the amino acid sequence shown in SEQ ID NO: 40, and optionally therein, the modified CRBN includes the I371N amino acid substitution, and the modified deglon includes the G30E amino acid substitution. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions. In some embodiments, the modified deglon includes the Q325R, N369Y, I371N, and H397F amino acid substitutions, and the modified deglon includes the Q28T, G30E, and L33I amino acid substitutions.

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

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

[0020] This disclosure also provides a method for modulating target gene expression, which comprises expressing an ACP complex provided herein in cells and exposing the cells to a ligand. In some embodiments, the ligand is an IMiD. In some embodiments, the IMiD is selected from the group consisting of thalidomide, iverdide, lenalidomide, and pomalidomide.

[0021] This disclosure also provides one or more nucleic acid sequences encoding modified degron, modified CRBN, inducible cell death systems, or ACP complexes provided herein.

[0022] This disclosure also provides vectors comprising nucleic acid sequences provided herein. In some embodiments, the vector includes a promoter. In some embodiments, the promoter is functional in mammalian cells.

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

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

[0025] This disclosure further relates to the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X 10 A modified degron is provided, in which X1 is I or R; X2 is F or W; X3 is C or T; X4 is R, D, H, or K; X5 is Q, R, or T; X6 is G or V; X7 is N, T, or A; X8 is L or I; X9 is K or G; and X 10 It is L or F, and therein, the modified degron has increased susceptibility to one or more IMiDs compared to degron containing the amino acid sequence shown in SEQ ID NO: 40 or 131.

[0026] The disclosure also provides 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, G30V, R27D, Q28R, N31T, F24W, R27K, I21R, G30V, and N31A, compared to a wild-type degron comprising the amino acid sequence shown in SEQ ID NO: 40.

[0027] In some embodiments, the modified deglon contains the amino acid sequence shown 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, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 140.

[0029] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 142.

[0030] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 144.

[0031] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 146.

[0032] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 148.

[0033] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 150.

[0034] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 152.

[0035] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 154.

[0036] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 156.

[0037] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 158.

[0038] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 160.

[0039] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 162.

[0040] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 164.

[0041] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 166.

[0042] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 226.

[0043] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 227.

[0044] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 228.

[0045] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 229.

[0046] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 230.

[0047] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 276.

[0048] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 280.

[0049] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 284.

[0050] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 288.

[0051] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 292.

[0052] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 296.

[0053] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 300.

[0054] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 304.

[0055] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 308.

[0056] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 312.

[0057] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 316.

[0058] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 320.

[0059] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 324.

[0060] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 328.

[0061] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 332.

[0062] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 336.

[0063] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 340.

[0064] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 344.

[0065] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 348.

[0066] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 352.

[0067] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 356.

[0068] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 360.

[0069] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 364.

[0070] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 368.

[0071] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 372.

[0072] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 376.

[0073] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 380.

[0074] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 384.

[0075] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 388.

[0076] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 392.

[0077] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 396.

[0078] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 400.

[0079] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 404.

[0080] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 408.

[0081] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 412.

[0082] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 416.

[0083] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 420.

[0084] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 424.

[0085] In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NO: 788.

[0086] In some embodiments, modified degron functionally associates with a cerebron domain or a variant thereof in an IMiD-responsive manner. In some embodiments, modified degron functionally associates with a cerebron domain or a variant thereof when exposed to lower concentrations of IMiD compared to unmodified degron. In some embodiments, the cerebron domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 127. In some embodiments, the cerebron domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 129. In some embodiments, the cerebron domain or a variant thereof is modified cerebron that functionally associates with modified degron when exposed to lower concentrations of IMiD compared to unmodified cerebron. In some embodiments, the cerebron domain or a variant thereof is modified cerebron as described herein. In some embodiments, the cerebron domain or a variant thereof is an E3 ubiquitin ligase or a component thereof, wherein the functional association induces the degradation of modified degron, a protein containing modified degron, or a polypeptide containing modified degron in an IMiD-responsive manner.

[0087] In some embodiments, the increased sensitivity is, or includes, increased degradation of proteins and / or polypeptides containing modified degron.

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

[0089] In some embodiments, modified degron has increased susceptibility to one or more IMiDs compared to degron containing the amino acid sequence of SEQ ID NO: 131.

[0090] This disclosure provides inducibly degradable proteins comprising a target polypeptide operably linked to a modified degron, as described herein.

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

[0092] In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 168, 175, or 199. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 169, 171, or 173. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 169. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 171. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 173. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 175. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NO: 199.

[0093] In some embodiments, the linker is a glycine-serine linker. In some embodiments, the linker contains one or more GGGS (SEQ ID NO: 252) motifs. In some embodiments, the linker contains one or more GGGGS (SEQ ID NO: 253) motifs. In some embodiments, the linker contains the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). In some embodiments, the linker contains the amino acid sequence shown in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193. In some embodiments, the linker does not contain a lysine residue.

[0094] This disclosure provides cells containing modified degrons as described herein, or inducibly degradable proteins as described herein.

[0095] This disclosure also provides a method for producing an inducibly degradable protein, the method comprising operably linking a modified degron described herein to a target polypeptide.

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

[0097] In some embodiments, the target polypeptide is fused directly to the modified degron or linked by a linker. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 168, 175, or 199. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 169, 171, or 173. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 169. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 171. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 173. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 175. In some embodiments, the linker includes the amino acid sequence shown in SEQ ID NOs. 199.

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

[0099] In some embodiments, the inducibly degradable protein is degraded upon expression in mammalian cells and upon cell exposure to IMiD.

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

[0101] This disclosure further provides a method for promoting the degradation of a targeted polypeptide, the method comprising expressing an inducibly degradable protein described herein in mammalian cells and exposing the cells to IMiD.

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

[0103] In some embodiments, the mammalian cells are human cells.

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

[0105] This disclosure provides an inducible cell death system comprising a first polypeptide comprising a modified degron as described herein, operably linked to a first cell death induction domain; and a second polypeptide comprising a ligand-binding domain, operably linked to a second cell death induction domain, wherein the first and 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, the first and second cell death induction domains are identical.

[0107] In some embodiments, the first and second cell death induction domains include caspases (e.g., any one of caspases 1-11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, etc.), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-related protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspase (SMAC), Omi, Bmf, Bid, Bim These proteins are derived from a group of proteins selected from the following: p53 upregulatory factor for apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-associated cell death-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamorase, liver cytochrome P450-2B1, purine nucleoside phosphorylase, and their variants and functional fragments.

[0108] In some embodiments, the first and second cell death induction domains are derived from caspase-9, and optionally, therein, caspase-9 does not contain a caspase activation and recruitment domain (CARD) domain sequence.

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

[0110] In some embodiments, the ligand-binding domain is or includes a cereblon domain or a variant thereof. In some embodiments, the cereblon domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 127. In some embodiments, the cereblon domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 129. In some embodiments, the cereblon domain or a variant thereof is a modified cereblon that functionally associates with modified degron when exposed to lower concentrations of ligand compared to unmodified cereblon. In some embodiments, the cereblon domain or a variant thereof is a modified cereblon as described herein. In some embodiments, modified cerebron (CRBN) includes one or more amino acid substitutions selected from the T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, compared to wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, wherein the Q325 amino acid substitution is selected from the Q325H substitution, Q325K substitution, and Q325R substitution, and / or, wherein the T359 amino acid substitution is selected from the T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, and wherein modified degron includes one or more amino acid substitutions selected from the Q28T amino acid substitution, G30E amino acid substitution, and L33I amino acid substitution, compared to wild-type degron having the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the modified CRBN includes the I371N amino acid substitution, wherein the modified deglon includes the G30E amino acid substitution. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions, wherein the modified deglon includes the Q28T, G30E, and L33I amino acid substitutions.

[0111] In some embodiments, the ligand is IMiD.

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

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

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

[0115] This disclosure provides cells including the inducible cell death system described herein.

[0116] In some embodiments, the cells are human cells.

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

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

[0119] In some embodiments, the ligand is IMiD.

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

[0121] The present invention provides an activation-condition-controlled 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 transcription effector domain, or (ii) the first polypeptide further comprises a transcription 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 to form an ACP complex, and wherein the ACP complex is capable of regulating the transcriptional expression of a gene of interest operably linked to an ACP complex-responsive promoter.

[0122] In some embodiments, the DNA-binding domain includes a DNA zinc finger protein domain. In some embodiments, the DNA-binding domain is a ZF10-1 domain. In some embodiments, the DNA zinc finger protein domain includes the amino acid sequence shown in SEQ ID NO: 57.

[0123] In some embodiments, the transcriptional effector domain is a transcriptional activation domain selected from the group consisting of the herpes simplex virus protein 16 (VP16) activation domain; an activation domain comprising four tandem copies of VP16; the VP64 activation domain; the p65 activation domain of NFκB; the Epstein-Barr virus R transactivator (Rta) activation domain; a tri-component activator comprising the VP64, p65, and Rta activation domains (VPR activation domain); a tri-component activator comprising the VP64, p65, and HSF1 activation domains (VPH activation domain); and the histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 HAT core activation domain), as well as their variants and functional fragments.

[0124] In some embodiments, the transcription effector domains include: Kruppel-associated box (KRAB) repressor domain; cleavage-type Kruppel-associated box (KRAB) repressor domain; histone deacetylase 4 (HDAC4) repressor domain; scleracsis (SCX) HLH domain; DNA binding inhibitor 1 (ID1) HLH domain; HECT domain and RCC1-like domain-containing protein 2 (HERC2) Cyt-b5 domain; twist-associated protein 1 (TWST1) HLH domain; homeobox protein Nkx-2.2 (NKX22) homeodomain; DNA binding inhibitor The transcriptional repressor domain is selected from the group consisting of the following: the harmful factor 1 (ID3) HLH domain, the twist-related protein 2 (TWST2) HLH domain, and the EED repressor domain; the repressor element silencing transcription factor (REST) ​​repressor domain; the WRPW motif of the hairy-related basic helix-loop helix repressor protein, the motif of which is known as a WRPW repressor domain; the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and the HP1 alpha-chromoshadow repressor domain.

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

[0126] In some embodiments, the ligand-binding domain is or includes a cereblon domain or a variant thereof. In some embodiments, the cereblon domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 127. In some embodiments, the cereblon domain or a variant thereof includes the amino acid sequence shown in SEQ ID NO: 129. In some embodiments, the cereblon domain or a variant thereof is a modified cereblon that functionally associates with modified degron when exposed to lower concentrations of ligand compared to unmodified cereblon. In some embodiments, the cereblon domain or a variant thereof is a modified cereblon as described herein. In some embodiments, modified cerebron (CRBN) includes one or more amino acid substitutions selected from the T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, compared to wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, wherein the Q325 amino acid substitution is selected from the Q325H substitution, Q325K substitution, and Q325R substitution, and / or, wherein the T359 amino acid substitution is selected from the T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, and wherein modified degron includes one or more amino acid substitutions selected from the Q28T amino acid substitution, G30E amino acid substitution, and L33I amino acid substitution, compared to wild-type degron having the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the modified CRBN includes the I371N amino acid substitution, wherein the modified deglon includes the G30E amino acid substitution. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions, wherein the modified deglon includes the Q28T, G30E, and L33I amino acid substitutions.

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

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

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

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

[0131] In some embodiments, the ligand is IMiD.

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

[0133] This disclosure provides cells containing the ACP complex described herein.

[0134] In some embodiments, the cells are human cells.

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

[0136] This disclosure provides a method for regulating the expression of a target gene, the method comprising expressing an ACP complex described herein in cells and exposing the cells to a ligand.

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

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

[0139] This disclosure provides one or more nucleic acid sequences encoding modified degrons as described herein; inducibly degradable proteins as described herein; inducibly degradable cell death systems as described herein; or ACP complexes as described herein. This disclosure provides nucleic acids as described herein and, optionally, vectors comprising promoters, preferably functional promoters in mammalian cells.

[0140] This disclosure provides a modified CRBN comprising one or more amino acid substitutions at positions Q325, V350, H353, Y355, N369, I371, R373, E377, S379, Q390, A395, S396, H397, or F402 compared to a wild-type CRBN comprising the amino acid sequence shown in SEQ ID NO: 127, wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to a wild-type CRBN.

[0141] This disclosure pertains to the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10 X 11 X 12 PX 13 TX 14 X 15 X 16 X 17 X 18 PGYAWTX 19 AX 20 CKICX21 X 22 X 23 IGWKX 24 TATKKDMSPQKFWX 25 Provides a modified CRBN including X1 being K, N, Q, or R; X2 being Q, H, K, or R; X3 being V, E, H, Q, R, or S; X4 being N, A, I, L, or V; X5 being H, P, Q, R, or S; X6 being Y, H, N, R, or S; X7 being H, Q, or W; X8 being T, F, I, L, V, W, or Y; X9 being N, D, or Y; X 10 is I, D, E, N, or Q; X 11 is G, N, or Q; X 12 is R, E, N, S, or Y; X 13 is X, D, K, N, Q, or R; X 14 is E, D, K, Q, S, or Y; X 15 is H, D, Q, or T; X 16 is S, N, or T; X 17 is W, F, or Y; X 18 is F or W; X 19 is V, D, E, N, Q, S, or L; X 20 is Q, D, H, K, or R; X 21 is A, D, E, N, or Q; X 22 is S, G, or Q; X 23 is H, D, F, K, Q, Y, I, L, or V; X 24 is F, H, N, Q, W, or Y; and X 25 The modified CRBN is either G or A, and therein, the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, or its cleaved form containing the amino acid sequence shown in SEQ ID NO: 129.

[0142] This disclosure pertains to the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10 X11 X 12 PX 13 TX 14 X 15 X 16 X 17 X 18 PGYAWTX 19 AX 20 CKICX 21 X 22 X 23 IGWKX 24 TATKKDMSPQKFWX 25 We provide modified cerebron (CRBN) containing X1, where X1 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 T, F, I, L, V, W, or Y; X9 is N, D, or Y; X 10 is I, D, E, N; Q, L, V, or M; X 11 is G, N, or Q; X 12 is R, E, N, S, or Y; X 13 The letters are S, D, K, N, Q, R, H, and P; X 14 is E, D, K, Q, S, or Y; X 15 is H, D, Q, or T; X 16 is S, N, or T; X 17 is W, F, or Y; X 18 is F or W; X 19 is V, D, E, N, Q, S, or L; X 20 is Q, D, H, K, or R; X 21 is A, D, E, N, or Q; X 22 is S, G, or Q; X 23 is H, D, F, K, Q, Y, I, L, or V; X 24 is F, H, N, Q, W, or Y; and X 25 is G, A, F, I, L, S, or V.

[0143] This disclosure provides a modified cerebron (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, I371, G372, R373, S375, E377, H378, S379, W380, F381, V388, Q390, A395, S396, H397, F402, and G416, compared to a wild-type CRBN comprising the amino acid sequence shown in SEQ ID NO: 127, wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to a wild-type CRBN.

[0144] In some embodiments, the modified CRBN does not contain the DDB1 interaction domain.

[0145] In some embodiments, the modification further includes the deletion of amino acids 194–247 compared to wild-type CRBN.

[0146] In some embodiments, the modified CRBNs are sequence numbers 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, The amino acid sequence includes any one of the following: 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, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 231.

[0148] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 232.

[0149] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 233.

[0150] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 234.

[0151] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 235.

[0152] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 236.

[0153] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 237.

[0154] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 238.

[0155] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 239.

[0156] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 240.

[0157] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 241.

[0158] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 242.

[0159] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 243.

[0160] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 244.

[0161] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 245.

[0162] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 246.

[0163] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 247.

[0164] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 248.

[0165] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 249.

[0166] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 256.

[0167] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 258.

[0168] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 260.

[0169] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 262.

[0170] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 264.

[0171] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 275.

[0172] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 279.

[0173] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 283.

[0174] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 287.

[0175] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 291.

[0176] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 295.

[0177] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 299.

[0178] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 303.

[0179] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 307.

[0180] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 311.

[0181] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 315.

[0182] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 319.

[0183] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 323.

[0184] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 327.

[0185] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 331.

[0186] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 335.

[0187] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 339.

[0188] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 343.

[0189] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 347.

[0190] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 351.

[0191] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 355.

[0192] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 359.

[0193] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 363.

[0194] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 367.

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

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

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

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

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

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

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

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

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

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

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

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

[0207] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 419.

[0208] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 423.

[0209] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 426.

[0210] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 428.

[0211] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 430.

[0212] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 432.

[0213] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 434.

[0214] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 436.

[0215] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 438.

[0216] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 440.

[0217] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 442.

[0218] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 444.

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

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

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

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

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

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

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

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

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

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

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

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

[0231] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 470.

[0232] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 472.

[0233] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 474.

[0234] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 476.

[0235] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 478.

[0236] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 480.

[0237] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 482.

[0238] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 484.

[0239] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 486.

[0240] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 488.

[0241] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 490.

[0242] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 492.

[0243] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 494.

[0244] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 496.

[0245] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 498.

[0246] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 500.

[0247] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 502.

[0248] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 504.

[0249] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 506.

[0250] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 508.

[0251] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 510.

[0252] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 512.

[0253] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 514.

[0254] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 516.

[0255] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 518.

[0256] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 520.

[0257] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 536.

[0258] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 538.

[0259] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 540.

[0260] In some embodiments, the modified CRBN includes the amino acid sequence shown in SEQ ID NO: 542.

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

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

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

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

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

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

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

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

[0269] In some embodiments, one or more amino acid substitutions in the modified CRBN include a T359 amino acid substitution, which may be selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, which may be the modified CRBN including a T359V substitution.

[0270] This disclosure pertains to the amino acid sequence X1X2CQETEITTKNEIFSLSLCGPMAAYX3X4PX5GX6VX7EX8LTVYKACNLX9LX 10 X 11 X 12 PX 13 TX 14 X 15 X 16 X 17 X 18 PGYAWTX 19 AX 20 CKICX 21 X 22 X 23 IGWKX 24 TATKKDMSPQKFWX 25 We provide modified cerebron (CRBN) containing X1, where X1 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; X 10 is I, D, E, N, or Q; X 11 is G, N, or Q; X 12 is R, E, N, S, or Y; X 13 is X, D, K, N, Q, or R; X 14is E, D, K, Q, S, or Y; X 15 is H, D, Q, or T; X 16 is S, N, or T; X 17 is W, F, or Y; X 18 is F or W; X 19 is V, D, E, N, Q, S, or L; X 20 is Q, D, H, K, or R; X 21 is A, D, E, N, or Q; X 22 is S, G, or Q; X 23 is H, D, F, K, Q, Y, I, L, or V; X 24 is F, H, N, Q, W, or Y; and X 25 It is either G or A.

[0271] In some embodiments, the modified CRBN contains an amino acid sequence that is 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, the modified CRBN includes, compared to a wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, (i) a Q325 amino acid substitution selected from Q325H substitution, Q325K substitution, and Q325R substitution; and (ii) one or more amino acid substitutions selected from I371N substitution, H397F substitution, and N369Y substitution.

[0273] In some embodiments, the modified CRBN contains, compared to the wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, (i) Q325H substitution and I371N substitution, optionally, the same amino acid sequence as SEQ ID NO: 351 by 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%; or (ii) Q325H substitution, I371N substitution, and H397F substitution, optionally, (iii) containing 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 amino acid sequence to sequence number 355; or (iii) having Q325H substitution and N369Y substitution, and in some cases containing 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 amino acid sequence to sequence number 359 Containing the same amino acid sequence; or (iv) Q325H substitution, N369Y substitution, and H397F substitution, and in some cases containing 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 amino acid sequence to SEQ ID NO: 363; or (v) Q325H substitution, N369Y substitution, and I371N substitution, and in some cases containing at least 80%, at least 85%, less identical amino acid sequence to SEQ ID NO: 367. (vi) Contains at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences with (vi) Q325H substitution, N369Y substitution, I371N substitution, and H397F substitution, which may contain 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 amino acid sequences with SEQ ID NO: 371;or (vii) Q325K substitution and H397F substitution, which may include 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 amino acid sequence to SEQ ID NO: 375; or (viii) Q325K substitution, I371N substitution, and H397F substitution, which may include at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97% identical amino acid sequence to SEQ ID NO: 383 , containing at least 98%, at least 99%, or 100% identical amino acid sequences; or (ix) Q325R substitution and H397F substitution, optionally containing 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 amino acid sequences to SEQ ID NO: 403; or (x) I371N substitution and H397F substitution, optionally containing at least 80%, at least 85%, at least 90%, Contains at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; or (xi)N369Y substitution and H397F substitution, optionally containing 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 amino acid sequences to SEQ ID NO: 335; or (xii)N369Y substitution and I371N substitution, optionally SEQ ID NO: 339 and containing 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 amino acid sequences; or (xiii)N369Y substitution, I371N substitution, and H397F substitution, which may contain 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 amino acid sequences to SEQ ID NO: 343;or (xiv) Q325H substitution and H397F substitution, which may contain 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 amino acid sequences to SEQ ID NO: 347; or (xv) Q325K substitution and I371N substitution, which may contain 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 containing 100% identical amino acid sequences; or (xvi) Q325K substitution and N369Y substitution, which may contain 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 amino acid sequences to SEQ ID NO: 387; or (xvii) Q325K substitution, N369Y substitution and H397F substitution, which may contain at least 80%, at least 85%, at least 90%, at least 95%, at least 96% identical amino acid sequences to SEQ ID NO: 391 , containing at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; or (xviii) Q325K substitution, N369Y substitution, and I371N substitution, optionally containing 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 amino acid sequences to SEQ ID NO: 395; or (xix) Q325K substitution, N369Y substitution, I371N substitution, and H397F substitution, optionally containing SEQ ID NO: 39 Contains 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 amino acid sequences to 9; or contains (xx)Q325R substitutions, N369Y substitutions, I371N substitutions, and H397F substitutions, which may include 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 amino acid sequences to SEQ ID NO: 315.

