Synthetic promoters for t cell expression

Abstract

Provided herein are synthetic promoters, including synthetic transcriptional regulatory sequences and variant MND promoters, that can be used for controlling expression of transcribable sequences, such as chimeric antigen receptors (CARs) and T cell receptors (TCRs), such as by allowing for induction, repression, or constitutive activation of transcription of the transcribable sequence by T cell stimulation. Also provided herein are polynucleotides and vectors containing the synthetic transcriptional regulatory sequences and variant MND promoters and transcribable sequences described herein. Also provided herein are engineered T cells containing the synthetic transcriptional regulatory sequences, variant MND promoters, polynucleotides, and vectors described herein, as well as methods of making and using same.

Description

SYNTHETIC PROMOTERS FOR T CELL EXPRESSIONCross-Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Application No. 63 / 676,277, filed July 26, 2024, entitled “SYNTHETIC PROMOTERS FOR T CELL EXPRESSION,” the contents of which is incorporated by reference in its entirety.Reference to An Electronic Sequence Listing

[0002] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042029540SeqList.xml, created on July 25, 2025, which is 141,539 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to synthetic promoters, including synthetic transcriptional regulatory sequences and variant MND promoters, that can be used for controlling expression of transcribable sequences, such as chimeric antigen receptors (CARs) and T cell receptors (TCRs). The present disclosure also relates to polynucleotides and vectors containing the synthetic transcriptional regulatory sequences and variant MND promoters described herein. The present disclosure also relates to engineered immune cells, e.g., T cells, containing the synthetic transcriptional regulatory sequences and variant MND promoters, polynucleotides, and vectors described herein, as well as methods of making and using the same.Background

[0004] Various cell therapy methods are available for treating diseases and conditions. Among cell therapy methods are methods involving immune cells, such as T cells, genetically engineered with a recombinant receptor, such as chimeric antigen receptors or T cell receptors. There is a need for improved tools and methods for enhancing the potency, persistence, and / or efficacy of such cell therapies, including with regards to controlling the expression of transcribable sequences expressed within the immune cells of such cell therapies.Summary

[0005] Provided herein is a synthetic transcriptional regulatory sequence, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and MOFO-9982734103, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

[0006] In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103. In some embodiments, the synthetic transcriptional regulatory sequence is constitutively active in T cells to control constitutive expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103.

[0007] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 111, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 71, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO:79 or 80; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 94, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 52, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

[0008] In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 104 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 107, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 104 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 108, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 19.

[0009] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of ZKSCAN comprising the nucleic acid sequence of SEQ ID NO: 112 and a core regulatory element motif of EFla comprising the nucleic acid sequence of SEQ ID NO: 113, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20.

[0010] In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 21. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 105 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 109, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 106 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 110, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 24. 21. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 103.

[0011] In some embodiments, the transcribable sequence comprises a chimeric antigen receptor (CAR), a decoy receptor, CD40L, 4-1BBL, a pro-inflammatory binder, a chemokine, or a safety switch. In some embodiments, the decoy receptor comprises TGFPRIIDN or a chimeric cytokine receptor. In some embodiments, the pro-inflammatory binder comprises an anti-PDl scFv, a CD40R agonist antibody, a CD47 antibody, or a bispecific T cell engager. In some embodiments, the chemokine comprises CCL19 or CXCL10.

[0012] In some embodiments, the synthetic transcriptional regulatory element is inducibly repressible in T cells to control inducible repression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent.

[0013] In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14.

[0014] In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatoryelement motif comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 91, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 86, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 71, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In some of any embodiments, the transcribable sequence comprises a chimeric antigen receptor (CAR) or a chimeric cytokine receptor.

[0015] In some of any embodiments, the synthetic transcriptional regulatory element is inducibly active in T cells to control inducible activation of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent.

[0016] In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101.

[0017] In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of NFkB comprising the nucleic acid sequencesof SEQ ID NO: 28 and a core regulatory element motif of NFkB comprising the nucleic acid sequencesof SEQ ID NO: 30, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7.

[0018] In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a plurality of a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

[0019] In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 55, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some of any embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101.

[0020] In some of any embodiments, the transcribable sequence comprises a chimeric antigen receptor (CAR), IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-36, TNFa, FTL3L, IFNy, and / or CCL21. In some of any embodiments, the transcribable sequence comprises IL-12.

[0021] In some of any embodiments, the transcribable sequence encodes an antibody or antigenbinding fragment thereof, an interleukin or variant thereof, or a cytokine or variant thereof. In some of any embodiments, the transcribable sequence encodes a recombinant receptor, an antibody or antigen binding fragment thereof, a cytokine of variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

[0022] Also provided herein is a synthetic transcriptional regulatory sequence, comprising a first core regulatory element motif and a second core regulatory element motif, wherein the first core regulatory element motif and the second core regulatory element motif each independently comprise a core regulatory element motif selected from the group consisting of FOS, NFkB, FLU, EFla, and ZKSC AN 1 ; and wherein the first core regulatory element motif and a second core regulatory element motif are separated by a spacer sequence.