[0274] In some embodiments, the modified CRBN includes one or more amino acid substitutions selected from the T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, compared to a wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, wherein the Q325 amino acid substitution is selected from the Q325H substitution, Q325K substitution, and Q325R substitution, and / or, wherein the T359 amino acid substitution is selected from the T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution.

[0275] This disclosure provides a polypeptide complex comprising a first polypeptide comprising degron and a second polypeptide comprising cerebron, wherein (i) degron is a modified degron as described herein; (ii) cerebron is a modified cerebron as described herein; or (iii) degron is a modified degron as described herein and cerebron is a modified cerebron as described herein.

[0276] In some embodiments, modified cerebron (CRBN) includes one or more amino acid substitutions selected from the T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, compared to wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, wherein the Q325 amino acid substitution is selected from the Q325H substitution, Q325K substitution, and Q325R substitution, and / or, wherein the T359 amino acid substitution is selected from the T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, and wherein modified degron includes one or more amino acid substitutions selected from the Q28T amino acid substitution, G30E amino acid substitution, and L33I amino acid substitution, compared to wild-type degron having the amino acid sequence shown in SEQ ID NO: 40. In some embodiments, the modified CRBN includes the I371N amino acid substitution, wherein the modified deglon includes the G30E amino acid substitution. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions. In some embodiments, the modified CRBN includes the Q325R, N369Y, I371N, and H397F amino acid substitutions, wherein the modified deglon includes the Q28T, G30E, and L33I amino acid substitutions.

[0277] In some embodiments, the modified CRBN includes, compared to a wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, (i) a Q325 amino acid substitution selected from Q325H substitution, Q325K substitution, and Q325R substitution; and (ii) one or more amino acid substitutions selected from T359V substitution, I371N substitution, H397F substitution, and N369Y substitution.

[0278] In some embodiments, the modified CRBN contains, compared to wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, (i) Q325H substitution, Q325H substitution, I371N substitution, and T359V substitution, and optionally, 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 amino acid sequence to SEQ ID NO: 544; or (ii) Q325R substitution, I371N substitution, H397F substitution, and T35 9V substitution, which may include 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 amino acid sequence to SEQ ID NO: 530; or (iii) Q325R substitution, N369Y substitution, H397F substitution, and T359V substitution, which may include at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and (iv) Q325R substitution, I371N substitution, and T359V substitution, in the case of SEQ ID NO: 524, contains 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 amino acid sequence; or (v) Q325K substitution, I371N substitution, H397F substitution, and T359V substitution, in the case of SEQ ID NO: 532, contains at least 80%, at least Containing 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences; or (vi) Q325H substitution, N369Y substitution, I371N substitution, H397F substitution, and T359V substitution, which may contain 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 amino acid sequences to SEQ ID NO: 528;or (vii) containing an amino acid sequence identical to 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% of SEQ ID NO: 534, including a Q325H substitution, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of SEQ ID NO: 522, including a Q325H substitution, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of SEQ ID NO: 522.

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

[0280] The disclosure further provides a modified degron comprising the amino acid sequence X1CGFTX2X3X4X5X6NLX7X8HIKX9, wherein X1 is I, H, V, or L; X2 is C, T, or S; X3 is R, T, N, K, Q, D, or F; X4 is Q, C, Y, S, L, T, P, or R; X5 is K, H, E, or L; X6 is G, E, or L; X7 is L, D, or I; X8 is R, V, or F; and X9 is L or A, wherein the modified degron exhibits reduced proteolysis when bound to one or more IMiDs compared to a degron comprising the amino acid sequence shown in SEQ ID NO: 40 or 131.

[0281] In some embodiments, the modified deglon includes the amino acid sequence shown in any one of SEQ ID NOs. 609 to 728. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 684. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 688. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 668. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 704. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 706. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 724. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 690. In some embodiments, the modified deglon includes the amino acid sequence shown in SEQ ID NOs. 702.

[0282] In some embodiments, the modified degron, compared to wild-type degron containing the amino acid sequence shown in SEQ ID NO: 40, further includes one or more amino acid substitutions 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, one or more amino acid substitutions confer enhanced binding of the modified degron to IMiD when compared to degron without one or more amino acid substitutions.

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

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

[0285] In some embodiments, the modified deglon does not contain the amino acid sequence shown in SEQ ID NO: 40. [Brief explanation of the drawing]

[0286] These and other features, aspects, and advantages of this disclosure will be better understood in relation to the following description and the accompanying drawings.

[0287] [Figure 1A] Figures 1A and 1B illustrate constructs for assaying libraries of modified degron polypeptides. Figure 1A details a screening vector containing EGFP-2a-mCherry operably linked to a degron polypeptide by a linker. [Figure 1B]Figures 1A and 1B illustrate constructs for assaying libraries of modified degron polypeptides. Figure 1B illustrates a degron-linker cassette for assaying lysine substitutions in the linker region. [Figure 2A] Figures 2A-2B detail an overview of library sorting and isolation of modified degron polypeptides with increased sensitivity to pomalidomide. Figure 2A illustrates the library sorting method. [Figure 2B] Figures 2A–2B detail an overview of library sorting and isolation of modified degron polypeptides with increased sensitivity to pomalidomide. Figure 2B shows an exemplary flow cytometry plot detailing sequential sorting and enrichment of modified degron polypeptides with increased sensitivity to pomalidomide. [Figure 3] Figure 3 is a graph showing the identification of modified degron polypeptides abundant in a pool treated with 1 nM pomalidomide. [Figure 4A] Figures 4A–4B detail the nanoluciferase degron assay for verifying degron identified in library screening. Figure 4A is a diagram of the dual luciferase construct containing the degron linker cassette. [Figure 4B] Figures 4A-4B detail the nanoluciferase degron assay for verifying degrons identified in the library screen. Figure 4B is a graph showing the degradation of luciferase using the degron cassette shown, as measured by the decrease in luciferase signal (luminescence). [Figure 5A] Figures 5A-5C illustrate dimerization assays of inducible cell death systems containing modified degron polypeptides. Figure 5A illustrates the induction of apoptosis by caspase-9 dimerization using a construct containing a small molecule binding domain. [Figure 5B]Figures 5A-5C illustrate dimerization assays of inducible cell death systems containing modified degron polypeptides. Figure 5B illustrates a construct containing caspase-9 and a small molecule binding domain, either cerebron (CRBN) or its variant (SB06097) or degron (SB06098). [Figure 5C] Figures 5A-5C illustrate the dimerization assay of an inducible cell death system containing modified degron polypeptides. Figure 5C shows the time course of the caspase-9 dimerization assay. [Figure 6] Figure 6 shows the killing efficiency of the modified degron polypeptides compared to that of wild-type degron. [Figure 7] Figure 7 shows an exemplary workflow for a split IMiD ON switch assay to screen for mutant CRIMP domains for improved sensitivity to pomalidomide. [Figure 8] Figure 8 shows the results of a split IMiD ON-switch assay to screen mutant CRIMP domains for improved sensitivity to pomalidomide. [Figure 9] Figure 9 shows an exemplary workflow for a split IMiD ON-switch assay to screen mutant del.CRBN domains for improved susceptibility to pomalidomide. [Figure 10] Figure 10 shows an exemplary heatmap of the reporter's standardized activation factor for selected mutant del.CRBN domains in response to dose settings of pomalidomide (100 nM, 10 nM, and 4 nM). [Figure 11] Figure 11 shows exemplary line graphs of the reporter's standardized activation ratios for selected mutant del.CRBN domains in response to dose settings of pomalidomide (100 nM, 10 nM, and 4 nM). [Figure 12] Figure 12 shows an exemplary bar graph of the standardized activation factor of the reporter for a selected mutant del.CRBN domain in response to 4nM pomalidomide. [Figure 13A] Figures 13A–13F show the results of assays to evaluate different 2A linkers between single-vector CRIMP / CRBN combinations and their switch components. [Figure 13B] Figures 13A–13F show the results of assays to evaluate different 2A linkers between single-vector CRIMP / CRBN combinations and their switch components. [Figure 13C] Figures 13A–13F show the results of assays to evaluate different 2A linkers between single-vector CRIMP / CRBN combinations and their switch components. [Figure 13D] Figures 13A–13F show the results of assays to evaluate different 2A linkers between single-vector CRIMP / CRBN combinations and their switch components. [Figure 13E] Figures 13A–13F show the results of assays to evaluate different 2A linkers between single-vector CRIMP / CRBN combinations and their switch components. [Figure 13F] Figures 13A–13F show the results of assays to evaluate different 2A linkers between single-vector CRIMP / CRBN combinations and their switch components. [Figure 14] Figure 14 shows the results of an assay to evaluate CRBN variants, including combinations of amino acid substitutions. [Figure 15] Figure 15 shows the results of an assay to evaluate CRBN variants, including combinations of amino acid substitutions. [Figure 16] Figure 16 shows an exemplary bar graph of the standardized activation factor of the reporter for a selected mutant del.CRBN domain in response to 10 nM pomalidomide. [Figure 17] Figure 17 shows the results of an assay for screening del.CRBN variants with improved sensitivity to pomalidomide. [Figure 18]Figure 18 shows an exemplary bar graph of the standardized activation factor of the reporter for a selected mutant del.CRBN domain in response to 4nM pomalidomide. [Figure 19] Figure 19 shows the results of an assay for screening del.CRBN variants with improved sensitivity to pomalidomide. [Figure 20] Figure 20 shows an exemplary bar graph of the standardized activation factor of the reporter for a selected mutant del.CRBN domain in response to 1 nM pomalidomide. [Figure 21A] Figures 21A–21C detail assays for identifying combinational mutants or variants of del.CRBN with improved sensitivity to pomalidomide. Figure 21A shows a diagram of a multicistronic construct containing del.CRBN variants (e.g., del.CRBN with combinations of amino acid substitutions), minVPR, Opt2A, CRIMP.mut1, and a ZF DNA-binding domain (DBD), as well as a diagram of a reporter construct containing a ZF-binding site (BS) and mCherry. [Figure 21B] Figures 21A–21C detail assays for identifying combinational mutants or variants of del.CRBN with improved sensitivity to pomalidomide. Figure 21B shows an exemplary workflow for evaluating del.CRBN variants with combinations of amino acid substitutions. [Figure 21C] Figures 21A–21C detail assays for identifying combinational mutants or variants of del.CRBN with improved sensitivity to pomalidomide. Figure 21C shows the results of assays for evaluating del.CRBN variants, including combinations of amino acid substitutions. [Figure 22A] Figures 22A-22B show the results of assays to evaluate del.CRBN variants, including combinations of amino acid substitutions. [Figure 22B] Figures 22A-22B show the results of assays to evaluate del.CRBN variants, including combinations of amino acid substitutions. [Figure 23A] Figures 23A–23C detail assays for identifying combinational mutants or variants of del.CRBN with improved sensitivity to pomalidomide. Figure 23A shows a diagram of a multicistronic construct containing del.CRBN variants (e.g., del.CRBN with combinations of amino acid substitutions), minVPR, Opt2A_2.0, CRIMP.mut1, and a ZF DNA-binding domain (DBD), as well as a diagram of a reporter construct containing a ZF-binding site (BS) and mCherry. [Figure 23B] Figures 23A–23C detail assays for identifying combinational mutants or variants of del.CRBN with improved sensitivity to pomalidomide. Figure 23B shows an exemplary workflow for evaluating del.CRBN variants with combinations of amino acid substitutions. [Figure 23C] Figures 23A–23C detail assays for identifying combinational mutants or variants of del.CRBN with improved sensitivity to pomalidomide. Figure 23C shows the results of assays for evaluating del.CRBN variants, including combinations of amino acid substitutions. [Figure 24A] Figures 24A-24B show the results of assays to evaluate del.CRBN variants, including combinations of amino acid substitutions. [Figure 24B] Figures 24A-24B show the results of assays to evaluate del.CRBN variants, including combinations of amino acid substitutions. [Figure 25] Figure 25 shows a graph illustrating a comparison of performance between structures, including Opt2A and Opt2A_2.0. [Figure 26A] Figures 26A–26B detail the assay for a CRIMP variant that is resistant to degradation in the presence of an IMiD (e.g., pomalidomide). Figure 26A is a schematic diagram detailing the design of constructs encoding the CRIMP variant and two fluorescent reporter genes. [Figure 26B]Figures 26A–26B detail assays for CRIMP variants that are resistant to degradation in the presence of IMiDs (e.g., pomalidomide). Figure 26B outlines an assay for evaluating the degradation of CRIMP variants in the presence of pomalidomide. [Figure 27A] Figures 27A–27D illustrate graphs showing the results of the degradation assays detailed in Figures 26A–26B for three batches of 20 constructs. Figure 27A illustrates the graph of the degradation assay results for batch 1. [Figure 27B] Figures 27A–27D illustrate graphs showing the results of the degradation assays detailed in Figures 26A–26B for three batches of 20 constructs. Figure 27B illustrates a graph showing the results of the degradation assay for batch 2. [Figure 27C] Figures 27A–27D illustrate graphs showing the results of the degradation assays detailed in Figures 26A–26B for three batches of 20 constructs. Figure 27C illustrates a graph showing the results of the degradation assay for batch 3. [Figure 27D] Figures 27A–27D illustrate graphs showing the results of the degradation assays detailed in Figures 26A–26B for three batches of the 20 constructs. Figure 27D illustrates a graph showing the combined results of the degradation assays for all three batches. [Figure 28] Figure 28 is a graph illustrating the degree of decomposition for the CRIMP variants shown at 0 nM, 0.1 nM, and 4.0 nM pomalidomide. [Figure 29] Figure 29 is a graph showing the activity of the IMiD-responsive transcription switch in a mouse model with 40 mg / kg pomalidomide. [Modes for carrying out the invention]

[0288] This disclosure generally relates to modified degron polypeptides with increased sensitivity to pomalidomide. Also provided are inducibly degradable proteins, inducible cell death systems, and activation condition control polypeptides using the modified degron polypeptides described herein and the methods and / or uses thereof.

[0289] Terms used in the claims and specification are defined as shown below unless otherwise specified.

[0290] The term "remission" refers to any therapeutically beneficial outcome in the treatment of a disease state, such as cancer, including prevention, reduction in severity or progression, remission, or cure.

[0291] The term "insights" refers to processes that occur in living cells that grow separately from a living organism, for example, in a tissue culture.

[0292] The term "in vivo" refers to a process that occurs within living organisms.

[0293] As used herein, the term “mammal” includes, but is not limited to, both humans and non-humans, including humans, non-human primates, dogs, cats, mice, cattle, horses, and pigs.

[0294] The term “percent identity” refers to two or more sequences or subsequences of nucleic acids or polypeptides that, when compared and aligned for the greatest match, have a specified percentage of identical nucleotide or amino acid residues, measured using a sequence comparison algorithm, e.g., one of those described herein (e.g., BLASTP and BLASTN, or other algorithms available to those skilled in the art), or by visual inspection. Depending on the application, percentage “identity” may exist across regions of the sequences being compared, e.g., across functional domains, or alternatively, across the entire length of the two sequences being compared.

[0295] In sequence comparison, typically, one sequence acts as the reference sequence compared to the test sequence. When using a sequence comparison algorithm, the test sequence and reference sequence are input into the computer, the coordinates of subsequences are specified if necessary, and the program parameters of the sequence algorithm are specified. The sequence comparison algorithm then calculates the sequence identity percentage for the test sequence relative to the reference sequence, based on the specified program parameters.

[0296] Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by searching for similarity methods of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized execution of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wisconsin), or by visual inspection (see Ausubel et al., hereafter).

[0297] One example of an algorithm 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 analysis is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov / ).

[0298] The term "sufficient amount" means an amount sufficient to produce the desired effect, for example, an amount sufficient to regulate protein aggregation in cells.

[0299] The term "therapeutic dose" refers to the amount of a substance that is effective in alleviating the symptoms of a disease. A therapeutic dose can also be called a "preventive dose," because prevention can be considered a form of treatment.

[0300] As used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” refer to multiple objects unless the context otherwise explicitly indicates otherwise.

[0301] The use of common terminology in the claims to modify the elements of the claims, such as “first,” “second,” “third,” etc., does not by itself indicate any priority, order, or sequence of one claim element over another, or a temporal order in which the actions of the method are performed, but is used simply as a label to distinguish one claim element having a particular name from another element having the same name (but due to the use of common terminology), thereby distinguishing the elements of the claims. Similarly, the use of these terms herein does not by itself imply any required priority, order, or sequence.

[0302] Manipulated nucleic acids and polypeptides This disclosure provides, in particular, engineered nucleic acids comprising one or more expression cassettes (e.g., one, two, three, four, five, or more expression cassettes), for example, any expression cassette described herein. In some embodiments, at least one expression cassette comprises a promoter and an exogenous polynucleotide sequence, as provided herein. In some embodiments, the promoter is operably and / or directly ligated to the exogenous polynucleotide sequence. In some embodiments, the exogenous polynucleotide sequence encodes one or more engineered polypeptides provided herein, for example, modified degron or CRIMP (e.g., fused or operably ligated to the polypeptide of interest), modified CRBN (e.g., fused or operably ligated to the polypeptide of interest), activation condition controlled polypeptide (ACP), inducibly degradable protein, inducibly cell death polypeptide monomer, regulated cell survival polypeptide, or chimeric polypeptide, as described herein.

[0303] In some embodiments, the engineered nucleic acid comprises a first expression cassette comprising a first promoter and a first exogenous polynucleotide sequence. In some embodiments, the first promoter is operably and / or directly ligated to the first exogenous polynucleotide sequence. In some embodiments, the first exogenous polynucleotide sequence encodes one or more engineered polypeptides provided by this disclosure, e.g., activated conditioned control polypeptides (ACPs), inducibly degradable proteins, inducibly cell death polypeptide monomers, regulatory cell survival polypeptides, or chimeric polypeptides, 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., multiple expression cassettes. In some embodiments, each expression cassette comprises a promoter operably ligated to a polynucleotide sequence encoding the protein of interest (e.g., any engineered polypeptide described herein).

[0304] In some embodiments of this disclosure, the engineered nucleic acid comprises an expression cassette containing an inducibly degradable protein. The inducibly degradable protein may contain one or more ligand-binding domains (e.g., degron polypeptide, e.g., the ligand-binding domain of a modified degron polypeptide) and at least one target polypeptide. When expressed in cells, the inducibly degradable protein can be targeted by an E3 ubiquitin ligase via a homologous ligand (e.g., a small molecule) that binds to its ligand-binding domain. In some embodiments, the E3 ubiquitin ligase may induce polyubiquitination upon binding to the inducibly degradable protein, which results in proteasomal degradation of the inducibly degradable protein.

[0305] In some embodiments, the engineered nucleic acid comprises an expression cassette comprising a promoter operably ligated to an exogenous polynucleotide sequence encoding at least one inducible cell death polypeptide monomer. The inducible cell death polypeptide monomer may contain one or more ligand-binding domains and at least one cell death-inducing domain. When expressed in cells, the cell death polypeptide monomer is oligomerizable via a homologous ligand (e.g., small molecule) that binds to the ligand-binding domain. If the ligand oligomerizes two or more cell death polypeptide monomers, a cell death-inducing signal may be generated in the cell. This generally results in cell death. In some embodiments, the engineered nucleic acid comprises an expression cassette comprising a promoter operably ligated to an exogenous polynucleotide sequence encoding (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, the engineered nucleic acid comprises a first expression cassette comprising (a) a first promoter operably ligated to a first exogenous polynucleotide sequence encoding 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 ligated to a second exogenous polynucleotide sequence encoding a second inducible cell death polypeptide monomer comprising a second ligand-binding domain and a cell death-inducing domain. In some embodiments, the engineered nucleic acid comprises an expression cassette comprising a promoter operably ligated to an exogenous polynucleotide sequence encoding a first inducible cell death polypeptide monomer comprising a first ligand-binding domain and a cell death-inducing domain. In some embodiments, the engineered nucleic acid comprises an expression cassette comprising a promoter operably ligated to an exogenous polynucleotide sequence encoding a second inducible cell death polypeptide monomer comprising a second ligand-binding domain and a cell death-inducing domain. In some embodiments, the first inducible cell death polypeptide monomer comprises degron (e.g., any degron or CRIMP described herein).In some embodiments, the second inducible cell death polypeptide monomer comprises a cereblon domain (e.g., any cereblon domain described herein).

[0306] In some embodiments, the manipulated nucleic acid comprises an expression cassette containing a promoter operably ligated to an exogenous polynucleotide sequence encoding at least one ACP. The ACP may contain one or more ligand-binding domains and at least one transcription factor comprising at least one nucleic acid-binding domain and at least one transcription effector domain. When expressed in cells, ACP may undergo nuclear localization upon binding of its ligand-binding domain to a homologous ligand. When localized to the nucleus of the cell, ACP can induce the transcriptional expression of a gene of interest operably ligated to an ACP-responsive promoter. In some embodiments, the gene of interest is associated with or induces cell death, for example, by apoptosis.

[0307] In some embodiments, the engineered nucleic acid comprises an expression cassette containing a promoter operably ligated to an exogenous polynucleotide sequence encoding at least one ACP. The ACP may contain at least one ligand-binding domain and at least one transcription effector domain. When expressed in cells and upon binding of the ligand-binding domain to a congenital ligand, ACP can regulate the transcriptional expression of a gene of interest operably ligated to an ACP-responsive promoter. For example, in some embodiments, when expressed in cells and upon binding of the ligand-binding domain to a congenital ligand, the activity of ACP regulates the transcriptional expression of a gene of interest operably ligated to an ACP-responsive promoter. Alternatively, in some embodiments, binding of the ligand-binding domain to a congenital ligand induces degradation of ACP, and thus, ACP-based regulation of the transcriptional expression of the gene of interest is suppressed by binding to the congenital ligand. In some embodiments, the gene of interest is associated with, or induces, cell death, for example, through apoptosis.

[0308] In some embodiments, the engineered nucleic acid comprises an expression cassette comprising a promoter operably ligated to an exogenous polynucleotide sequence encoding at least one modulotable cell survival polypeptide containing at least one ligand-binding domain. When expressed in cells, at least one cell survival polypeptide is capable of inhibiting at least one cell death-inducing polypeptide, and upon binding to a congenital ligand, the congenital ligand inhibits at least one survival-promoting polypeptide, which can lead to cell death.

[0309] In some embodiments, the manipulated nucleic acid comprises an expression cassette comprising a promoter operably ligated to an exogenous polynucleotide sequence having the formula: C1-L-C2, where C1 comprises a polynucleotide sequence encoding at least a first chimeric polypeptide comprising at least a first ligand-binding domain and at least a transcriptional activation domain, L comprises at least a linker polynucleotide sequence, and 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 cells, the first and second chimeric polypeptides can functionally associate (e.g., directly or indirectly bind, oligomerize, multimerize, etc.) and form an ACP via homologous ligands that bind to each ligand-binding domain. The multimeric ACP can then induce the transcriptional expression of a gene of interest operably ligated 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.

[0310] In some embodiments, the engineered nucleic acid comprises: (a) a first expression cassette comprising a first promoter operably ligated to a first exogenous polynucleotide sequence encoding a first chimeric polypeptide, wherein the first chimeric polypeptide comprises a first ligand-binding domain and a transcription effector domain; and (b) a second expression cassette comprising a second promoter operably ligated 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 cells, the first and second chimeric polypeptides may functionally associate (e.g., directly or indirectly binding, oligomerizing, multimerizing, etc.) and form an ACP via homologous ligands that bind to each ligand-binding domain. The ACP can then induce or repress the expression of a gene of interest operably ligated to an ACP-responsive promoter. In some embodiments, the transcription effector domain is a transcription activator domain. In some embodiments, the transcription effector domain is a transcription repressor domain.

[0311] In some embodiments, the engineered nucleic acid comprises: (a) a first expression cassette comprising a first promoter operably ligated 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; and (b) a second expression cassette comprising a second promoter operably ligated 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 cells, the first and second chimeric polypeptides can functionally associate to form an ACP in a ligand-dependent manner (e.g., binding directly or indirectly, oligomerizing, multimerizing, etc.). The ACP can then induce transcriptional expression of a gene of interest operably ligated to an ACP-responsive promoter in cells. Depending on the gene of interest, this can result in one or more biological responses or events, including, but not limited to, cell death.

[0312] In some embodiments, the chimeric polypeptide (e.g., the first, second, third, etc. chimeric polypeptides described herein) includes a ligand-binding domain and a transcription effector domain (e.g., a transcription activation domain). In some embodiments, the ligand-binding domain and the transcription effector domain are linked by a linker sequence (e.g., any linker sequence described herein). In some embodiments, the chimeric polypeptide includes, from the N-terminus to the C-terminus, a ligand-binding domain, a linker sequence, and a transcription effector domain. In some embodiments, the chimeric polypeptide (e.g., the first, second, third, etc. chimeric polypeptides described herein) includes a ligand-binding domain and a nucleic acid-binding domain (e.g., a DNA-binding domain). In some embodiments, the ligand-binding domain and the nucleic acid-binding domain are linked by a linker sequence (e.g., any linker sequence described herein). In some embodiments, the chimeric polypeptide includes, from the N-terminus to the C-terminus, a ligand-binding domain, a linker sequence, and a nucleic acid-binding domain.

[0313] One or more linkers can be used between various domains of a provided, engineered nucleic acid. A suitable polypeptide linker may include a glycine-serine linker comprising one or more serine residues and one or more glycine residues. An exemplary glycine-serine linker sequence may include one or more GGGS (SEQ ID NO: 252) motifs, one or more GGGGGS (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 may include 1 to 20 GGGS (SEQ ID NO: 252) motifs and / or GGGGS (SEQ ID NO: 253) motifs. In some embodiments, a glycine-serine linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 254). This disclosure identifies certain polypeptide linker sequences that, depending on the presence or absence of certain amino acids (e.g., one or more lysine residues) within the linker sequence, can increase or decrease the degradation (e.g., via the ubiquitin-proteosome pathway) of a particular engineered polypeptide comprising the polypeptide linker. In some embodiments, the polypeptide linker sequence includes one or more lysine residues. In some embodiments, the polypeptide linker sequence including one or more lysine residues has increased degradation compared to the polypeptide linker sequence not having the one or more lysine residues (e.g., when the lysine residues are substituted with another amino acid, e.g., alanine). In some embodiments, one or more lysine residues in the polypeptide linker sequence are substituted with another amino acid residue (e.g., alanine). In some embodiments, the polypeptide linker sequence having one or more lysine residues substituted with another amino acid (e.g., alanine) has decreased degradation compared to the polypeptide linker sequence in which the one or more lysine residues are not substituted with another amino acid. In some embodiments, the polypeptide linker encoded by the manipulated nucleic acid includes an amino acid sequence, e.g., one or more of the following: GGGGSGGGGSGGGGSVDGF (SEQ ID NO: 91) and ASGGGGSAS (SEQ ID NO: 92).In some embodiments, the polypeptide linker encoded by the manipulated nucleic acid may include the amino acid sequence AEAAAKEAX1AKEAX2AKA (SEQ ID NO: 168), where X1 is A or K; and X2 is A or K. In some embodiments, the linker includes the amino acid sequence shown in any one of SEQ ID NOs: 169, 171, 173, or 175. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 169. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 171. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 173. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 175. In some embodiments, the linker includes the amino acid sequence of SEQ ID NO: 199.

[0314] 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-recognition 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 target protein (e.g., an engineered polypeptide as described herein) can be linked to a nucleotide sequence encoding a second target protein, e.g., in the 5' to 3' direction of first gene:linker:second gene. Features and options of multicistronicity are described, for example, in the section "Multicistronic and Multiple Promoter Systems".

[0315] In some embodiments, the manipulated nucleic acid is selected from DNA, cDNA, RNA, mRNA, and naked plasmids (linear or circular). Also provided herein are expression vectors containing the manipulated nucleic acid.

[0316] In some embodiments, the engineered nucleic acid further comprises an insulator. The insulator can be localized between a first expression cassette and a second expression cassette. The insulator is a cis-regulatory element having an enhancer-blocking or barrier function. Enhancer-blocker insulators block enhancers from acting on the promoter of a proximal gene. Barrier insulators prevent euchromatin silencing. An example of a suitable insulator of this disclosure is the A2 insulator described in Liu M, et al., Nat Biotechnol. 2015 Feb;33(2):198-203. Additional insulators are described in West et al, Genes & Dev, 002.16:271-288, both of which are incorporated by reference in their entirety. Other examples of suitable insulators include, without limitation, A1 insulators, CTCF insulators, Gypsy insulators, HS5 insulators, and β-globin locus insulators, such as cHS4. In some embodiments, the insulator is an A2 insulator, an A1 insulator, a CTCF insulator, an HS5 insulator, a Gypsy insulator, a β-globin locus insulator, or a cHS4 insulator.

[0317] Ligand-binding domain In some embodiments of this disclosure, the manipulated polypeptide (e.g., degron, CRIMP, CRBN, etc., provided) includes a ligand-binding domain. In some embodiments, the ligand-binding domain interacts with a ligand, such as a homologous ligand. In some embodiments, the interaction between the ligand-binding domain and the ligand results in oligomerization, e.g., dimerization, of the ligand-binding domain and one or more other polypeptides. In some embodiments, the interaction between the ligand-binding domain and the ligand results in oligomerization, e.g., dimerization, of multiple ligand-binding domains. In some embodiments, the interaction between the ligand-binding domain and the ligand results in a functional association between multiple ligand-binding domains, thereby resulting in a biologically relevant output (e.g., an increase or decrease in gene expression, an increase or decrease in biological signaling, etc.). In some embodiments, the functional association between multiple ligand-binding domains is direct in that the multiple ligand-binding domains are in direct contact with each other. In some embodiments, the functional association between multiple ligand-binding domains is indirect in that the multiple ligand-binding domains do not come into direct contact with each other (e.g., the functional association between the multiple ligand-binding domains may be facilitated by one or more intermediate agents or molecules, such as polypeptides, nucleic acids, etc.). In some embodiments, the functional association includes oligomerization, polymerization, and / or complex formation between two or more manipulated polypeptides of the Disclosure.

[0318] In some embodiments, the ligand-binding domain includes a degron. The terms “degron” and “degron domain” refer to a protein or a portion thereof involved in regulating the rate of proteolysis, as used herein. A variety of degrons, including short amino acid sequences, structural motifs, and exposed amino acids, may be used in various embodiments of this disclosure, but are not limited to those described herein. Degrons identified from a variety of organisms may be used. For example, see Varshazsky A., PNAS 2019 Jan 8;116(2):358-366, incorporated herein by reference.

[0319] As used herein, the term “degradation sequence” refers to a sequence that promotes the degradation of an attached protein via either the proteasome or the autophagy-lysosome pathway. Any degradation sequence, e.g., those described herein, may be used for various embodiments of this disclosure. In some embodiments, the degradation sequence includes degron. Degron from any organism may be used in accordance with this disclosure, including its derivatives and variants. In some embodiments, the degradation sequence is a polypeptide that, when fused to a protein, destabilizes the protein such that the protein’s half-life is reduced by at least twofold (e.g., at least twofold, at least threefold, at least fourfold, at least fivefold, etc.). Any degradation sequence / signal (e.g., of the ubiquitin-proteasome system) may be used in accordance with this disclosure. The degradation sequence may be operably ligated to a cell receptor, but does not need to be adjacent to it, as long as the degradation sequence still functions to direct the degradation of the cell receptor. In some embodiments, the degradation sequence induces rapid degradation of the cell receptor. For discussions on degradation sequences and their functions in protein degradation, see, for example, Kanemaki et al. (2013) Pflugers Arch. 465(3): 419-425, Erales et al. (2014) Biochim Biophys Acta 1843(l): 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): 520-528, Meinnel et al. (2006) Biol. Chem. 387(7): 839-851, Kim et al. (2013) Autophagy See 9(7):1100-1103, Varshavsky (2012) Methods Mol. Biol. 832:1-11, and Fayadat et al. (2003) Mol Biol Cell. 14(3):1268-1278; incorporated herein by reference.

[0320] In some embodiments, the degron used in accordance with this disclosure is a CeReblon IMiD binding partner ("CRIMP"). As used herein, "CRIMP" may refer to a degron, in particular a degron used in a system in which binding of a congener ligand (e.g., IMiD) does not result in directional degradation of the degron. Thus, in some embodiments, the degron or modified degron used in accordance with this disclosure is a CRIMP.

[0321] In some embodiments, degron can bind to or functionally associate with CRBN or its variants (e.g., modified CRBN) in response to immunomodulatory drugs (IMiDs). Such functional association can promote ubiquitin-mediated degradation of target polypeptides (e.g., engineered polypeptides, ACP, etc., as described herein). In some embodiments, the CRBN polypeptide substrate domain is selected from: IKZF1, IKZF3, Ck1a, ZFP91, GSPT1, MEIS2, GSS E4F1, ZN276, ZN517, ZN582, ZN653, ZN654, ZN692, ZN787, and ZN827, or fragments thereof, which are capable of drug-induced binding to or functional association with CRBN or its variants. In some embodiments, the CRBN polypeptide substrate domain is a chimeric fusion product of a native CRBN polypeptide sequence. In some embodiments, the CRBN polypeptide substrate domain is an IKZF3 / ZFP91 / IKZF3 chimeric fusion product having the amino acid sequence FNVLMVHKRSHTGERPLQCEICGFTCRQKGNLLRHIKLHTGEKPFKCHLCNYACQRRDAL (SEQ ID NO: 93).

[0322] In some embodiments, degron (e.g., modified degron, e.g., CRIMP) can bind to or functionally associate with CRBN or its variant (e.g., modified CRBN) in response to IMiD (e.g., IMiD binding at the CRBN ligand-binding domain and / or degron ligand-binding domain). In some embodiments, such functional association between modified degron or CRIMP and modified CRBN may not lead to targeted degradation of the target polypeptide, but rather produce biologically relevant outputs as a result of the functional association (e.g., increased or decreased gene expression, increased or decreased biological signaling, etc.). In some embodiments, the biologically relevant output is produced when CRIMP and modified CRBN functionally associate, bringing together their effector domain and DNA-binding domain. For example, in some embodiments, CRIMP may be operably ligated to a DNA-binding domain, and modified CRBN may be operably ligated to an effector domain, and in the presence of a suitable ligand (e.g., IMiD), the CRIMP component and the modified CRBN component functionally associate to bring together the effector domain and the DNA-binding domain to produce an increase or decrease in gene expression at the target gene.

[0323] In some embodiments, degron includes the amino acid sequence of SEQ ID NO: 40. In some embodiments, degron includes the amino acid sequence of SEQ ID NO: 131. In some embodiments, degron includes modified d913 degron. In some embodiments, degron includes modified d913 degron which includes one or more amino acid substitutions to the amino acid sequence of SEQ ID NO: 131. In some embodiments, degron does not include the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, degron does not include the amino acid sequence of SEQ ID NO: 40. In some embodiments, modified degron includes the amino acid sequence of SEQ ID NO: 133. In some embodiments, modified d913 degron includes one or more modifications / mutations that reduce ubiquitination compared to unmodified d913 degron having the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, modified d913 degron includes one or more modifications / mutations that reduce ubiquitination compared to unmodified d913 degron having the amino acid sequence of SEQ ID NO: 131. In some embodiments, the modification of a modified d913 degron includes substituting one or more lysine residues compared to an unmodified d913 degron containing, for example, the amino acid sequence of SEQ ID NO: 131. In some embodiments, the modification of a modified d913 degron includes substituting all lysine residues compared to an unmodified d913 degron containing, for example, the amino acid sequence of SEQ ID NO: 131. In some embodiments, the modification of a modified d913 degron includes substituting one or more lysine residues with arginine residues compared to an unmodified d913 degron containing, for example, the amino acid sequence of SEQ ID NO: 131. In some embodiments, the modification of a modified d913 degron includes substituting all lysine residues with arginine residues compared to an unmodified d913 degron containing, for example, the amino acid sequence of SEQ ID NO: 131, such as a modified degron containing, for example, the amino acid sequence of SEQ ID NO: 133 or 139.

[0324] In some embodiments, the modified degron includes 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, one or more amino acid sequences confer increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to degron containing the amino acid sequence shown in SEQ ID NO: 40 or 131.

[0325] In some embodiments, the modified deglon has the amino acid sequence X1CGX2TX3X4X5KX6X7LX8RHIX9X 10 This includes, where X1 is I or R; X2 is F or W; X3 is C or T; X4 is R, D, H, or K; X5 is Q, R, or T; X6 is G or V; X7 is N, T, or A; X8 is L or I; X9 is K or G; and X 10 It is either L or F.

[0326] In some embodiments, the modified deglon contains the amino acid sequence of SEQ ID NO: 139. In some embodiments, the modified deglon contains the amino acid sequence 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, 31 The modified degron includes the amino acid sequence shown in any one of 6, 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 includes the amino acid sequence of SEQ ID NO: 140. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 142. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 144. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 146. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 148. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 150. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 152. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 154. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 156. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 158. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 160. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 162. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 164. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 166. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 226. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 227. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 228. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 229. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 230.In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 276. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 280. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 284. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 288. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 292. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 296. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 300. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 304. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 308. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 312. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 316. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 320. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 324. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 328. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 332. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 336. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 340. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 344. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 348. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 352. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 356. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 360. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 364. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 368. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 372. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 376. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 380.In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 384. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 388. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 392. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 396. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 400. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 404. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 408. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 412. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 416. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 420. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 424. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 788.

[0327] In some embodiments, the modified degron includes the amino acid sequence shown in any one of SEQ ID NOs: 177, 179, 181, 183, 185, or 187. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 177. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 179. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 181. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 183. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 185. In some embodiments, the modified degron includes the amino acid sequence of SEQ ID NO: 187.

[0328] In some embodiments, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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: 162. In some embodiments, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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 some embodiments, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon is a sequence. The modified deglon includes 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 sequence number 400. In some embodiments, the modified deglon includes 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 sequence number 404. In some embodiments, the modified deglon includes 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 sequence number 408. In some embodiments, the modified deglon includes 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, the modified deglon includes 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, the modified deglon includes 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, the modified deglon contains 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, the modified deglon contains 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, the modified deglon does not contain an N-terminal methionine (M) residue.

[0329] In some embodiments, modified degron (e.g., CRIMP) has increased sensitivity to ligands (e.g., IMiD, e.g., pomalidomide) compared to degron without modification. In some embodiments, modified degron (e.g., CRIMP) has increased sensitivity to ligands (e.g., IMiD, e.g., pomalidomide) compared to degron containing the amino acid sequence of SEQ ID NO: 40 or 131. In some embodiments, the increased sensitivity results in increased degradation of the protein containing the modified degron. In some embodiments, the increased degradation is measured by the extinction of a detectable signal produced by the protein. In some embodiments, the detectable signal is fluorescence or luminescence. In some embodiments, the detectable signal is fluorescence. In some embodiments, the detectable signal is luminescence. In some embodiments, the increased sensitivity results in increased association with another polypeptide (e.g., CRBN or modified CRBN as described herein). In some embodiments, the increased sensitivity results in increased binding of modified degron (e.g., CRIMP) to ligands (e.g., IMiD, e.g., pomalidomide) compared to degron containing the amino acid sequence of SEQ ID NO: 40 or 131.

[0330] In some embodiments, the degron used in accordance with this disclosure (e.g., modified degron) includes an amino acid sequence or nucleic acid sequence shown in Table C-1, Table C-2, or Table C-4. In some embodiments, the degron used in accordance with this disclosure (e.g., modified degron) includes 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 the amino acid sequence or nucleic acid sequence shown in Table C-1, Table C-2, or Table C-4.

[0331] In some embodiments, modified degron exhibits reduced proteolysis upon binding to a ligand (e.g., IMiD, e.g., pomalidomide). In some embodiments, reduced proteolysis is the reduced degradation of a protein containing modified degron. In some embodiments, reduced degradation is measured by the reduced quenching of a detectable signal produced by the protein compared to a protein without modified degron. In some embodiments, the detectable signal is fluorescence or emission. In some embodiments, the detectable signal is fluorescence. In some embodiments, the detectable signal is emission.

[0332] In some embodiments, modified degron polypeptides exhibiting reduced proteolysis upon binding to a ligand (e.g., IMiD, e.g., pomalidomide) include 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.

[0333] In some embodiments, a modified degron exhibiting reduced proteolysis upon binding to a ligand (e.g., IMiD, e.g., pomalidomide) comprises the amino acid sequence X1CGFTX2X3X4X5X6NLX7X8HIKX9, where X1 is I, H, V, or L; X2 is C, T, or S; X3 is R, T, N, K, Q, D, or F; X4 is Q, C, Y, S, L, T, P, or R; X5 is K, H, E, or L; X6 is G, E, or L; X7 is L, D, or I; X8 is R, V, or F; and X9 is L or A.

[0334] In some embodiments, the modified degron exhibiting reduced proteolysis upon binding to a ligand (e.g., IMiD, e.g., pomalidomide) includes 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 the amino acids shown in any one of SEQ ID NOs.607-728.

[0335] In some embodiments, the modified degron exhibiting reduced proteolysis upon binding to a ligand (e.g., IMiD, e.g., pomalidomide) is encoded by a nucleic acid sequence that includes an amino acid sequence or has at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with the sequences shown in Table C-6.