[0023] In some embodiments, the first core regulatory element motif and the second core regulatory element motif are different. In some of any embodiments, the first core regulatory element motif is a core regulatory element motif of FOS, and the second core regulatory element motif is a core regulatory element motif of NFkB. In some of any embodiments, the core regulatory element motif of FOS comprises the nucleic acid sequence of SEQ ID NO: 38 and the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28. In some of any embodiments, the first core regulatory element motif is a core regulatory element motif of NFkB, and the second core regulatory element motif is a core regulatory element motif of FLU. In some of any embodiments, the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28, and the core regulatory element motif of FLU comprises the nucleic acid sequence of SEQ ID NO: 32. In some of any embodiments, the first core regulatory element motif is a core regulatory element motif of ZKSCAN, and the second core regulatory element motif is a core regulatory element motif of EFla. In some of any embodiments, the core regulatory element motif of ZKSCAN comprises the nucleic acid sequence of SEQ ID NO: 112, the core regulatory element motif of EFla comprises the nucleic acid sequence of SEQ ID NO: 113.

[0024] In some of any embodiments, the first core regulatory element motif is a first core regulatory element motif of NFkB, and the second core regulatory element motif is a second core regulatory element motif of NFkB, wherein the first core regulatory element motif and the second core regulatory element motif are different. In some of any embodiments, the first core regulatory element motif ofNFkB comprises the nucleic acid sequence of SEQ ID NO: 28, the second core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 30.

[0025] Also provided herein is a synthetic transcriptional regulatory sequence, comprising two or more core regulatory element motifs, wherein each of the two or more core regulatory element motifs is the same and is a core regulatory element motif selected from the group consisting of PRDM1, FOS, and NFkB ; wherein each of the two or more core regulatory element motifs are separated from one another by a spacer sequence. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of BEIMP l(PRDMl).In some embodiments, the core regulatory element motif of BEIMP l(PRDMl) comprises the nucleic acid sequence of SEQ ID NO: 111. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of FOS. In some embodiments, the core regulatory element motif of FOS comprises the nucleic acid sequence of SEQ ID NO: 38. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of NFkB. In some embodiments, the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28 or 30.

[0026] In some of any embodiments, the spacer sequence has a length of at least 1 nucleotide and up to 20, 19, 18, 17, 16, 15, 14, 13, or 12 nucleotides. In some of any embodiments, the spacer sequence has a length of 1 to 12 nucleotides, 1 to 10 nucleotides, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 4 to 12 nucleotides, 4 to 10 nucleotides, 4 to 9 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides, or 4 to 6 nucleotides. In some of any embodiments, the spacer sequence has a length of 1 to 12 nucleotides. In some of any embodiments, the spacer sequence has a length of 4 to 6 nucleotides.

[0027] In some of any embodiments, the synthetic transcriptional regulatory sequence further comprises a minimal promoter or variant thereof. In some embodiments, the minimal promoter or variant thereof is a minimal promoter variant. In some embodiments, the minimal promoter variant is an SCP2 minimal promoter. In some embodiments, the minimal promoter or variant thereof is an EFla minimal promoter.

[0028] In some of any embodiments, the T cell stimulatory agent is or comprises an agent that activates a T cell receptor. In some embodiments, the T cell receptor comprises an immunoreceptor tyrosine-based activation motif (IT AM).

[0029] In some of any embodiments, the synthetic transcriptional regulatory sequence is not naturally occurring.

[0030] Also provided herein is a variant MND promoter, comprising one or more core regulatory element motifs inserted into a parental MND promoter, wherein each of the one or more core regulatorymotifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT:AP1, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, TP53, STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1. In some embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53. In some of any embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACHL

[0031] Also provided herein is a variant MND promoter, comprising one or more core regulatory element motifs inserted into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some of any embodiments, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some of any embodiments, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB.

[0032] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a coreregulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NF YA and a core regulatory element motif of PATZE. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NF YA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatory element motif of FLU ; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of CEBPg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1 ; and a core regulatory element motif of RUNX1. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEBPg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4. In some of any embodiments, the one or more core regulatory element motifscomprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52.

[0033] In some of any embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some of any embodiments, the parental MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some of any embodiments, the parental MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some of any embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5.

[0034] In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 11, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 11.

[0035] In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 12, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0036] In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 12.

[0037] In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:13. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 13.

[0038] In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:14. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14.

[0039] In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:15. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 15.

[0040] In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 16, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:16. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 16.

[0041] In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO:17. In some of any embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 17. In some of any embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 17.

[0042] In some of any embodiments, the variant MND promoter is not naturally occurring.