[0336] In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 608. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 610. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 612. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 614. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 616. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 618. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 620.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 622. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 624. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 626. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 628. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 630. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 632. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 634.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 636. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 638. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 640. In some embodiments, the modified deglon includes 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, the modified deglon includes the amino acid sequence of SEQ ID NO: 642. In some embodiments, the modified deglon includes 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, the modified deglon includes the amino acid sequence of SEQ ID NO: 644. In some embodiments, the modified deglon includes 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, the modified deglon includes the amino acid sequence of SEQ ID NO: 646. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 648.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 650. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 652. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 654. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 656. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 658. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 660. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 662.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 664. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 666. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 668. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 670. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 672. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 674. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 676.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 678. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 680. In some embodiments, the modified degron includes... Ron contains 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, modified degron contains the amino acid sequence of SEQ ID NO: 682. In some embodiments, modified degron contains 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, modified degron contains the amino acid sequence of SEQ ID NO: 684. In some embodiments, modified degron contains 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, modified degron contains the amino acid sequence of SEQ ID NO: 686. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 688. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 690. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 692. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 694.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 696. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 698. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 700. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 702. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 704. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 706. In some embodiments, the modified deglon contains 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, the modified deglon contains the amino acid sequence of SEQ ID NO: 708.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 710. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 712. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 714. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 716. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 718. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 720. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 722.In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 724. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 726. In some embodiments, the modified degron includes 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, the modified degron includes the amino acid sequence of SEQ ID NO: 728. In some embodiments, the modified deglon further comprises an N-terminal methionine (M) residue.

[0337] In some embodiments, a modified degron exhibiting reduced proteolysis upon binding to a ligand (e.g., IMiD, e.g., pomalidomide) further includes one or more amino acid substitutions that confer enhanced binding of the modified degron to IMiD when compared to a degron without one or more amino acid substitutions. In some embodiments, one or more amino acid substitutions that confer enhanced binding of the modified degron to IMiD are selected from the group consisting of: I21R, F24W, C26T, R27D, R27H, R27K, Q28R, Q28T, G30V, G30E, N31T, N31A, L33I, K37G, L38F, compared to wild-type degron containing the amino acid sequence shown in SEQ ID NO: 40.

[0338] In some embodiments, cerebron (CRBN) is a wild-type CRBN polypeptide, e.g., a CRBN comprising the amino acid sequence of SEQ ID NO: 127, as used in accordance with this disclosure. In some embodiments, the CRBN is a modified CRBN. In some embodiments, the modified CRBN comprises mutations that reduce ubiquitination compared to wild-type CRBN. In some embodiments, the DDB1 interaction domain is deleted in the modified CRBN. In some embodiments, amino acids 194-247 are deleted in the modified CRBN, e.g., a modified CRBN comprising the amino acid sequence of SEQ ID NO: 129. In some embodiments, the 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, the modified CRBN includes 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.

[0339] In some embodiments, the modified CRBN is sequence numbers 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, 42 3, 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, The modified CRBN includes the amino acid sequence shown in any one of 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 includes the amino acid sequence of SEQ ID NO: 231. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 232. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 233. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 234. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 235. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 236. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 237. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 238. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 239. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 240. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 241. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 242.In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 243. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 244. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 245. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 246. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 247. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 248. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 249. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 256. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 258. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 260. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 262. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 264. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 275. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 279. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 283. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 287. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 291. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 295. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 299. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 303. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 307. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 311. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 315. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 319. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 323. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 327. In some embodiments, the modified CRBN contains the amino acid sequence of SEQ ID NO: 331.In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 335. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 339. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 343. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 347. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 351. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 355. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 359. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 363. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 367. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 371. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 375. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 379. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 383. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 387. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 391. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 395. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 399. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 403. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 407. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 411. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 415. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 419. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 423. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 426. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 428. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 430. In some embodiments, the modified CRBN contains the amino acid sequence of SEQ ID NO: 43.In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 434. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 436. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 438. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 440. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 442. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 444. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 446. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 448. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 450. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 452. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 454. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 456. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 458. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 460. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 462. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 464. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 466. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 468. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 470. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 472. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 474. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 476. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 478. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 480. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 482. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 484. In some embodiments, the modified CRBN contains the amino acid sequence of SEQ ID NO: 486.In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 488. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 490. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 492. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 494. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 496. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 498. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 500. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 502. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 504. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 506. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 508. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 510. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 512. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 514. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 516. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 518. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 520. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 522. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 524. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 526. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 528. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 530. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 532. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 534. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 536. In some embodiments, the modified CRBN includes the amino acid sequence of SEQ ID NO: 538. In some embodiments, the modified CRBN contains the amino acid sequence of SEQ ID NO: 540.In some embodiments, the modified CRBN contains the amino acid sequence of SEQ ID NO: 542. In some embodiments, the modified CRBN contains the amino acid sequence of SEQ ID NO: 544.

[0340] In some embodiments, the modified CRBNs are sequence numbers 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, 30 3, 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, The modified CRBN contains 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 514, 516, 518, 520, 522, 524, 530, 532, 534, 536, 538, 540, 542, or 544. In some embodiments, the modified CRBN does not contain an N-terminal methionine (M) residue.

[0341] In some embodiments, the CRBN used in accordance with this disclosure (e.g., modified CRBN) comprises an amino acid sequence or nucleic acid sequence shown in Table C-1, Table C-3, Table C-4, or Table C-5. In some embodiments, the CRBN used in accordance with this disclosure (e.g., modified CRBN) 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 the amino acid sequence or nucleic acid sequence shown in Table C-1, Table C-3, Table C-4, or Table C-5.

[0342] Ligand and homologous ligand pairs This disclosure identifies certain ligands that are useful for binding to ligand-binding domains, as discussed herein. In many embodiments of this disclosure, ligands may bind to specific ligand-binding domains (e.g., the ligand domain in the provided degron or CRIMP, or the provided CRBN). A given ligand that consistently binds to a given ligand-binding domain may be referred to as a homologous ligand pair.

[0343] In some embodiments, the ligand used in accordance with this disclosure is an immunomodulatory imide drug (IMiD). In some embodiments, the IMiD is an FDA-approved drug. In some embodiments, the IMiD is thalidomide. In some embodiments, the IMiD is a variant or derivative of thalidomide. In some embodiments, the IMiD is selected from the group consisting of thalidomide, iverdamide, lenalidomide, and pomalidomide. In some embodiments, the IMiD is pomalidomide. In some embodiments, the IMiD is thalidomide. In some embodiments, and the IMiD is iverdamide. In some embodiments, the IMiD is lenalidomide.

[0344] Inducible degradation proteins In some embodiments, the manipulated polypeptides of the Disclosure comprise inducibly degradable polypeptides or proteins. In some embodiments, the inducibly degradable protein comprises at least one ligand-binding domain and at least one target polypeptide targeted for degradation. When expressed in cells, the inducibly degradable protein can be targeted by an E3 ubiquitin ligase via a homologous ligand (e.g., small molecule) that binds to the ligand-binding domain. In some embodiments, the E3 ubiquitin ligase can induce polyubiquitination upon binding to the inducibly degradable protein, thereby resulting in proteasomal degradation of the inducibly degradable protein and subsequent degradation of the target polypeptide.

[0345] In some embodiments, the inducibly degradable protein comprises a degron (e.g., any degron described herein, e.g., any of those in Table C-1, Table C-2, or Table C-4) operably linked to the target polypeptide. In some embodiments, the provided degron or modified degron is directly fused to the target polypeptide, for example, as a fusion protein. In some embodiments, the provided degron or modified degron is operably linked to the target polypeptide using a linker (e.g., any linker described herein).

[0346] Cell death induction domain In some embodiments, the manipulated polypeptides of the Disclosure include inducible cell death polypeptides. An inducible cell death polypeptide may include one or more ligand-binding domains and at least one cell death-inducing domain. When expressed in cells, the provided inducible cell death polypeptides can be used as components of an inducible cell death system, where a homologous ligand that binds to the ligand-binding domain of one inducible cell death polypeptide may be provided, resulting in the ligand-binding domain functionally associating with another inducible cell death polypeptide to form a complex that induces or promotes cell death. For example, in some embodiments, a first polypeptide containing a modified degron (e.g., any modified degron provided herein, e.g., CRIMP) containing a first ligand-binding domain is operatively linked to a first cell death-inducing domain, and a second polypeptide containing a second ligand-binding domain is operatively linked to a second cell death-inducing domain, and the first and second polypeptides form a complex when at least one ligand-binding domain is bound by a suitable homologous ligand (e.g., IMiD), and the formed complex induces or promotes cell death. In some embodiments, the CRBN (e.g., any modified CRBN described herein) includes a second ligand-binding domain. In some embodiments, the first and second cell death induction domains are the same (e.g., the inducible cell death system forms a homodimer complex in the presence of a suitable homologous ligand).

[0347] Exemplary cell death-inducing domains can originate from proteins, such as caspases (e.g., any one of caspases 1-11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, etc.), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-related protein with death domain (FADD), tumor necrosis factor receptor 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, caspase's second mitochondrial-derived activator (SMAC), Omi, Bmf, and Bi. d, Bim, p53 upregulatory factor for apoptosis (PUMA), Noxa, Blk, Hrk, cytochrome c, Arts, TNF-associated apoptosis-inducing ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamorase, liver cytochrome P450-2B1, purine nucleoside phosphorylase, or one or more of their variants or functional fragments. As used herein, “variant” can refer to a biomolecule, e.g., a variant of a polynucleotide or polypeptide, a splice variant, a three-dimensional structure, an isoform, an allele variant, a species variant, and a species homolog. In some embodiments, the variant biomolecule is a native variant. In some embodiments, the variant biomolecule is an engineered variant. In some embodiments, the 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, that contains one or more different nucleotides compared to a reference (or “parent”) sequence. Thus, a variant polynucleotide sequence may contain at least one mutation, substitution, insertion, or deletion compared to their respective reference sequences.A “variant” may have 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% sequence identity (e.g., nucleic acid sequence identity or amino acid sequence identity) compared to a reference sequence (e.g., a naturally occurring sequence, fragment or derivative thereof, or an engineered sequence). In some embodiments, depending on the 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 the reference biomolecule (i.e., shorter and / or smaller) that has the same or similar functional activity as the reference biomolecule. The functional activity of a similar biomolecule is intended to be greater than, approximately equal to, or less than, the functional activity of the reference biomolecule, insofar as the functional fragment achieves at least a portion of the activity of the reference biomolecule. If the reference biomolecule is a polypeptide, the polypeptide fragment is intended to retain 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, if the reference biomolecule is a polypeptide, the polypeptide fragment may be a polypeptide that is shorter and / or smaller than the reference polypeptide, or otherwise modified, but is still intended to retain the functional activity of the reference polypeptide, such as binding to a specific receptor; if the reference biomolecule is a polynucleotide, the polynucleotide fragment is intended to retain a portion of the same activity of the reference polynucleotide. For example, in the case of a protein-coding polynucleotide, the polynucleotide fragment is intended to encode a protein having 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 protein encoded by the reference polynucleotide in a suitable assay.In the case of polynucleotides acting as regulatory elements (e.g., promoters or enhancers), the polynucleotide fragment is intended to have 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 polynucleotide in the appropriate assay. Exemplary cell death-inducing domain sequences can be found in Table C-1.

[0348] In some embodiments, the cell death induction domain is derived from caspase 9. In some embodiments, the cell death induction domain includes the amino acid sequence of SEQ ID NO: 39. In some embodiments, the cell death induction domain includes the amino acid sequence of SEQ ID NO: 123. The cell death induction domain may include or be derived from caspase 9, for example, the amino acid sequence shown in SEQ ID NO: 39 or 123. In some embodiments, a derivative of caspase 9 includes inducible caspase 9 ("iCasp-9"), which is capable of inducing apoptosis due to drug-based dimerization, for example, the amino acid sequence shown in SEQ ID NO: 48 or 125. In some embodiments, the caspase domain or its derivatives or functional fragments, such as inducible Casp-9, do not include the caspase activation and recruitment domain (CARD) domain sequence.

[0349] In some embodiments, the 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, wherein the first and second caspase-9 polypeptide monomers form a complex (e.g., dimerize) in the presence of a suitable homologous ligand (e.g., when the ligand domain is linked by an IMiD).

[0350] The cell death induction domain may include the amino acid sequence shown in BAX, for example, SEQ ID NO: 32.

[0351] Regulatory cell survival polypeptides In some embodiments, the manipulated polypeptides of the Disclosure include regulatory cell survival polypeptides. The regulatory cell survival polypeptides may include at least one ligand-binding domain. When expressed in cells, the provided regulatory cell survival polypeptides can be used as components of a regulatory cell survival system, where a homologous ligand that binds to the ligand-binding domain of one regulatory cell survival polypeptide may be provided, the ligand-binding domain of which functionally associates with another regulatory cell survival polypeptide, thereby forming a complex that promotes cell survival.

[0352] For example, in some embodiments, a first polypeptide comprising a modified degron (e.g., any modified degron provided herein, e.g., CRIMP) containing 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 and second polypeptides form a complex when at least one ligand-binding domain is bound with a suitable homologous ligand (e.g., IMiD), and the formed complex promotes cell survival. In some embodiments, a CRBN (e.g., any modified CRBN described herein) comprises a second ligand-binding domain.

[0353] Exemplary cell survival polypeptides include one or more of XIAP, Bcl-2, Bcl-xL, Bcl-w, Bcl-2-related protein A1 (BCL2A1), Mcl-1, FLICE-like inhibitory protein (c-FLIP), and adenovirus E1B-19K protein. A cell survival polypeptide may include XIAP. For example, a cell survival polypeptide may include wild-type XIAP having amino acid sequence number 107. A cell survival polypeptide may also include modified XIAP. Modified XIAP may include one or more amino acid substitutions with reference to sequence number 107.

[0354] Activated Conditionally Regulated Polypeptide (ACP) In some embodiments, the manipulated polypeptides of the Disclosure comprise ACPs. In some embodiments, the manipulated polypeptides of the 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 operably linked to a transcriptional effector domain (e.g., a transcriptional repressor or transcriptional activator), and a second ACP comprising a second polypeptide comprising a second ligand-binding domain operably linked to a DNA-binding domain, form an ACP complex when at least one ligand-binding domain is bound with a suitable homologous ligand, and the formed ACP complex promotes a biologically relevant output (e.g., an increase or decrease in gene expression, an increase or decrease in biological signaling, etc.).

[0355] In some embodiments, ACP is or includes a transcription modulator. In some embodiments, ACP is or includes a transcription repressor. In some embodiments, ACP is or includes a transcription activator. In some embodiments, ACP is or includes a transcription factor. In some embodiments, ACP includes a DNA-binding domain. In some embodiments, ACP includes a transcription effector domain. In some embodiments, ACP includes both a DNA-binding domain and a transcription 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 includes a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the DNA-binding domain includes a tetracycline (or derivative thereof) repressor (TetR) domain. In some embodiments, ACP may include one or more ligand-binding domains.

[0356] In some embodiments, the ACP used in accordance with this disclosure comprises a polypeptide comprising a ligand-binding domain operably ligated to a transcription effector domain. In some embodiments, the ACP comprises a degron or modified degron (e.g., any degron or modified degron provided herein, e.g., CRIMP) operably ligated to a transcription effector domain (e.g., any transcription effector domain described herein). In some embodiments, the ACP comprises a degron or modified degron comprising a sequence shown in Table C-1, Table C-2, or Table C-4, operably ligated to a transcription effector domain comprising a sequence shown in Table 1, Table 2, Table C-1, or Table C-2. In some embodiments, the ACP comprises a CRBN (e.g., any CRBN or modified CRBN provided herein) operably ligated to a transcription effector domain (e.g., any transcription effector domain described herein). In some embodiments, the ACP includes a CRBN containing the sequence shown in Table C-1, Table C-3, Table C-4, or Table C-5, which is operably linked to a transcription effector domain containing the sequence shown in Table 1, Table 2, Table C-1, or Table C-2.

[0357] nucleic acid binding domain In some embodiments, this disclosure provides engineered polypeptides comprising at least one nucleic acid-binding domain (e.g., a DNA-binding domain). In some embodiments, the engineered polypeptide comprises at least one ligand-binding domain and at least one nucleic acid-binding domain.

[0358] In some embodiments, the manipulated polypeptide comprises at least one transcription factor. In some embodiments, the transcription factor comprises at least one nucleic acid-binding domain. In some embodiments, the transcription factor comprises at least one nucleic acid-binding domain and at least one transcription effector domain.

[0359] In some embodiments, the nucleic acid binding domain includes a DNA-binding zinc finger protein domain (ZF protein domain). In some embodiments, the ZF protein domain is a modular design and consists of zinc finger sequences (ZFAs). In some embodiments, the transcription effector domain includes a herpes simplex virus protein 16 (VP16) activation domain; an activation domain containing four tandem copies of VP16; a VP64 activation domain; a p65 activation domain of NFκB; an Epstein-Barr virus R transactivator (Rta) activation domain; a tri-component activator containing VP64, p65, and Rta activation domains (VPR activation domain); a tri-component activator containing VP64, p65, and HSF1 activation domains (VPH activation domain); and a histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300). The following are selected from the group consisting of: HAT core activating domain; Kruppel association box (KRAB) repression domain; repressor element silencing transcription factor (REST) ​​repression domain; WRPW motif of Hairy-associated basic helix-loop-helix repressor protein, which is known as the WRPW repression domain; DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; HP1 alpha-chromoshadow repression domain, and variants or functional fragments thereof. In some embodiments, the transcription effector domain is a tri-component activator comprising VP64, p65, and Rta activating domains (VPR activating domains), or variants or functional fragments thereof. In some embodiments, the transcription effector domain comprises a p65 domain, or a variant or functional fragment thereof.

[0360] In some embodiments, the ZF protein domain is a modular design and consists of a zinc finger array (ZFA). The zinc finger sequence contains multiple zinc finger protein motifs linked together. Each zinc finger motif binds to a different nucleic acid motif. This results in a ZFA with specificity to any desired nucleic acid sequence. The ZF motifs may be directly adjacent to each other or separated by a mobile linker sequence. In some embodiments, the ZFA is an array, string, or chain of ZF motifs arranged in tandem. The ZFA may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 zinc finger motifs. ZFA can have zinc finger motifs numbered 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.

[0361] In some embodiments, the ZF protein domain contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more ZFAs. In some embodiments, the ZF domain contains 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 contains 1-10 ZFAs. In some embodiments, the ZF protein domain contains at least one ZFA. In some embodiments, the ZF protein domain contains at least two ZFAs. In some embodiments, the ZF protein domain contains at least three ZFAs. In some embodiments, the ZF protein domain contains at least four ZFAs. In some embodiments, the ZF protein domain contains at least five ZFAs. In some embodiments, the ZF protein domain contains at least ten ZFAs.

[0362] An exemplary ZF protein domain is shown in the sequence SRPGERPFQCRICMRNFSRRHGLDRHTRTHTGEKPFQCRICMRNFSDHSSLKRHLRTHTGSQKPFQCRICMRNFSVRHNLTRHLRTHTGEKPFQCRICMRNFSDHSNLSRHLKTHTGSQKPFQCRICMRNFSQRSSLVRHLRTHTGEKPFQCRICMRNFSESGHLKRHLRTHLRGS (SEQ ID NO: 57). In some embodiments, the ZF protein domain includes the amino acid sequence of SEQ ID NO: 57.

[0363] Transcription Effector Domain This disclosure provides, in some embodiments, engineered polypeptides comprising at least one transcription effector domain. In some embodiments, the engineered polypeptide may comprise at least one ligand-binding domain and at least one transcription effector domain.

[0364] In some embodiments, the inducible cell death polypeptide provided herein may include at least one transcription effector domain. In some embodiments, the ACP provided herein may include at least one transcription effector domain. In some embodiments, the inducible cell death polypeptide comprises at least one ligand-binding domain and at least one transcription effector domain. In some embodiments, the ACP comprises at least one ligand-binding domain and at least one transcription effector domain.

[0365] In some embodiments, the transcription effector domain includes one or more of the following: herpes simplex virus protein 16 (VP16) activation domain; activation domain including four tandem copies of VP16, VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; tricomponent activator including VP64, p65, and Rta activation domains (VPR activation domain); tricomponent activator including VP64, p65, and HSF1 activation domains (VPH activation domain); histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 HAT core activating domain; Kruppel association box (KRAB) repression domain; repressor element silencing transcription factor (REST) ​​repression domain; WRPW motif of Hairy-associated basic helix-loop-helix repressor protein, this motif is known as the WRPW repression domain; DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and HP1 alpha chromoshadow repression domain. In some embodiments, the transcription effector domain is a three-component activator comprising VP64, p65, and Rta activating domains (VPR activating domain). In some embodiments, the transcription effector domain comprises the p65 domain.

[0366] In some embodiments, the transcription effector domain includes a transcription repressor domain. In some embodiments, the transcription repressor domain includes a Kruppel-associated box (KRAB) repressor domain; a cleaved Kruppel-associated box (KRAB) repressor domain; a histone deacetylase 4 (HDAC4) repressor domain; a scleracsis (SCX) HLH domain; a DNA binding inhibitor 1 (ID1) HLH domain; a HECT domain and an RCC1-like domain-containing protein 2 (HERC2) Cyt-b5 domain; a twist-associated protein 1 (TWST1) HLH domain; and a homeobox protein Nkx-2.2 (NKX22) homeodomain. The following are selected from the group consisting of a DNA-binding inhibitor 1 (ID3) HLH domain, a Twist-related protein 2 (TWST2) HLH domain, and an EED repressor domain; a repressor element silencing transcription factor (REST) ​​repressor domain; a WRPW motif of the hairy-related basic helix-loop helix repressor protein, which is known as a WRPW repressor domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and an HP1 alpha-chromoshadow repressor domain. In some embodiments, the transcription effector domain includes a transcription activation domain. In some embodiments, the transcriptional activation domain is selected from the group consisting of: herpes simplex virus protein 16 (VP16) activation domain; activation domain containing four tandem copies of VP16; VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; ternary activator containing VP64, p65, and Rta activation domains (VPR activation domain); ternary activator containing VP64, p65, and HSF1 activation domains (VPH activation domain); and histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 HAT core activation domain). In some embodiments, the transcriptional activator domain is a ternary activator containing VP64, p65, and Rta activation domains (VPR activation domain).In some embodiments, the transcriptional activator domain includes a p65 domain. The transcriptional activating domain can also be referred to as the transcriptional activator domain.

[0367] In some embodiments, the manipulated nucleic acid of the Disclosure comprises a polynucleotide encoding an inducible cell death polypeptide. In some embodiments, the manipulated nucleic acid comprises a polynucleotide encoding ACP. In some embodiments, the inducible cell death polypeptide provided herein comprises at least one transcription factor comprising at least one transcription effector domain. In some embodiments, the ACP provided herein comprises at least one transcription factor comprising at least one transcription effector domain. In some embodiments, the inducible cell death polypeptide provided herein comprises at least one transcription factor comprising at least one nucleic acid binding domain and at least one transcription effector domain. In some embodiments, the ACP provided herein comprises at least one transcription factor comprising at least one nucleic acid binding domain and at least one transcription effector domain. Furthermore, the ACP may comprise at least one ligand binding domain, as well as at least one transcription factor comprising at least one nucleic acid binding domain and at least one transcription effector domain.

[0368] The manipulated nucleic acids provided in this disclosure may encode effector domains, such as transcription effector domains. In some embodiments, the transcription effector domains include effector domains of transcription factors (e.g., activator domains or repressor domains). Transcription factor effector domains are also known as transactivation domains and act as scaffolding domains for proteins, such as transcription coregulators that act to activate or repress the transcription of genes. Any suitable transcription effector domain may be used, but is not limited to, the herpes simplex virus protein 16 (VP16) activation domain; an activation domain consisting of four tandem copies of VP16; the VP64 activation domain; the p65 activation domain of NFκB; the Epstein-Barr virus R transactivator (Rta) activation domain; a tri-component activator including the VP64, p65, and Rta activation domains; the tri-component activator is known as the VPR activation domain; and the histone acetyltransferase (HAT) core of human E1A-related protein p300. The domains include the p300HAT core activating domain, known as the p300HAT core activating domain; the Kruppel association box (KRAB) repression domain; the cleavage-type Kruppel association box (KRAB) repression domain; the repressor element silencing transcription factor (REST) ​​repression domain; the WRPW motif of the Hairy-associated basic helix-loop-helix repressor protein, known as the WRPW repression domain; the DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repression domain; and the HP1 alpha-chromoshadow repression domain, or any combination thereof. In some embodiments, the transcription effector domain is a three-component activator comprising the VP64, p65, and Rta activating domains (VPR activating domain). In some embodiments, the transcription effector domain includes the p65 domain.

[0369] In some embodiments, the transcription effector domain used in accordance with this disclosure is a VPR domain. In some embodiments, the transcription effector domain used in accordance with this disclosure is a minVPR domain. In some embodiments, the minVPR domain includes 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 minVPR domain includes the amino acid sequence shown in SEQ ID NO: 197. In some embodiments, the minVPR domain includes 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 minVPR domain includes the amino acid sequence shown in SEQ ID NO: 545.

[0370] Exemplary transcription effector domain protein sequences are shown in Table 1. Exemplary transcription effector domain nucleotide sequences are shown in Table 2. In some embodiments, the transcription effector domains used in accordance with this disclosure include sequences shown in Table 1 or Table 2. In some embodiments, the transcription effector domains used in accordance with this disclosure include sequences having at least 80% (e.g., at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) sequence identity with sequences shown in Table 1 or Table 2. [Table 1] [Table 2]

[0371] promoter In some embodiments, the engineered nucleic acids of the Disclosure include, for example, one or more promoters in one or more expression cassettes. In some embodiments, the engineered nucleic acids of the Disclosure include a first expression cassette including a first promoter operably ligated to an exogenous polynucleotide sequence. In some embodiments, the engineered nucleic acids of the Disclosure include a second expression cassette including a second promoter operably ligated to a second exogenous polynucleotide sequence encoding one or more effector molecules. In some embodiments, the first and second expression cassettes are each encoded by separate engineered nucleic acids of the Disclosure. In some embodiments, the first and second expression cassettes are encoded by the same engineered nucleic acid of the Disclosure.

[0372] In some embodiments, the promoter is an ACP-responsive promoter. In some embodiments, the ACP-responsive promoter of this disclosure comprises an ACP-binding domain and a promoter sequence. In some embodiments, the ACP-responsive promoter is operably ligated to a nucleotide sequence encoding an effector molecule (e.g., a protein of interest, e.g., any engineered polypeptide described herein).

[0373] A “promoter” refers to a regulatory region of a nucleic acid sequence that controls the initiation and rate of the rest of the transcription of that sequence. In some embodiments, a promoter includes a small region to which regulatory proteins or other molecules, such as RNA polymerase, ACP, and / or transcription factors, can be bound. A promoter can be constitutive, inductive, repressive, tissue-specific, or any combination thereof. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inductive promoter. A promoter drives the transcription of a nucleic acid sequence that it drives expression or regulates. As used herein, a promoter is considered “operably linked” if it is in the correct functional location and orientation in relation to the nucleic acid sequence it regulates to control (“drive”) the transcription initiation and / or expression of that sequence.

[0374] In some embodiments, promoters may be naturally associated with a particular gene or sequence, but may be obtained by isolating a 5' non-coding sequence located upstream of the coding segment of that gene or sequence. Such promoters may be referred to as “endogenous.” In some embodiments, coding nucleic acid sequences may be located under the control of recombinant or heterologous promoters, which refer to promoters that do not normally associate with coding sequences in their native environment. Such promoters may include promoters of other genes; promoters isolated from any other cell; and synthetic promoters or enhancers that do not “occur naturally,” such as those involving mutations that alter expression through different elements of different transcriptional regulatory regions and / or genetic engineering methods. In addition to synthetically producing promoter and enhancer nucleic acid sequences, sequences may also be produced using nucleic acid amplification techniques, including recombinant cloning and / or polymerase chain reaction (PCR) (see, e.g., U.S. Patents 4,683,202 and 5,928,906).

[0375] The promoters of the engineered nucleic acids described herein may be “inducible promoters,” which are characterized by modulating (e.g., initiating or activating) transcriptional activity in the presence of a signal, or upon contact with a signal. This signal may be an endogenous or usually exogenous state (e.g., light), a compound (e.g., a chemical or non-chemical compound), or a protein (e.g., an engineered polypeptide, e.g., ACP) that comes into contact with the inducible promoter in a manner that is active in modulating transcriptional activity from the inducible promoter. Activation of transcription may include acting directly on the promoter to drive transcription, or acting indirectly on the promoter by inactivating a repressor (e.g., an engineered polypeptide, e.g., an ACP) that prevents the promoter from driving transcription. Conversely, inactivation of transcription may include acting directly on the promoter to prevent transcription, or acting indirectly on the promoter by activating a repressor that then acts on the promoter.

[0376] In some embodiments, the promoter is “responsive to” or “regulated by” a signal if, in the presence of a local tumor condition (e.g., inflammation or hypoxia), or such condition or signal, transcription from the promoter is activated, inactivated, increased, or decreased. In some embodiments, the promoter includes a “response element,” which is a short sequence of DNA within the promoter region that binds from the promoter to a specific molecule (e.g., a transcription factor, or an engineered polypeptide as described herein, e.g., ACP) that modulates (regulates) gene expression. Response elements that may be used in accordance with this disclosure include, but are not limited to, the phloretin-adjustable regulatory element (PEACE), zinc finger DNA-binding domain (DBD), interferon-gamma activating sequence (GAS) (Decker, T. et al. J Interferon Cytokine Res. 1997 Mar; 17(3): 121-34, incorporated herein by reference), interferon-stimulated response element (ISRE) (Han, K. et al. J Biol Chem. 2004 Apr 9; 279(15): 15652-61, incorporated herein by reference), NF-kappa B response element (Wang, V. et al. Cell Reports. 2012; 2(4): 824-839, incorporated herein by reference), and STAT3 response element (Zhang, D. et al. J of Biol This includes Chem. 1996;271:9503-9509 (incorporated herein by reference). Other response elements are incorporated herein. Response elements may also include tandem repeats (e.g., consecutive repeats of the same nucleotide sequence encoding the response element) to generally increase the sensitivity of the response element to its homozygous molecules. Tandem repeats may be labeled as 2×, 3×, 4×, 5×, etc., to indicate the number of repeats present.

[0377] Table 3 lists non-limiting examples of responsive promoters (also referred to as "inducible promoters") (e.g., TGF-beta responsive promoter). In addition, Table 3 provides exemplary promoter and transcription factor combinations that can be used in accordance with this disclosure, as well as the effects of selective inducing molecules on such promoter and transcription factor combinations. For example, the transcription factor response to an inducer is shown in the TF column (where A represents activation; DA represents inactivation; and DR represents decompression), and the transgene transcription response to an inducer is shown in the T column (where B represents binding; D represents dissociation; and nd represents undetermined) (see Horner, M. & Weber, W. FEBS Letters 586 (2012) 20784-2096m and the references cited therein). Table 4 shows non-limiting examples of components that may be included in inducible promoters (e.g., minimal promoters and responsive elements). [Table 3] TIFF2026516229000005.tif252167 [Table 4]

[0378] Other non-limiting examples of promoters include the cytomegalovirus (CMV) promoter, the elongation factor 1-alpha (EF1a) promoter, the elongation factor (EFS) promoter, the MND promoter (a synthetic promoter containing the U3 region of a modified MoMuLV LTR with a myeloproliferative sarcoma virus enhancer), the phosphoglycerate kinase (PGK) promoter, the splenic fociforming 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] TIFF2026516229000008.tif250163TIFF2026516229000009.tif249163TIFF2026516229000010.tif250163TIFF2026516229000011.tif106163

[0379] In some embodiments, the promoter sequence is derived from a promoter selected from: minP, NFkB response element, CREB response element, NFAT response element, SRF response element 1, SRF response element 2, AP1 response element, TCF-LEF response element promoter fusion, hypoxia-responsive element, SMAD binding element, STAT3 binding site, minCMV, YB_TATA, minTK, inducer molecule-responsive promoter, and their tandem repeats.

[0380] In some embodiments, the first promoter is a constitutive promoter, an inducible promoter, or a synthetic promoter. In some embodiments, the constitutive promoter is selected from: CMV, EFS, SFFV, SV40, MND, PGK, UbC, hEF1aV1, hCAGG, hEF1aV2, hACTb, heIF4A1, hGAPDH, hGRP78, hGRP94, hHSP70, hKINb, and hUBIb.

[0381] In some embodiments, the ACP-responsive promoter is a synthetic promoter. In some embodiments, the ACP-responsive promoter includes a minimal promoter. In some embodiments, the ACP-binding domain includes one or more zinc finger binding sites. In some embodiments, the ACP-binding domain includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more zinc finger binding sites. In some embodiments, the ACP-binding domain includes one zinc finger binding site. In some embodiments, the ACP-binding domain includes two zinc finger binding sites. In some embodiments, the ACP-binding domain includes three zinc finger binding sites. In some embodiments, the ACP-binding domain includes four zinc finger binding sites. In some embodiments, the ACP-binding domain includes the polynucleotide sequence cgggtttcgtaacaatcgcatgaggattcgcaacgccttcGGCGTAGCCGATGTCGCGctcccgtctcagtaaaggtcGGCGTAGCCGATGTCGCGcaatcggactgccttcgtacGGCGTAGCCGATGTCGCGcgtatcagtcgcctcggaacGGCGTAGCCGATGTCGCGcattcgtaagaggctcactctcccttacacggagtggataACTAGTTCTAGAGGGTATATAATGGGGGCCA (SEQ ID NO: 100).

[0382] In some embodiments, the ACP-responsive promoter includes an enhancer that promotes transcription when the antigen-recognizing receptor binds to a congener antigen, e.g., an antigen expressed on a target cell. The enhancer may include, but is not limited to, an enhancer enhanced in the ATAC sequence of activated T cells (Gate et al. Nat Genet. Author manuscript; available at PMC 2019 Jan 9; incorporated herein by reference for all purposes) or an enhancer associated with upregulated cells in single-cell RNA sequence data (Xhangolli et al. Genomics Proteomics Bioinformatics. 2019 Apr;17(2):129-139. Doi:10.1016 / j.gpb.2019.03.002; incorporated herein by reference for all purposes). In some embodiments, the enhancer is or includes a synthetic enhancer. The synthetic enhancer may include multiple repeats of a transcription factor binding site. In some embodiments, the synthetic enhancer comprises one or more (e.g., one, two, three, four, five, or more) repeats of one or more different transcription factor binding sites. In some embodiments, the synthetic enhancer comprises four repeats of two different transcription factor binding sites in an aaaabbbb or abababab configuration. Exemplary, non-exclusive examples of genes from which enhancers may 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, FOXA1, FOXA2, GATA3, HIF1A, IKZF1, IKZF2, IRF4, JUN, JUNB, JUND, Lef1, NFAT, NFIA, NFIB, NFKB, NR2F1, Nur77, PU.1, RELA, RUNX3, SCRT1, SCRT2, SP1, STAT4, STAT5A, T-Bet, Tcf7, ZBED1, ZNF143, or ZNF217.

[0383] Multi-cistronic and multiple promoter systems In some embodiments, the manipulated nucleic acid is configured to produce multiple polypeptides. For example, the nucleic acid may be configured to produce 2 to 20 different polypeptides.In some embodiments, the manipulated nucleic acids are 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 It is configured to produce polypeptides of ~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.In some embodiments, the engineered nucleic acid is configured to produce polypeptides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the engineered nucleic acid is configured to produce two polypeptides. In some embodiments, the engineered nucleic acid is configured to produce three polypeptides. In some embodiments, the engineered nucleic acid is configured to produce four polypeptides. In some embodiments, the engineered nucleic acid is configured to produce five polypeptides.

[0384] In some embodiments, the engineered nucleic acid provided can be multicistronic, i.e., it can produce more than one separate polypeptide (e.g., multiple exogenous polynucleotides or effector molecules, or engineered polypeptides, as described herein) from a single transcript. In some embodiments, the engineered nucleic acid can be multicistronic through the use of various linkers, for example, a polynucleotide sequence encoding a first exogenous polynucleotide or effector molecule can be linked to a nucleotide sequence encoding a second exogenous polynucleotide or effector molecule, for example, in the first gene:linker:second gene 5'-3' direction. In some embodiments, the linker polynucleotide sequence includes a polynucleotide sequence encoding a 2A ribosome skipping element, e.g., T2A. In some embodiments, the 2A ribosome skipping element includes an E2A, P2A, or F2A ribosome skipping element. In some embodiments, the 2A ribosome skipping element includes a P2A ribosome skipping element. In some embodiments, the P2A ribosome skipping element includes the amino acid sequence shown in SEQ ID NO: 195. In some embodiments, the P2A ribosome skipping element includes the amino acid sequence shown in SEQ ID NO: 271. In some embodiments, the 2A ribosome skipping element includes the T2A ribosome skipping element. In some embodiments, the T2A ribosome skipping element includes the amino acid sequence shown in SEQ ID NO: 267. In some embodiments, the 2A ribosome skipping element includes the E2A G4S T2A(Opt2A) ribosome skipping element. In some embodiments, the E2A G4S T2A(Opt2A) ribosome skipping element includes the amino acid sequence shown in SEQ ID NO: 265. In some embodiments, the 2A ribosome skipping element includes the P2A 3-T2A 2(Opt2A 2.0) ribosome skipping element.In some embodiments, the P2A 3-T2A 2 (Opt2A 2.0) ribosome skipping element comprises the amino acid sequence shown in SEQ ID NO: 269. The 2A ribosome skipping element enables the production of separate polypeptides encoded by the first and second genes, which are produced during translation. In some embodiments, the linker is or comprises a cleavable linker polypeptide sequence, such as a furin cleavage site or a TEV cleavage site, where, following expression, the cleavable linker polypeptide is cleaved to produce separate polypeptides encoded by the first and second genes. In some embodiments, the cleavable linker sequence comprises a mobile linker sequence that further facilitates cleavage. (In some embodiments, the mobile linker comprises a Gly-Ser-Gly sequence).

[0385] In some embodiments, the linker polynucleotide sequence includes a polynucleotide sequence encoding an internal ribosome entry site (IRES) so that separate polypeptides encoded by the first and second genes are produced during translation. In some embodiments, the linker polynucleotide sequence includes a polynucleotide sequence encoding a splice acceptor, such as a viral splice acceptor.

[0386] In some embodiments, the linker includes a 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, allowing for the complete removal of the 2A residue. In some embodiments, the linker combination may include a furin sequence, a mobile linker, and a 2A linker. Thus, in some embodiments, the linker is a furin-Gly-Ser-Gly-2A fusion polypeptide. In some embodiments, the linker is a furin-Gly-Ser-Gly-T2A fusion polypeptide.

[0387] Generally, a multicistron system used in accordance with this disclosure may include any number or combination of linkers to express any number of genes or any portion thereof (e.g., an engineered nucleic acid may encode the first, second, and third effector molecules or engineered polypeptides described herein, each of which is separated by a linker to produce separate polypeptides encoded by the first, second, and third effector molecules).

[0388] "Linker" may refer to a polypeptide that links a first polypeptide sequence and a second polypeptide sequence, as used herein, or to a multi-cistronic linker as described herein.

[0389] Post-transfer regulatory elements In some embodiments, the manipulated nucleic acids of the Disclosure include post-transcriptional regulatory elements (PREs). In some embodiments, PREs can enhance gene expression by enabling tertiary RNA structure stability and 3' end formation. Non-limiting examples of PREs include hepatitis B virus PREs (HPREs) and woodchuck hepatitis virus PREs (WPREs). In some embodiments, the post-transcriptional regulatory element is a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). In some embodiments, the WPRE includes the alpha, beta, and gamma components of the WPRE element. In some embodiments, the WPRE includes the alpha component of the WPRE element.

[0390] Payload molecule In some embodiments, the engineered nucleic acid of the present invention (e.g., engineered nucleic acid encoding ACP) comprises one or more nucleic acid sequences encoding a payload molecule. In some embodiments, ACP controls the expression of one or more payload molecules.

[0391] In some embodiments, the payload molecule is an effector molecule. Any suitable effector molecule known in the art can be encoded by an engineered nucleic acid or expressed by an engineered cell. Suitable effector molecules can be grouped into therapeutic classes based on structural similarity, sequence similarity, or function. The therapeutic classes of effector molecules include, but are not limited to, cytokines, chemokines, homing molecules, growth factors, co-activating molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0392] In some embodiments, each effector molecule is independently selected from a therapeutic class, where the therapeutic class is selected from: cytokines, chemokines, homing molecules, growth factors, co-activating molecules, tumor microenvironment modifiers, receptors, ligands, antibodies, polynucleotides, peptides, and enzymes.

[0393] In some embodiments, the effector molecule is a chemokine. Chemokines are small cytokines or signaling proteins secreted by cells that can induce directional chemotaxis in cells. Chemokines can be classified into four main subfamilies: CXC, CC, CX3C, and XC, all of which exert their biological effects by selectively binding to chemokine receptors located on the surface of target cells. Non-limiting examples of chemokines that may be encoded by engineered nucleic acids in this disclosure include: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1, any combination thereof. In some embodiments, the chemokine is selected from: CCL21a, CXCL10, CXCL11, CXCL13, CXCL10-CXCL11 fusion protein, CCL19, CXCL9, and XCL1.

[0394] In some embodiments, the effector molecule is a cytokine. Non-limiting examples of cytokines that may be encoded by the manipulated nucleic acids of this disclosure include: IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha, or any combination thereof. In some embodiments, the cytokine is selected from: IL1-beta, IL2, IL4, IL6, IL7, IL10, IL12, IL12p70 fusion protein, IL15, IL17A, IL18, IL21, IL22, type I interferon, interferon-gamma, and TNF-alpha.

[0395] In some embodiments, the payload molecule is a homing molecule. "Homing" refers to the active navigation (migration) of a cell to a target site (e.g., a cell, a tissue (e.g., a tumor), or an organ). "Homing molecule" refers to a molecule that directs a cell toward a target site. In some embodiments, the homing molecule functions to recognize and / or initiate an interaction of the manipulated cell toward the target site. Non-limiting examples of homing molecules include CXCR1, CCR9, CXCR2, CXCR3, CXCR4, CCR2, CCR4, FPR2, VEGFR, IL6R, CXCR1, CSCR7, PDGFR, anti-integrin alpha-4, beta-7; anti-MAdCAM; CCR9; CXCR4; SDFl; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15, or any combination thereof. In some embodiments, the homing molecule is selected from: anti-integrin alpha 4, beta 7; anti-MAdCAM; CCR9; CXCR4; SDFl; MMP-2; CXCR1; CXCR7; CCR2; CCR4; and GPR15.

[0396] In some embodiments, the payload molecule is a growth factor. Suitable growth factors for use as effector molecules include, but are not limited to, FLT3L and GM-CSF, or any combination thereof. In some embodiments, the growth factors are selected from: FLT3L and GM-CSF.

[0397] In some embodiments, the payload molecule is a co-activating molecule. Suitable co-activating molecules for use as effector molecules include, but are not limited to, c-Jun, 4-1BBL, and CD40L, or any combination thereof. In some embodiments, the co-activating molecule is selected from: c-Jun, 4-1BBL, and CD40L.

[0398] The "tumor microenvironment" is the cellular environment in which a tumor resides, including the surrounding blood vessels, immune cells, fibroblasts, myeloid-derived inflammatory cells, lymphocytes, signaling molecules, and the extracellular matrix (ECM) (see, e.g., Pattabiraman, DR & Weinberg, RANature Reviews Drug Discovery 13, 497-512 (2014); Balkwill, F 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 effector molecules include, but are not limited to, adenosine deaminase, TGF-beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2, or any combination thereof. In some embodiments, the tumor microenvironment modifiers are selected from: adenosine deaminase, TGF-beta inhibitors, immune checkpoint inhibitors, VEGF inhibitors, and HPGE2.

[0399] In some embodiments, the payload molecule is a TGF beta inhibitor. Suitable TGF beta inhibitors for use as effector molecules include, but are not limited to, anti-TGF beta peptides, anti-TGF beta antibodies, TGFb-TRAPs, or combinations thereof. In some embodiments, the TGF beta inhibitor is selected from: anti-TGF beta peptides, anti-TGF beta antibodies, TGFb-TRAPs, and combinations thereof.

[0400] In some embodiments, the payload molecule is an immune checkpoint inhibitor. Suitable immune checkpoint inhibitors for use as effector molecules include, but are not limited to, anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFa antibody, anti-TREM1 antibody, and anti-TREM2 antibody, or any combination thereof. In some embodiments, the immune checkpoint inhibitor is selected from: anti-PD-1 antibody, anti-PD-L1 antibody, anti-PD-L2 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, anti-TIM-3 antibody, anti-TIGIT antibody, anti-VISTA antibody, anti-KIR antibody, anti-B7-H3 antibody, anti-B7-H4 antibody, anti-HVEM antibody, anti-BTLA antibody, anti-GAL9 antibody, anti-A2AR antibody, anti-phosphatidylserine antibody, anti-CD27 antibody, anti-TNFa antibody, anti-TREM1 antibody, and anti-TREM2 antibody.

[0401] Exemplary immune checkpoint inhibitors include pembrolizumab (anti-PD-1; MK-3475 / Keytruda®-Merck), nivolumab (anti-PD-1; Opdivo®-BMS), pidilizumab (anti-PD-1 antibody; CT-011-Teva / CureTech), AMP224 (anti-PD-1; NCI), avelumab (anti-PD-L1; Bavencio®-Pfizer), and durvalumab (anti-PD-L1; MEDI4736 / Imf Includes inzi(registered trademark)-Medimmune / AstraZeneca), atezolizumab (anti-PD-L1; Tecentriq(registered trademark)-Roche / Genentech), BMS-936559 (anti-PD-L1-BMS), tremelimumab (anti-CTLA-4; Medimmune / AstraZeneca), ipilimumab (anti-CTLA-4; Yervoy(registered trademark)-BMS), lirirumab (anti-KIR; BMS), and monalizumab (anti-NKG2A; Innate Pharma / AstraZeneca).

[0402] In some embodiments, the payload molecule is a VEGF inhibitor. Suitable VEGF inhibitors for use as effector molecules include, but are not limited to, anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof. In some embodiments, the VEGF inhibitor includes anti-VEGF antibodies, anti-VEGF peptides, or combinations thereof.

[0403] In some embodiments, each payload molecule is a human-derived payload molecule.

[0404] Secretion markers Generally, one or more payload molecules contain a secretion signal peptide (also referred to as a signal peptide or signal sequence) at the N-terminus of the payload molecule, thereby directing a newly synthesized protein destined for secretion or membrane insertion into an appropriate protein processing pathway. In embodiments involving two or more payload molecules, each payload molecule may contain a secretion signal (S). In embodiments involving two or more payload molecules, each payload molecule may contain a secretion signal, causing each payload molecule to be secreted from the manipulated cell. In embodiments, (LE) X A second expression cassette comprising one or more units further comprises a polynucleotide sequence encoding a secretory signal peptide (S). In embodiments, for each X, the corresponding secretory signal peptide is operably associated with the payload molecule. In embodiments, an ACP-responsive promoter and formula:(LSE) X A second expression cassette containing a second exogenous polynucleotide sequence having [a specific characteristic].

[0405] The secretory signal peptide operably associated with the payload molecule can be a native secretory signal peptide (e.g., a secretory signal peptide that is generally endogenously associated with a given payload molecule). The secretory signal peptide operably associated with the payload molecule can also be a non-native secretory signal peptide. Non-native secretory signal peptides can promote improved expression and function, such as maintained secretion, in specific environments, such as the tumor microenvironment. Non-specific examples of non-native secretory signal peptides are shown in Table 14. [Table 14] TIFF2026516229000013.tif230166

[0406] Manipulated cells Also provided herein are engineered cells comprising one or more engineered nucleic acids, and methods for producing engineered cells. Thus, the engineered cells provided typically comprise one or more engineered nucleic acids, as described herein, and do not occur naturally. In some embodiments, the engineered cells are isolated (e.g., engineered cells may be substantially isolated from other cell types, including unengineered cells and / or other different engineered cells). In some embodiments, engineered cells are isolated for a specific use (e.g., any use described herein). In some embodiments, the engineered cells comprise one or more vectors comprising one or more engineered nucleic acids, as described herein. In some embodiments, the engineered cells comprise one or more loci within their genome comprising one or more loci comprising one or more engineered nucleic acids, as described herein. In some embodiments, the engineered cells comprise at least one promoter operably ligated to an exogenous polynucleotide sequence (e.g., a polynucleotide sequence encoding any engineered polypeptide described herein).

[0407] In some embodiments, the manipulated cells of this disclosure include manipulated nucleic acids that are incorporated into the cell's genome. In some embodiments, the manipulated cells include manipulated nucleic acids that can be expressed without being incorporated into the cell's genome. In some embodiments, the manipulated cells include a vector containing the manipulated nucleic acids. In some embodiments, the manipulated cells are brought into contact with the manipulated nucleic acids (e.g., in the form of mRNA) so that the manipulated nucleic acids are expressed in the manipulated cells.

[0408] Manipulated cell types In some embodiments, the manipulated cells or isolated, manipulated cells of this disclosure are human cells. In some embodiments, the manipulated cells or isolated, manipulated cells are human primary cells. In some embodiments, the primary cells are somatic cells. In some embodiments, the primary cells are stem cells. In some embodiments, the primary cells are induced pluripotent stem cells (iPSCs). In some embodiments, the manipulated cells are derived from a subject. In some embodiments, the manipulated cells are homogeneous with respect to a subject.

[0409] In some embodiments, the manipulated cells of this disclosure can be prepared using cells isolated from a subject. In some embodiments, the subject is known to have or suspected to have cancer. In some embodiments, the manipulated cells are prepared using cancer cells isolated from the subject. Cell isolation methods include, but are not limited to, positive isolation techniques such as sorting techniques based on cell surface marker expression, e.g., FACS sorting, and negative isolation, magnetic isolation, and combinations thereof. The manipulated cells may be allogeneic with respect to the subject being treated. Allogeneic modified cells can be HLA-matched to the subject being treated. The manipulated cells may be cultured cells, e.g., ex vivo cultured cells. The manipulated cells may be ex vivo cultured cells, e.g., primary cells isolated from a subject. Cultured cells can be cultured with one or more cytokines.

[0410] In some embodiments of the Disclosure, the manipulated or isolated cells are selected from: T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma delta T cells), cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, natural killer (NK) cells, B cells, tumor-infiltrating lymphocytes (TILs), innate lymphoid cells, mast cells, eosinophils, basophils, neutrophils, myeloid cells, macrophages (e.g., M1 macrophages or M2 macrophages), monocytes, dendritic cells, erythrocytes, platelet cells, neurons, oligodendrocytes, astrocytes, placode-derived cells, Schwann cells, cardiomyocytes, endothelial cells, nodular cells, microglia, hepatocytes, cholangiocarcinomas, beta cells, human embryonic stem cells (ESCs), ESC-derived cells, pluripotent stem cells, mesenchymal stromal cells (MSCs), induced pluripotent stem cells (iPSCs), and iPSC-derived cells.

[0411] In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are T cells (e.g., CD8+ T cells, CD4+ T cells, or gamma delta T cells). In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are cytotoxic T lymphocytes (CTLs). In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are regulatory T cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are natural killer T cells (NTKs). In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are natural killer cells (NKs). In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are B cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are tumor-infiltrating lymphocytes (TILs). In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are innate lymphoid cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are mast cells. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are eosinophils. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are basophils. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are neutrophils. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are myeloid cells. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are macrophages (e.g., M1 macrophages or M2 macrophages). In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are monocytes. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are dendritic cells. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are erythrocytes. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are platelet cells. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are neurons. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are oligodendrocytes.In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are astrocytes. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are placode-derived cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are Schwann cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are cardiomyocytes. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are endothelial cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are nodular cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are microglia cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are hepatocytes. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are cholangiocarcinomas. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are beta cells. In some embodiments, the manipulated or isolated and manipulated cells of the Disclosure are human embryonic stem cells (ESCs). In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are ESC-derived cells. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are pluripotent stem cells. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are mesenchymal stromal cells (MSCs). In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are induced pluripotent stem cells (iPSCs). In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are iPSC-derived cells. In some embodiments, the manipulated cells are autologous. In some embodiments, the manipulated cells are allogeneic. In some embodiments, the manipulated or isolated, manipulated cells of the Disclosure are CD34+ cells, CD3+ cells, CD8+ cells, CD16+ cells, and / or CD4+ T cells.

[0412] In some embodiments, the manipulated cells of this disclosure are a cell therapy modality. In some embodiments, the cell therapy modality may express one or more therapeutic proteins. In some embodiments, the therapeutic proteins are chimeric antigen receptors (CARs) or modified T cell receptors (TCRs). In some embodiments, the cell therapy modality is CAR-T cells or CAR-NK cells. In some embodiments, the cell therapy modality is CAR-T cells. In some embodiments, the cell therapy modality is CAR-NK cells.

[0413] In some embodiments, the manipulated cells of the Disclosure are tumor cells. In some embodiments, the manipulated cells of the Disclosure are selected from adenocarcinoma cells, bladder tumor cells, brain tumor cells, breast tumor cells, cervical tumor cells, colorectal tumor cells, esophageal tumor cells, glioma cells, kidney tumor cells, liver tumor cells, lung tumor cells, melanoma cells, mesothelioma cells, ovarian tumor cells, pancreatic tumor cells, prostate tumor cells, skin tumor cells, thyroid tumor cells, and uterine tumor cells.

[0414] In some embodiments, the manipulated cells of the Disclosure are bacterial cells. In some embodiments, the manipulated cells of the Disclosure are selected from: Clostridium beijerinckii, Clostridium sporogenes, Clostridium novyi, Escherichia coli, Pseudomonas aeruginosa, Listeria monocytogenes, Salmonella typhimurium, and Salmonella choleraesuis.

[0415] Also provided herein are methods for culturing the manipulated cells of the Disclosure. The manipulated cells provided may be cultured using any suitable method. Those skilled in the art will recognize that the culture conditions depend on the manipulated cells for a particular purpose. Those skilled in the art will recognize that the culture conditions depend on the specific culture conditions for the specific downstream use of the manipulated cells, for example, the subsequent administration of the manipulated cells to a subject.

[0416] Methods for manipulating cells Also provided herein are compositions and methods for manipulating cells using any nucleic acids described herein.

[0417] The provided, manipulated cells may be manipulated through the introduction (i.e., delivery) of one or more polynucleotides (e.g., manipulated nucleic acids) of the Disclosure. Delivery methods include, but are not limited to, virus-mediated delivery, lipid-mediated transfection, nanoparticle delivery, electroporation, sonication, and cell membrane deformation by physical means. Those skilled in the art will understand that the choice of delivery method may depend on the specific cell type being manipulated.

[0418] In some embodiments, the manipulated cells are transduced using an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indianabecyclovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replication retrovirus, oncolytic rhabdovirus, oncolytic seneca-valley virus, oncolytic sindobisvirus, and any variants or derivatives thereof. In some embodiments, the oncolytic virus is a recombinant oncolytic virus. In some embodiments, the recombinant oncolytic virus comprises an engineered nucleic acid, as provided herein. In some embodiments, the oncolytic virus is a recombinant oncolytic virus comprising a first expression cassette and a second expression cassette. In some embodiments, the oncolytic virus further comprises a third expression cassette.

[0419] In some embodiments, the virus provided pursuant to this disclosure is an oncolytic virus. In some embodiments, the virus is a recombinant virus encoding one or more transgenes encoding one or more payload molecules, such as any of the engineered nucleic acids described herein. In some embodiments, the engineered cells are engineered via transduction using an oncolytic virus.

[0420] Virus-borne delivery The manipulated cells described herein can be constructed using any suitable viral vector-based delivery platform. Generally, viral vector-based delivery platforms manipulate cells by introducing (i.e., delivering) a nucleic acid payload (e.g., any manipulated nucleic acid described herein) into the host cell. Viral vector-based delivery platforms may include manipulated or recombinant viruses for delivering the manipulated nucleic acids described herein to cells.

[0421] A viral vector-based delivery platform can deliver one or more nucleic acid payloads, e.g., engineered nucleic acids, genes, or transgenes as described herein, within the same nucleic acid. In some embodiments, a viral vector-based delivery platform (e.g., a platform using recombinant or engineered viruses) can deliver one or more transgenes, including, but not limited to, any engineered nucleic acid as described herein that encodes one or more payload molecules. In some embodiments, one or more transgenes encoding one or more payload molecules can be configured to express one or more payload molecules. In addition to one or more transgenes (e.g., transgenes encoding one or more payload molecules), a viral vector-based delivery platform can encode one or more genes, e.g., viral genes required for viral infectivity and / or viral production, referred to as cis-acting elements or genes (e.g., capsid proteins, envelope proteins, viral polymerases, viral transcriptases, etc.).

[0422] A viral vector-based delivery platform may include one or more viral vectors, such as separate viral vectors encoding engineered nucleic acids, genes, or transgenes, as described herein and referred to as transactive elements or genes. For example, a helper-dependent viral vector-based delivery platform may provide additional genes required for viral infectivity and / or viral production on one or more additional separate vectors, in addition to a vector encoding one or more effector molecules. One viral vector may deliver one or more engineered nucleic acids, such as one vector delivering engineered nucleic acids configured to produce two or more effector molecules. One or more viral vectors may deliver one or more engineered nucleic acids, such as one or more vectors delivering one or more engineered nucleic acids configured to produce one or more effector molecules. The number of viral vectors used may depend on the packaging capacity of the viral vector-based vaccine platform mentioned above, but those skilled in the art can select a suitable number of viral vectors.

[0423] Generally, any viral vector-based system can be used for the in vitro production of molecules, such as effector molecules, or in in vivo and ex vivo gene therapy procedures for the in vitro delivery of engineered nucleic acids encoding one or more effector molecules. The selection of a suitable viral vector-based system will depend on various factors, such as cargo / payload size, immunogenicity of the viral system, target cells of interest, intensity and timing of gene expression, and other factors understood by those skilled in the art.

[0424] Viral vector-based delivery platforms may be RNA-based viruses or DNA-based viruses. Exemplary viral vector-based delivery platforms include, but are not limited to, herpes simplex virus, adenovirus, measles virus, influenza virus, Indiana beshiclovirus, Newcastle disease virus, vaccinia virus, poliovirus, myxoma virus, reovirus, mumps virus, maraba virus, rabies virus, rotavirus, hepatitis virus, rubella virus, dengue virus, chikungunya virus, respiratory syncytial virus, lymphocytic choriomeningitis virus, morbillivirus, lentivirus, replication retrovirus, rhabdovirus, seneca-valley virus, sindobis virus, and any variants or derivatives thereof.Other exemplary viral vector-based delivery platforms have been described in the Art, including, but are not limited to, vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (see, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616-629), or lentiviruses, including second, third, or hybrid second / third generation lentiviruses and recombinant lentiviruses of any generation designed to target specific cell types or receptors (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).

[0425] This sequence may be preceded by one or more sequences that target intracellular compartments. Upon introduction (i.e., delivery) into host cells, infected cells (i.e., engineered cells) may express and, in some cases, secrete one or more effector molecules. Useful vaccinia vectors and methods in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacille Calmette Guerin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the introduction (i.e., delivery) of engineered nucleic acids, such as salmonellar vectors and similar, will be apparent to those skilled in the art from the description herein.

[0426] The viral vector-based delivery platform can be a virus that targets tumor cells, and is referred to herein as an oncolytic virus. Examples of oncolytic viruses include, but are not limited to, oncolytic herpes simplex virus, oncolytic adenovirus, oncolytic measles virus, oncolytic influenza virus, oncolytic Indianabecyclovirus, oncolytic Newcastle disease virus, oncolytic vaccinia virus, oncolytic poliovirus, oncolytic myxoma virus, oncolytic reovirus, oncolytic mumps virus, oncolytic maraba virus, oncolytic rabies virus, oncolytic rotavirus, oncolytic hepatitis virus, oncolytic rubella virus, oncolytic dengue virus, oncolytic chikungunya virus, oncolytic respiratory syncytial virus, oncolytic lymphocytic choriomeningitis virus, oncolytic morbillivirus, oncolytic lentivirus, oncolytic replication retrovirus, oncolytic rhabdovirus, oncolytic seneca-valley virus, oncolytic sindobisvirus, and any variants or derivatives thereof. Any of the oncolytic viruses described herein may be recombinant oncolytic viruses comprising another transgene (e.g., engineered nucleic acid) encoding one or more effector molecules. The transgene encoding one or more effector molecules may be configured to express one or more effector molecules.

[0427] In some embodiments, the virus is selected from lentiviruses, retroviruses, oncolytic viruses, adenoviruses, adeno-associated viruses (AAVs), and virus-like particles (VLPs).

[0428] Viral vector-based delivery platforms can be retroviral-based. Generally, retroviral vectors consist of cis-acting long terminal repeats with packaging ability for exogenous sequences up to 6–10 kb. A minimum cis-acting LTR is sufficient for vector replication and packaging, which is then used to incorporate one or more manipulated nucleic acids (e.g., a transgene encoding one or more effector molecules) into target cells to provide persistent transgene expression. Retrovirus-based delivery systems include, but are not limited to, delivery systems based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (e.g., Buchscher et al., J.Virol.66:2731-2739(1992); Johann et al., J.Virol.66:1635-1640(1992); Sommnerfelt et al., Virol.176:58-59(1990); Wilson et al., J.Virol.63:2374-2378(1989); Miller et al, J.Virol.65:2220-2224(1991); see PCT / US94 / 05700). Other retrovirus systems include the Phoenix retrovirus system.

[0429] Viral vector-based delivery platforms can be lentiviral-based. Generally, lentiviral vectors are retroviral vectors that can transduce or infect non-dividing cells and typically produce high viral titers. Lentiviral-based delivery platforms can be HIV-based, such as the ViraPower system (ThermoFisher) or the pLenti system (Cell Biolabs). Lentiviral-based delivery platforms can also be SIV or FIV-based. Other exemplary lentivirus-based delivery platforms are described in more detail in U.S. Patents Nos. 7,311,907; 7,262,049; 7,250,299; 7,226,780; 7,220,578; 7,211,247; 7,160,721; 7,078,031; 7,070,993; 7,056,699; and 6,955,919, each incorporated herein by reference for all purposes.

[0430] Viral vector-based delivery platforms can be adenovirus-based. Generally, adenovirus-based vectors offer very high transduction efficiency in many cell types, do not require cell division, achieve high titers and expression levels, and can be produced in large quantities in relatively simple systems. Adenoviruses can generally be used for transient expression of transgenes within infected cells because they are typically not integrated into the host genome. Adenovirus-based delivery platforms are described in more detail in Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO94 / 12649, WO93 / 03769; WO93 / 19191; WO94 / 28938; WO95 / 11984; and WO95 / 00655, each incorporated herein by reference for all purposes. Other exemplary adenovirus-based delivery platforms are described in more detail in U.S. Patents Nos. 5,585,362; 6,083,716, 7,371,570; 7,348,178; 7,323,177; 7,319,033; 7,318,919; and 7,306,793, as well as in International Patent Application No. 96 / 13597, each incorporated herein by reference for all purposes.

[0431] The viral vector-based delivery platform may be adeno-associated virus (AAV) based. The adeno-associated virus ("AAV") vector may be used to transduce cells using engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). The AAV system can be used for the in vitro production of effector molecules, or for the in vivo delivery of engineered nucleic acids encoding one or more effector molecules in in vivo and ex vivo gene therapy procedures (e.g., West et al., Virology 160:38-47 (1987); U.S. Patent Nos. 4,797,368; 5,436,146; 6,632,670; 6,642,051; 7,078,387; 7,314,912; 6,498,244; 7,906,111; U.S. Patent Publications US2003-0138772, US2007 / 0036760, and US2009 / 0197338; Gao, et al.). al., J. Virol, 78(12):6381-6388 (June 2004); Gao, et al, Proc Natl Acad Sci USA, 100(10):6081-6086 (May 13, 2003); and international patent applications WO2010 / 138263 and WO93 / 24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994), each incorporated herein by reference for all purposes. Exemplary methods for constructing recombinant AAV vectors are described in more detail in U.S. Patent 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 of which is incorporated herein by reference for all purposes.Generally, AAV-based vectors contain a capsid protein having an amino acid sequence corresponding to one of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.Rh10, AAV11, or any of their variants.

[0432] Viral vector-based delivery platforms can be virus-like particle (VLP) platforms. Generally, VLPs are constructed by producing viral structural proteins and purifying the resulting viral particles. After purification, the cargo / payload (e.g., any of the engineered nucleic acids described herein) is then ex vivo encapsulated within the purified particles. Thus, VLP production maintains the separation of the nucleic acids encoding the viral structural proteins and those 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 translational expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo using any suitable method to produce VLPs. VLP production is described in more detail in Seow et al. (Mol Ther. 2009 May;17(5):767-777).

[0433] Viral vector-based delivery platforms can be manipulated to target (i.e., infect) a range of cells, a narrow subset of cells, or specific cells. Generally, the tropism of the virus will be determined by the envelope protein chosen for the viral vector-based delivery platform. The virus used in a viral vector-based delivery platform can be pseudotyped to target specific cells of interest. A viral vector-based delivery platform can be pan-affinity and capable of infecting a range of cells. For example, a pan-affinity viral vector-based delivery platform may contain the VSV-G envelope. A viral vector-based delivery platform can be ambidextrous and capable of infecting mammalian cells. Therefore, those skilled in the art can select the appropriate tropism, pseudotype, and / or envelope protein to target the desired cell type.

[0434] Lipid structure delivery system The engineered nucleic acids described herein (e.g., any of the engineered nucleic acids described herein) can be introduced into cells using lipid-mediated delivery systems. Generally, lipid-mediated delivery systems use structures consisting of an outer lipid membrane enclosing an internal compartment. Examples of lipid-based structures include, but are not limited to, lipid-based nanoparticles, liposomes, micelles, exosomes, vesicles, extracellular vesicles, cells, or tissues. Lipid structure delivery systems can deliver cargo / payloads (e.g., any of the engineered nucleic acids described herein) in vitro, in vivo, or ex vivo.

[0435] Lipid-based nanoparticles may include, but are not limited to, monolayer liposomes, multilayer liposomes, and lipid preparations. As used herein, “liposome” is a general term encompassing in vitro preparations of lipid media formed by encapsulating a desired cargo, e.g., engineered nucleic acids, e.g., any of the engineered nucleic acids described herein, within a lipid shell or lipid aggregate. Liposomes may generally be characterized as having a vesicular structure with a phospholipid-containing bilayer membrane and an internal medium generally containing an aqueous composition. Liposomes may include, but are not limited to, emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, layered layers, and the like. Liposomes may be monolayer liposomes. Liposomes may be multilayer liposomes. Liposomes may be multi-vesicular liposomes. Liposomes may be positively charged, negatively charged, or neutrally charged. In certain embodiments, liposomes are neutrally charged. Liposomes can generally be formed from standard vesicle-forming lipids, including neutral and negatively charged phospholipids, as well as sterols, such as cholesterol. Lipid selection is generally guided by consideration of the desired purpose, e.g., criteria for in vivo delivery, such as liposome size, acid instability, and liposome stability in the bloodstream. A variety of methods are available for preparing liposomes, as described, for example, in Szokan et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Patents No. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369, each incorporated herein by reference for all purposes.

[0436] Multilayer liposomes are spontaneously formed when phospholipid-containing lipids are suspended in an excess aqueous solution, such that multiple lipid layers are separated by an aqueous medium. The water and dissolved solute are encapsulated in a closed structure between the lipid bilayers after the self-reconstitution of the lipid components. The desired cargo (e.g., polypeptides, nucleic acids, small molecule drugs, engineered nucleic acids, such as any of the engineered nucleic acids described herein, viral vectors, virus-based delivery systems, etc.) can be encapsulated within the aqueous interior of the liposome, attached to the liposome via linking molecules that associate with both the liposome and the polypeptide / nucleic acid, dispersed within the lipid bilayer of the liposome, encapsulated within the liposome, complexed with the liposome, or otherwise associated with the liposome, so that it can be delivered to the target entity. Lipophilic molecules or molecules with lipophilic regions can also dissolve in or associate with the lipid bilayer.

[0437] Liposomes used according to this embodiment can be prepared using any suitable method. The preparation of liposomes is described in more detail in WO2016 / 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 incorporated herein by reference for all purposes.

[0438] Liposomes may be cationic liposomes. Examples of cationic liposomes are described in more detail in U.S. Patents 5,962,016; 5,030,453; 6,680,068, U.S. Patent Application 2004 / 0208921, and International Patent Applications WO03 / 015757A1, WO04029213A2, and WO02 / 100435A1, each incorporated herein by reference in whole.

[0439] Lipid-mediated gene delivery methods are described, for example, in WO96 / 18372, WO93 / 24640, Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988); U.S. Patent No. 5,279,833 Rose; WO91 / 06309; and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987), each incorporated herein by reference for all purposes.

[0440] Exosomes are small membrane vesicles of endocytosis origin that are released into the extracellular environment after fusion of the polyendoplasmic reticulum with the plasma membrane. Exosomes vary in size between 30 and 100 nm in diameter. Their surfaces consist of a lipid bilayer from the donor cell membrane, they contain cytosol from the exosome-producing cell, and exhibit membrane proteins from the parent cell on their surface. Any exosome useful for nucleic acid delivery can be used in accordance with this disclosure, for example, the exosome described in more detail in U.S. Patent No. 9,889,210, which is incorporated herein by reference for all purposes.

[0441] As used herein, the term “extracellular vesicle” or “EV” refers to a cell-derived vesicle containing a membrane that encloses an internal space. Generally, extracellular vesicles include all membrane-bound vesicles having a diameter smaller than the cell from which they originate. Generally, extracellular vesicles range in diameter from 20 nm to 1000 nm and may contain a variety of macromolecular cargoes, either within the internal space presented on the outer surface of the extracellular vesicle and / or across the membrane. The cargoes may include nucleic acids (e.g., any of the manipulated nucleic acids described herein), proteins, carbohydrates, lipids, small molecules, and / or combinations thereof. As an example, but not limited to, extracellular vesicles include apoptotic bodies, cell fragments, vesicles derived from cells by direct or indirect manipulation (e.g., by serial extrusion or treatment with alkaline solutions), vesicled organelles, and vesicles produced by living cells (e.g., by direct plasma membrane budding or fusion of late endosomes with the plasma membrane). Extracellular vesicles may originate from living or dead organisms, explanted tissues or organs, and / or cultured cells.

[0442] As used herein, the term “exosome” refers to a small, cell-derived vesicle (20–300 nm in diameter, more preferably between 40–200 nm in diameter) that contains a membrane surrounding its internal space and is produced from a cell by direct plasma membrane budding or by fusion of late endosomes with the plasma membrane. Exosomes contain lipids or fatty acids and polypeptides and, optionally, a payload (e.g., a therapeutic agent), a receiver (e.g., a targeted portion), polynucleotides (e.g., nucleic acids, RNA, or DNA, such as any of the engineered nucleic acids described herein), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Exosomes originate from the producing cell and can be isolated from the producing cell based on their size, density, biochemical parameters, or combinations thereof. Exosomes are a type of extracellular vesicle. Generally, exosome production / biosynthesis does not result in the destruction of the producing cell. The exosomes and their preparation are described in further detail in WO2016 / 201323, which is incorporated herein by reference in its entirety.

[0443] As used herein, the term “nanopes” (also referred to as “microvesicles”) refers to small, cell-derived vesicles (20–250 nm in diameter, more preferably between 30–150 nm in diameter) that contain a membrane surrounding their internal space and are produced by a cell through direct or indirect manipulation, thereby preventing the nanovesicles from being produced by the producing cell without such manipulation. Generally, nanovesicles are a subspecies of extracellular vesicles. Appropriate manipulation of the producing cell includes, but is not limited to, serial extrusion, treatment with alkaline solutions, sonication, or a combination thereof. Nanovesicle production may, in some cases, result in the destruction of the producing cell. Preferably, the nanovesicle population is substantially free of vesicles originating from the producing cell by direct budding from the plasma membrane or fusion of late endosomes with the plasma membrane. Nanovesicles contain lipids or fatty acids and polypeptides, and optionally include a payload (e.g., a therapeutic agent), a receiver (e.g., a targeting portion), polynucleotides (e.g., nucleic acids, RNA, or DNA, such as any of the engineered nucleic acids described herein), sugars (e.g., monosaccharides, polysaccharides, or glycans), or other molecules. Once the nanovesicles are induced from the producing cells according to the said operation, they can be isolated from the producing cells based on their size, density, biochemical parameters, or a combination thereof.

[0444] Lipid nanoparticles (LNPs) are generally synthetic lipid structures that form membrane and vesicle-like structures relying on the amphiphilic nature of lipids (Riley 2017). Typically, these vesicles deliver cargo / payloads, such as any of the engineered nucleic acids or viral systems described herein, by being absorbed into the membrane of a target cell and releasing the cargo into the cytosol. The lipids used for LNP formation may be cationic, anionic, or neutral. Lipids may be synthetic or naturally occurring and, in some cases, biodegradable. Lipids may include fats, cholesterol, phospholipids, lipid conjugates (including, but not limited to, polyethylene glycol (PEG) conjugates (PEGylated lipids)), waxes, oils, glycerides, and fat-soluble vitamins. Lipid compositions generally include defined mixtures of materials, such as cationic lipids, neutral lipids, anionic lipids, and amphiphilic lipids. In some cases, specific lipids are included to provide functional chemical groups that prevent LNP aggregation, prevent lipid oxidation, or promote the attachment of additional parts. Lipid compositions can affect the overall LNP size and stability. For example, lipid compositions may include dilinoleylmethyl-4-dimethylaminobutyrate (MC3) or MC3-like molecules. MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as PEG or PEG-conjugated lipids, sterols, or neutral lipids. Furthermore, LNPs can be further manipulated or functionalized to enhance targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity.

[0445] A micelle is generally a spherical synthetic lipid structure formed using single-chain lipids, where the hydrophilic head of the single-chain lipid forms the outer layer or membrane, and the hydrophobic tail of the single-chain lipid forms the micelle center. Typically, a micelle refers to a lipid structure containing only a lipid monolayer. Micelles are described in detail by Quader et al. (Mol Ther. 2017 Jul 5;25(7):1501-1513).

[0446] Nucleic acid vectors directly exposed to serum, such as expression vectors, may have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by free nucleic acids. Similarly, viral delivery systems directly exposed to serum may induce undesirable immune responses and / or neutralization of the viral delivery system. Therefore, encapsulation of engineered nucleic acids and / or viral delivery systems can be used to avoid degradation while also avoiding potential off-target effects. In certain examples, engineered nucleic acids and / or viral delivery systems are completely encapsulated within a delivery medium, such as within the aqueous interior of an LNP. Encapsulation of engineered nucleic acids and / or viral delivery systems within an LNP can be carried out by any suitable method, such as droplet generation performed on a microfluidic mixing and microfluidic droplet generation device. Such devices include, but are not limited to, standard T-junction devices or flow focusing devices. In one example, a desired lipid formulation, such as MC3 or an MC3-like composition, is supplied to a droplet-generating device in parallel with an engineered nucleic acid or viral delivery system and any other desired drug, such that the delivery vector and the desired drug are completely encapsulated within the MC3 or MC3-like LNP. In one example, the droplet-generating device can control the size range and size distribution of the produced LNPs. For example, the LNPs may have sizes ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers. After droplet generation, the delivery medium (e.g., engineered nucleic acid and / or viral delivery system) encapsulating the cargo / payload can be further processed or manipulated to prepare them for administration.

[0447] Nanoparticle delivery Nanomaterials can be used to deliver engineered nucleic acids (e.g., any of the engineered nucleic acids described herein). Importantly, the nanomaterial medium can be made from non-immunogenic materials and generally avoid inducing immunity to its delivery vector itself. These materials may include, but are not limited to, lipids (as previously described), inorganic nanomaterials, and other polymer 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), which is incorporated herein by reference for all purposes.

[0448] Genome editing systems Genome editing systems can be used to manipulate the host genome to encode a modified nucleic acid, such as the modified nucleic acid described herein. Generally, “genome editing system” refers to any system for incorporating an exogenous gene into the genome of a host cell. Genome editing systems include, but are not limited to, transposon systems, nuclease genome editing systems, and viral vector-based delivery platforms.

[0449] Transposon systems can be used to integrate engineered nucleic acids, such as those described herein, into the host genome. Transposons typically consist of a cargo / payload nucleic acid and a terminal inversion repeat (TIR) ​​adjacent to the transposase. Transposon systems can provide transposons in cis or trans orientation, accompanied by a TIR-adjacent cargo. Transposon systems can be retrotransposon systems or DNA transposon systems. Generally, transposon systems randomly integrate the cargo / payload (e.g., engineered nucleic acid) into the host genome. Examples of transposon systems include systems using transposons from the Tc1 / mariner transposon superfamily, such as the Sleeping Beauty transposon system, which are described in more detail in Hudecek et al. (Crit Rev Biochem Mol Biol. 2017 Aug;52(4):355-380), and U.S. Patents 6,489,458, 6,613,752, and 7,985,739. Another example of a transposon system is the PiggyBac transposon system, which is described in more detail in U.S. Patents 6,218,185 and 6,962,810, each of which is incorporated herein by reference for all purposes.

[0450] Nuclease-mediated gene editing systems can be used to manipulate the host genome to encode an engineered nucleic acid, for example, the engineered nucleic acid described herein. While we do not wish to be constrained by theory, nuclease-mediated gene editing systems used to introduce exogenous genes generally utilize the cell's innate DNA repair mechanisms, particularly the homologous recombination (HR) repair pathway. Simply put, following damage to genomic DNA (typically a double-strand break), the cell can resolve the damage by using another DNA source having identical or substantially identical sequences at both its 5' and 3' ends as a template during DNA synthesis to repair the damage. In natural contexts, HDR can use other chromosomes present in the cell as templates. In gene editing systems, an exogenous polynucleotide is introduced into the cell and used as a homologous recombination template (HRT or HR template). Generally, any additional exogenous sequences not naturally found in the chromosome, involving damage between the 5' and 3' complementary ends of an HRT (e.g., a gene or a portion of a gene), can be incorporated (i.e., “integrated”) into a given genomic locus in a templated HDR. Thus, a typical HR template for a given genomic locus has a nucleotide sequence identical to the first region of the endogenous genomic target locus, a nucleotide sequence identical to the second region of the endogenous genomic target locus, and a nucleotide sequence encoding a cargo / payload nucleic acid (e.g., any of the engineered nucleic acids described herein, such as any of the engineered nucleic acids encoding one or more effector molecules).

[0451] In some cases, HR templates can be linear. Examples of linear HR templates include, but are not limited to, linear plasmid vectors, ssDNA, synthetic DNA, and PCR-amplified DNA. In certain cases, HR templates can be circular, such as plasmids. Circular templates can include supercoiled templates.

[0452] Identical or substantially identical sequences found at the 5' and 3' ends of the HR template are generally referred to as arms (HR arms) with respect to the introduced exogenous sequence. HR arms can be identical (i.e., 100% identical) to the region of the endogenous genomic target locus. In some cases, HR arms may be substantially identical to the region of the endogenous genomic target locus. While substantially identical HR arms can be used, it may be advantageous for HR arms to be identical, as the efficiency of the HDR pathway may be affected by HR arms with less than 100% identity.

[0453] Each HR arm, i.e., the 5' and 3' HR arms, can be the same size or different sizes. Each HR arm may be longer than or equal to 50, 100, 200, 300, 400, or 500 bases. While HR arms can generally be of any length, practical considerations, such as the effect of HR arm length and overall template size on overall editing efficiency, may also be taken into account. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus directly adjacent to the cleavage site. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus directly adjacent to the cleavage site. Each HR arm may be identical or substantially identical to a region of an endogenous genomic target locus located within a specific distance from the cleavage site, for example, one 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.

[0454] Nuclease genome editing systems can cleave target genomic loci using a variety of nucleases, including, but not limited to, clustered, regularly spaced, short-interval palindromic repeat (CRISPR) family nucleases or derivatives, transcriptional activator-like effector nucleases (TALENs) or derivatives, zinc finger nucleases (ZFNs) or derivatives, and homing endonucleases (HEs) or derivatives.

[0455] CRISPR-mediated gene editing systems can be used to manipulate the host genome to encode manipulated nucleic acids, such as manipulated nucleic acids 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), all of which are incorporated herein by reference. Generally, CRISPR-mediated gene editing systems include a CRISPR-associated (Cas) nuclease and RNA that directs cleavage to a specific target sequence. An exemplary CRISPR-mediated gene editing system is the CRISPR / Cas9 system, which consists of a Cas9 nuclease and RNA having a CRISPR RNA (crRNA) domain and a transactivating CRISPR (tracrRNA) domain. crRNA typically has two RNA domains: a guide RNA (gRNA) that directs specificity to a target sequence ("defined nucleotide sequence"), e.g., a genomic sequence, through base-pair hybridization; and an RNA domain that hybridizes to tracrRNA. TracrRNA can interact with nucleases (e.g., Cas9) to facilitate its recruitment to genomic loci. The crRNA and tracrRNA polynucleotides can be separate polynucleotides. The crRNA and tracrRNA polynucleotides can also be a single polynucleotide and are referred to as a single guide RNA (sgRNA). While the Cas9 system is exemplified herein, other CRISPR systems can be used, e.g., the Cpf1 system. Nucleases may include their derivatives, e.g., Cas9 functional mutants, such as Cas9 "nickase" mutants, which generally mediate single-strand breaks of a defined nucleotide sequence, in contrast to the complete double-strand breaks typically produced by the Cas9 enzyme.

[0456] Generally, components of a CRISPR system interact with each other to form ribonucleoprotein (RNP) complexes, which mediate sequence-specific cleavage. In some CRISPR systems, each component can be produced separately and used to form the RNP complex. In some CRISPR systems, each component can be produced separately in vitro and brought into contact with each other (i.e., "complexed") in vitro to form the RNP complex. The RNP produced in vitro can then be introduced (i.e., "delivered") into the cytosol and / or nucleus of a cell, for example, the cytosol and / or nucleus of a T cell. The RNP complex produced in vitro can be delivered to cells by a variety of means, not limited to electroporation, lipid-mediated transfection, cell membrane deformation by physical means, lipid nanoparticles (LNPs), virus-like particles (VLPs), and sonication. In certain cases, the RNP complex produced in vitro can be delivered to cells using the Nucleofactor / Nucleofection® electroporation-based delivery system (Lonza®). Other electroporation systems include, but are not limited to, the MaxCyte electroporation system, the Miltenyi CliniMACS electroporation system, the Neon electroporation system, and the BTX electroporation system. CRISPR nucleases, such as Cas9, can be produced (i.e., synthesized and purified) in vitro using any suitable method. CRISPR RNAs, such as sgRNA, can be produced (i.e., synthesized and purified) in vitro using various RNA production techniques, such as in vitro transcription or chemosynthesis.

[0457] RNP complexes produced in vitro can be complexed with nucleases and gRNAs in different ratios. RNP complexes produced in vitro can also be used in different amounts in CRISPR-mediated editing systems. For example, the total amount of RNP added can be adjusted depending on the number of cells to be edited, such as reducing the amount of RNP complex added when editing a large number of cells in the reaction.

[0458] In some CRISPR systems, each component (e.g., Cas9 and sgRNA) can be encoded separately by polynucleotides, and each polynucleotide can be introduced into the cell together or separately. In some CRISPR systems, each component can be encoded by a single polynucleotide (i.e., a multi-promoter or multi-cistronic vector; see the description of the exemplary multi-cistronic system below) and introduced into the cell. Following the expression of the CRISPR components encoded by each polynucleotide in the cell (e.g., translation of nucleases and transcription of CRISPR RNA), an RNP complex can be formed in the cell and then directed for site-specific cleavage.

[0459] Some RNPs can be engineered to have a portion that facilitates the delivery of RNPs into the nucleus. For example, Cas9 nuclease may have a nuclear localization signaling (NLS) domain, and if the Cas9 RNP complex is delivered into the cell cytosol, or after Cas9 translation and subsequent RNP formation, the NLS can facilitate further transport of Cas9 RNPs into the nucleus.

[0460] The manipulated cells described herein can be manipulated using nonviral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using nonviral methods. The manipulated cells described herein can be manipulated using viral methods, for example, the nucleases and / or CRISPR-mediated gene editing systems described herein can be delivered to cells using viral methods, such as adenoviruses, retroviruses, lentiviruses, or any other virus-based delivery method described herein.

[0461] Some CRISPR systems can provide two or more CRISPR compositions, each independently targeting the same gene or common genomic locus at multiple target nucleotide sequences. For example, two separate CRISPR compositions can be provided, directed to cleave at two different target nucleotide sequences within a specific distance of each other. Some CRISPR systems can provide two or more CRISPR compositions, each independently targeting the reverse strand of the same gene or common genomic locus. For example, two separate CRISPR "nickase" compositions can be provided, directed to cleave at the same gene or common genomic locus in the reverse strand.

[0462] In general, the characteristics of the CRISPR-mediated editing systems described herein can be applied to other nuclease-based genome editing systems. TALENs are engineered site-specific nucleases comprising a DNA-binding domain of a TALE (transcriptional activator-like effector) and a catalytic domain of the restriction endonuclease Fokl. Different artificial TALENs can be created by altering the amino acids present in the highly variable residue region of the monomer of the DNA-binding domain, thereby targeting various nucleotide sequences. The DNA-binding domain then directs the nuclease to the target sequence, creating a double-strand break. TALEN-based systems are described in more detail in U.S. Patent Nos. 12 / 965,590; 8,450,471; 8,440,431; 8,440,432; 10,172,880; and 13 / 738,381, all of which are incorporated herein by reference in their entirety. The ZFN-based editorial 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 Publications 2005 / 0064474; 2007 / 0218528; and 2005 / 0267061, all of which are incorporated herein by reference in their entirety for all purposes.

[0463] Other operational delivery systems Various additional means are available to those skilled in the art for introducing engineered nucleic acids (e.g., any of the engineered nucleic acids described herein) into cells or other target recipient entities, such as any of the lipid structures described herein. Those skilled in the art will understand which methods are appropriate for introducing engineered nucleic acids into cells or other biological systems.

[0464] In some embodiments, electroporation is used to deliver polynucleotides (e.g., any engineered nucleic acids described herein) to a recipient entity. Electroporation is a method of internalizing cargo / payload into the internal compartment of a target cell or entity by applying an electric field to transiently permeate the outer membrane or shell of the target cell or entity. Generally, the method involves placing the cell or target entity between two electrodes in a solution containing the cargo of interest (e.g., any of the engineered nucleic acids described herein). The lipid membrane of the cell is then broken, i.e., made permeable, by applying a transient set voltage, thereby allowing the cargo to enter the interior of the entity, such as the cytoplasm of the cell. In the case of cells, at least some, but not the majority, of the cell remain viable. Cells and other entities can be electroporated in vitro, in vivo, or ex vivo. Electroporation conditions (e.g., cell number, cargo concentration, recovery conditions, voltage, time, capacitance, pulse type, pulse length, volume, cuvette length, electroporation solution composition, etc.) vary depending on several factors, i...

Claims

1. The amino acid sequence shown in SEQ ID NO: 139, or amino acid sequence X １ CGX ２ TX ３ X ４ X ５ KX ６ X ７ LX ８ RHIX ９ X １０ A modified degron comprising １ where X ２ is I or R; X ３ is F or W; X ４ is C or T; X ５ is Q, R, or T; X ６ is G or V; X ７ is N, T, or A; X ８ is L or I; X ９ is K or G; and X １０ is L or F, and the modified degron has an increased sensitivity to one or more IMiDs compared to a degron comprising the amino acid sequence shown in SEQ ID NO: 40 or 131, optionally, the modified degron has one or more amino acid substitutions at one or more positions selected from G30E, Q28T, L33I, R27H, K37G, L38F, I21R and G30V, R27D, Q28R, N31T, F24W, R27K, I21R, G30V, and N31A compared to the wild-type degron comprising the amino acid sequence shown in SEQ ID NO: 40, Modified degron, in which the IMiD is at least one of thalidomide, iverdide, lenalidomide, and pomalidomide, and in which the IMiD is pomalidomide.

2. The aforementioned modified degrons are sequence numbers 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, 3 The modified degron according to claim 1, comprising an amino acid sequence represented in any one of 48, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, 400, 404, 408, 412, 416, 420, 424, or 788, wherein the modified degron optionally comprises SEQ ID NO:

148.

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

4. The modified degron according to any one of claims 1 to 3, wherein the cerebron domain or a variant comprises the amino acid sequence shown in SEQ ID NO: 127 or 129, and optionally the cerebron domain or a variant is a modified cerebron that functionally associates with the modified degron when exposed to a lower concentration of IMiD compared to unmodified cerebron, and optionally the cerebron domain or a variant is the modified cerebron according to any one of claims 5 to 8.

5. Modified cereblon (CRBN), a. Compared to a wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, the modified CRBN comprises one or more amino acid substitutions at positions Q325, V350, H353, Y355, N369, I371, R373, E377, S379, Q390, A395, S396, H397, or F402, wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to the wild-type CRBN; b. Amino acid sequence X １ X ２ CQETEITTKNEIFSLSLCGPMAYX ３ X ４ PX ５ GX ６ VX ７ EX ８ LTVYKACNLX ９ LX １０ X １１ X １２ PX １３ TX １４ X １５ X １６ X １７ X １８ PGYAWTX １９ AX ２０ CKICX ２１ X ２２ X ２３ IGWKX ２４ TATKKDMSPQKFWX ２５ Includes X １ is K, N, Q, or R; X ２ is Q, H, K, or R; X ３ is V, E, H, Q, R, or S; X ４ is N, A, I, L, or V; X ５ is H, P, Q, R, or S; X ６ is Y, H, N, R, or S; X ７ is H, Q, or W; X ８ is T, F, I, L, V, W, or Y; X ９ is N, D, or Y; X １０ is I, D, E, N, or Q; X １１ is G, N, or Q; X １２ is R, E, N, S, or Y; X １３ is X, D, K, N, Q, or R; X １４ is E, D, K, Q, S, or Y; X １５ is H, D, Q, or T; X １６ is S, N, or T; X １７ is W, F, or Y; X １８ is F or W; X １９ is V, D, E, N, Q, S, or L; X ２０ is Q, D, H, K, or R; X ２１ is A, D, E, N, or Q; X ２２ is S, G, or Q; X ２３ is H, D, F, K, Q, Y, I, L, or V; X ２４ is F, H, N, Q, W, or Y; and X ２５ is G or A, and the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to wild-type CRBN having the amino acid sequence shown in SEQ ID NO: 127, or its cleaved form having the amino acid sequence shown in SEQ ID NO: 129; c. Amino acid sequence X １ X ２ CQETEITTKNEIFSLSLCGPMAAYX ３ X ４ PX ５ GX ６ VX ７ EX ８ LTVYKACNLX ９ LX １０ X １１ X １２ PX １３ TX １４ X １５ X １６ X １７ X １８ PGYAWTX １９ AX ２０ CKICX ２１ X ２２ X ２３ IGWKX ２４ TATKKDMSPQKFVX ２５ comprising, X １ is K, N, Q, or R; X ２ is Q, H, K, or R; X ３ is V, E, H, Q, R, or S; X ４ is N, A, I, L, or V; X ５ is H, P, Q, R, or S; X ６ is Y, H, N, R, or S; X ７ is H, Q, or W; X ８ is T, F, I, L, V, W, or Y; X ９ is N, D, or Y; X １０ is I, D, E, N; Q, L, V, or M; X １１ is G, N, or Q; X １２ is R, E, N, S, or Y; X １３ is S, D, K, N, Q, R, H, P; X １４ is E, D, K, Q, S, or Y; X １５ is H, D, Q, or T; X １６ is S, N, or T; X １７ is W, F, or Y; X[[ID=​​is V, D, E, N, Q, S, or L; X ２０ is Q, D, H, K, or R; X ２１ is A, D, E, N, or Q; X ２２ is S, G, or Q; X ２３ is H, D, F, K, Q, Y, I, L, or V; X ２４ is F, H, N, Q, W, or Y; and X ２５ is G, A, F, I, L, S, or V; d. Compared to a wild-type CRBN containing the amino acid sequence shown in Sequence ID No. 127, the modified CRBN comprises one or more amino acid substitutions at any one or more positions selected from K324, Q325, V350, N351, H353, Y355, H357, T359, N369, I371, G372, R373, S375, E377, H378, S379, W380, F381, V388, Q390, A395, S396, H397, F402, and G416, wherein the modified CRBN has increased sensitivity to one or more immunomodulatory drugs (IMiDs) compared to the wild-type CRBN; e. Amino acid sequence X １ X ２ CQETEITTKNEIFSLSLCGPMAYX ３ X ４ PX ５ GX ６ VX ７ EX ８ LTVYKACNLX ９ LX １０ X １１ X １２ PX １３ TX １４ X １５ X １６ X １７ X １８ PGYAWTX １９ AX ２０ CKICX ２１ X ２２ X ２３ IGWKX ２４ TATKKDMSPQKFWX ２５ Includes X １ is K, N, Q, or R; X ２ is Q, H, K, or R; X ３ is V, E, H, Q, R, or S; X ４ is N, A, I, L, or V; X ５ is H, P, Q, R, or S; X ６ is Y, H, N, R, or S; X ７ is H, Q, or W; X ８ is V; X ９ is N, D, or Y; X １０ is I, D, E, N, or Q; X １１ is G, N, or Q; X １２ is R, E, N, S, or Y; X １３ is X, D, K, N, Q, or R; X １４ is E, D, K, Q, S, or Y; X １５ is H, D, Q, or T; X １６ is S, N, or T; X １７ is W, F, or Y; X １８ is F or W; X １９ is V, D, E, N, Q, S, or L; X ２０ is Q, D, H, K, or R; X ２１ is A, D, E, N, or Q; X ２２ is S, G, or Q; X ２３ is H, D, F, K, Q, Y, I, L, or V; X ２４ is F, H, N, Q, W, or Y; and X ２５ is G or A; or f. A modified CRBN comprising a T359 amino acid substitution, wherein the T359 amino acid substitution is selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, wherein the modified CRBN comprises a T359V substitution.

6. The modified CRBN according to claim 5, wherein the modified CRBN does not contain a DDB1 interaction domain, optionally the modified CRBN further includes a deletion of amino acids 194 to 247 compared to the wild-type CRBN, and optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NOs. 231 to 249, 256, 258, 260, 262, or 264.

7. The modified CRBN, compared to wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, a) Q325 amino acid substitutions selected from Q325H substitution, Q325K substitution, and Q325R substitution; and one or more amino acid substitutions selected from I371N substitution, H397F substitution, and N369Y substitution; or b) comprising Q325H substitutions and I371N substitutions, and optionally comprising an amino acid sequence that is 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) comprising Q325H substitution, I371N substitution, and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325H substitution and N369Y substitution, and optionally comprising an amino acid sequence that is 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) comprising Q325H substitution, N369Y substitution, and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325H substitution, N369Y substitution, and I371N substitution, and optionally containing an amino acid sequence that is 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) comprising Q325H substitution, N369Y substitution, I371N substitution, and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325K substitution and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325K substitution, I371N substitution, and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325R substitutions and H397F substitutions, and optionally containing an amino acid sequence that is 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) comprising I371N substitution and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising N369Y substitution and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising N369Y substitution and I371N substitution, and optionally containing an amino acid sequence that is 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) comprising N369Y substitution, I371N substitution, and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325H substitutions and H397F substitutions, and optionally containing an amino acid sequence that is 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) comprising Q325K substitution and I371N substitution, and optionally comprising an amino acid sequence that is 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) comprising Q325K substitution and N369Y substitution, and optionally comprising an amino acid sequence that is 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) comprising Q325K substitution, N369Y substitution, and H397F substitution, and optionally containing an amino acid sequence that is 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) comprising Q325K substitution, N369Y substitution, and I371N substitution, and optionally containing an amino acid sequence that is 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) comprising an amino acid sequence which includes a Q325K substitution, an N369Y substitution, an I371N substitution, and an H397F substitution, and which may be 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) comprising an amino acid sequence which includes a Q325R substitution, an N369Y substitution, an I371N substitution, and an H397F substitution, and which may be 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) comprising one or more amino acid substitutions selected from T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, wherein the Q325 amino acid substitution is selected from Q325H substitution, Q325K substitution, and Q325R substitution, and / or, wherein the T359 amino acid substitution is selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution; w) comprising an amino acid sequence which includes a Q325H substitution, a Q325H substitution, an I371N substitution, and a T359V substitution, and which may be 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) comprising an amino acid sequence which includes Q325R substitution, I371N substitution, H397F substitution, and T359V substitution, and which may be 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) comprising an amino acid sequence which includes a Q325R substitution, an N369Y substitution, an H397F substitution, and a T359V substitution, and which may be 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) comprising an amino acid sequence which includes a Q325R substitution, an I371N substitution, and a T359V substitution, and which may be 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) comprising an amino acid sequence which includes a Q325K substitution, an I371N substitution, an H397F substitution, and a T359V substitution, and which may be 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) comprising an amino acid sequence which includes Q325H substitution, N369Y substitution, I371N substitution, H397F substitution, and T359V substitution, and which may be 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) comprising an amino acid sequence which includes Q325H substitution, N369Y substitution, H397F substitution, and T359V substitution, and which may be 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) comprising an amino acid sequence which includes a Q325H substitution, an I371N substitution, an H397F substitution, and a T359V substitution, and which may be 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) comprising an amino acid sequence which includes a Q325K substitution, an I371N substitution, and a T359V substitution, and which may be 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) Q325 amino acid substitution selected from Q325H substitution, Q325K substitution, and Q325R substitution; and (ii) one or more amino acid substitutions selected from T359V substitution, I371N substitution, H397F substitution, and N369Y substitution, according to claim 5 or 6.

8. The aforementioned modified CRBNs are sequence numbers 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, Ami shown with one of the following numbers: 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. A modified CRBN according to any one of claims 5 to 7, wherein the modified CRBN includes an amino acid sequence, and optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 544, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 530, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 526, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 524, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 532, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 528, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 534, optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 522, and optionally the modified CRBN includes the amino acid sequence shown in SEQ ID NO 542 compared to a wild-type CRBN that includes the amino acid sequence shown in SEQ ID NO 127.

9. An activated conditionally controlled polypeptide (ACP) complex, A first polypeptide comprising the modified degron described in any one of claims 1 to 4, and It contains a second polypeptide that includes a ligand-binding domain, (i) the first polypeptide further comprises a DNA-binding domain and the second polypeptide further comprises a transcription effector domain, or (ii) the first polypeptide further comprises a transcription effector domain and the second polypeptide further comprises a DNA-binding domain, and 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 the ACP complex is capable of regulating the transcriptional expression of a target gene operably linked to an ACP complex-responsive promoter, wherein the DNA-binding domain optionally comprises a DNA zinc finger protein domain, and optionally the DNA zinc finger protein domain comprises the amino acid sequence of SEQ ID NO: 57; An ACP complex wherein, in some cases, the ligand is IMiD, and in some cases, the IMiD is selected from the group consisting of thalidomide, iverdide, lenalidomide, and pomalidomide, and in some cases, the IMiD is pomalidomide.

10. The aforementioned transcription effector domain A transcriptional activation domain selected from the group consisting of: herpes simplex virus protein 16 (VP16) activation domain; activation domain containing four tandem copies of VP16; VP64 activation domain; p65 activation domain of NFκB; Epstein-Barr virus R transactivator (Rta) activation domain; tricomponent activator containing VP64, p65, and Rta activation domains (VPR activation domain); tricomponent activator containing VP64, p65, and HSF1 activation domains (VPH activation domain); and the histone acetyltransferase (HAT) core domain of human E1A-related protein p300 (p300 HAT core activation domain); or Kruppel-associated box (KRAB) repressor domain; cleaved Kruppel-associated box (KRAB) repressor domain; histone deacetylase 4 (HDAC4) repressor domain; scleracsis (SCX) HLH domain, DNA binding inhibitor 1 (ID1) HLH domain, HECT domain and RCC1-like domain-containing protein 2 (HERC2) Cyt-b5 domain, twist-associated protein 1 (TWST1) HLH domain, homeobox protein Nkx-2.2 (NKX22) homeodomain, DNA binding inhibitor 1 (ID3) HLH domain, and twist The ACP complex according to claim 9, wherein the transcriptional repressor domain is selected from the group consisting of a str-related protein 2 (TWST2) HLH domain and an EED repressor domain; a repressor element silencing transcription factor (REST) ​​repressor domain; a WRPW motif of hairy-related basic helix-loop helix repressor protein, wherein the motif is known as a WRPW repressor domain; a DNA (cytosine-5)-methyltransferase 3B (DNMT3B) repressor domain; and an HP1 alpha-chromoshadow repressor domain.

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

12. The modified degron and the DNA-binding domain or the transcription effector domain are linked by a first linker, and optionally the ligand-binding domain and the transcription effector domain or the DNA-binding domain are linked by a second linker, and optionally the first linker and / or the second linker comprises the amino acid sequence shown in SEQ ID NOs: 169, 171, 173, 265, or 269, 168, 175, or 199, and optionally the first linker and / or the second linker is a glycine-serine linker, and optionally the glycine-serine linker comprises one or more GG The ACP complex according to any one of claims 9 to 11, comprising a GS (SEQ ID NO: 252) motif, optionally the glycine-serine linker comprising one or more GGGGS (SEQ ID NO: 253) motifs, optionally the linker comprising the amino acid sequence GGGGSGGGGGSGGGGGS (SEQ ID NO: 254), optionally the linker comprising the amino acid sequence shown in any one of SEQ ID NOs: 45, 46, 91, 92, 191, or 193, optionally the linker not comprising a lysine residue, and optionally the first linker and / or the second linker comprising the amino acid sequence shown in SEQ ID NO: 265 or 269.

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

14. The first and second cell death induction domains include caspases (e.g., any one of caspases 1-11, e.g., caspase 3, caspase 6, caspase 7, caspase 8, caspase 9, etc.), diphtheria toxin fragment A (DTA), Bax, Bak, Bok, Bad, Bcl-xS, Bak, Bik, Bcl-2 interacting protein 3 (BNIP3), Fas, Fas-related protein with death domain (FADD), tumor necrosis factor receptor type 1-associated death domain protein (TRADD), TNF receptor (TNF-R), APAF-1, granzyme B, second mitochondrial-derived activator of caspase (SMAC), Omi, Bmf, Bid, Bim, p53 upregulator of apoptosis (PUMA), Noxa, Blk, Hrk, An inducible cell death system according to claim 13, derived from a protein selected from the group consisting of cytochrome c, Arts, TNF-associated cell death induction ligand (TRAIL), herpes simplex virus thymidine kinase (HSV-TK), varicella-zoster virus thymidine kinase (VZV-TK), viral spike protein, carboxylesterase, cytosine deaminase, nitroreductase Fksb, carboxypeptidase G2, carboxypeptidase A, horseradish peroxidase, linamorase, liver cytochrome P450-2B1, purine nucleoside phosphorylase, and variants and functional fragments thereof, wherein the first and second cell death induction domains are derived from caspase 9, and wherein the caspase 9 does not contain a caspase activation and recruitment domain (CARD) domain sequence.

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

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

17. The modified CRBN, compared to a wild-type CRBN containing the amino acid sequence shown in SEQ ID NO: 127, includes one or more amino acid substitutions selected from T359 amino acid substitution, Q325 amino acid substitution, N369Y amino acid substitution, I371N amino acid substitution, and H397F amino acid substitution, and optionally the Q325 amino acid substitution is selected from Q325H substitution, Q325K substitution, and Q325R substitution, and / or optionally the T359 amino acid substitution is selected from T359V substitution, T359F substitution, T359I substitution, T359L substitution, T359W substitution, and T359Y substitution, and the modified degron, compared to a wild-type degron containing the amino acid sequence shown in SEQ ID NO: 40, includes Q28T amino acid substitution, G30E amino acid substitution An inducible cell death system according to any one of claims 13 to 16, comprising one or more amino acid substitutions selected from acid substitutions and L33I amino acid substitutions, wherein the modified CRBN comprises an I371N amino acid substitution and the modified degron comprises a G30E amino acid substitution, wherein the modified CRBN comprises Q325R, N369Y, I371N, and H397F amino acid substitutions, or wherein the modified degron comprises Q325R, N369Y, I371N, and H397F amino acid substitutions and the modified degron comprises Q28T, G30E, and L33I amino acid substitutions, an ACP complex according to any one of claims 89 to 102 or 163 to 168, or a polypeptide complex according to claim 149.

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

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

20. A method for regulating the expression of a target gene, the method comprising expressing an ACP complex according to any one of claims 9 to 12 in cells, and exposing the cells to the ligand, wherein the ligand is optionally an IMiD, and optionally the IMiD is selected from the group consisting of thalidomide, iverdamide, lenalidomide, and pomalidomide.

21. One or more nucleic acid sequences encoding a modified degron according to any one of claims 1 to 4, a modified CRBN according to any one of claims 5 to 8, an inducible cell death system according to any one of claims 13 to 17, or an ACP complex according to any one of claims 9 to 12.

22. A vector comprising the nucleic acid sequence described in claim 21, and optionally a promoter, preferably a promoter that functions in mammalian cells.

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

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