[0043] Also provided herein is a method of generating a synthetic transcriptional regulatory sequence, the method comprising operably linking a first core regulatory element motif and a second core regulatory element motif via a spacer sequence, wherein the first core regulatory element motif and the second core regulatory element motif each independently comprise a core regulatory element motif selected from the group consisting of FOS, NFkB, FLU, EFla, and ZKSCAN1. Also provided herein is a method of generating a polynucleotide comprising a synthetic transcriptional regulatory sequence, a vector comprising a synthetic transcriptional regulatory sequence, a lipid particle comprising a synthetic transcriptional regulatory sequence, a cell comprising a synthetic transcriptional regulatory sequence, an engineered immune cell comprising a synthetic transcriptional regulatory sequence, or a pharmaceutical composition comprising an engineerined immune cell comprising a synthetic transcriptional regulatory sequence, in which the synthetic transcriptional regulatory element of such methods is generated by operably linking a first core regulatory element motif and a second core regulatory element motif via a spacer sequence, wherein the first core regulatory element motif and the second core regulatory element motif each independently comprise a core regulatory element motif selected from the group consisting of FOS, NFkB, FLU, EFla, and ZKSCAN1. In some embodiments, the first core regulatory element motif and the second core regulatory element motif are different.

[0044] In some of any embodiments, the first core regulatory element motif is a core regulatory element motif of FOS, and the second core regulatory element motif is a core regulatory element motif of NFkB. In some of any embodiments, the core regulatory element motif of FOS comprises the nucleic acid sequence of SEQ ID NO: 38 and the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28.

[0045] In some of any embodiments, the first core regulatory element motif is a core regulatory element motif of NFkB, and the second core regulatory element motif is a core regulatory element motif of FLU. In some of any embodiments, core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28, and the core regulatory element motif of FLU comprises the nucleic acid sequence of SEQ ID NO: 32

[0046] In some of any embodiments, the first core regulatory element motif is a core regulatory element motif of ZKSCAN 1 , and the second core regulatory element motif is a core regulatory element motif of EFla. In some of any embodiments, the core regulatory element motif of ZKSCAN comprises the nucleic acid sequence of SEQ ID NO: 112, the core regulatory element motif of EFla comprises the nucleic acid sequence of SEQ ID NO: 113

[0047] In some of any embodiments, the first core regulatory element motif is a first core regulatory element motif of NFkB, and the second core regulatory element motif is a second core regulatory element motif of NFkB, wherein the first core regulatory element motif and the second core regulatory element motif are different. In some of any embodiments, the first core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28, the second core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 30

[0048] Also provided herein is a method of generating a synthetic transcriptional regulatory sequence, the method comprising operably linking two or more core regulatory element motifs from one another via a spacer sequence, wherein each of the two or more core regulatory element motifs is the same and is a core regulatory element motif selected from the group consisting of PRDM1, FOS, and NFkB. Also provided herein is a method of generating a polynucleotide comprising a synthetic transcriptional regulatory sequence, a vector comprising a synthetic transcriptional regulatory sequence, a lipid particle comprising a synthetic transcriptional regulatory sequence, a cell comprising a synthetic transcriptional regulatory sequence, an engineered immune cell comprising a synthetic transcriptional regulatory sequence, or a pharmaceutical composition comprising an engineerined immune cell comprising a synthetic transcriptional regulatory sequence, in which the synthetic transcriptional regulatory element of such methods is generated by operably linking two or more core regulatory element motifs from one another via a spacer sequence, wherein each of the two or more core regulatory element motifs is the same and is a core regulatory element motif selected from the group consisting of PRDM1, FOS, and NFkB. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of PRDM1. In some of any embodiments, the core regulatory element motif of PRDM1 comprises the nucleic acid sequence of SEQ ID NO: 111. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of FOS. In some of any embodiments, the core regulatory element motif of FOS comprises the nucleic acid sequence of SEQID NO: 38. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of NFkB. In some of any embodiments, the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28 or 30.

[0049] In some of any embodiments, the spacer sequence has a length of at least 1 nucleotide and up to 20, 19, 18, 17, 16, 15, 14, 13, or 12 nucleotides. In some of any embodiments, the spacer sequence has a length of 1 to 12 nucleotides, 1 to 10 nucleotides, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 4 to 12 nucleotides, 4 to 10 nucleotides, 4 to 9 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides, or 4 to 6 nucleotides. In some of any embodiments, the spacer sequence has a length of 1 to 12 nucleotides. In some of any embodiments, the spacer sequence has a length of 4 to 6 nucleotides.

[0050] In some of any embodiments, the method further comprises operably linking a minimal promoter or variant thereof to the first core regulatory element motif and / or the second core regulatory element motif. In some of any embodiments, the method further comprises operably linking a minimal promoter or variant thereof to at least one of the two or more core regulatory element motifs. In some of any embodiments, the minimal promoter or variant thereof is a minimal promoter variant. In some embodiments, the minimal promoter variant is an SCP2 minimal promoter. In some of any embodiments, the minimal promoter or variant thereof is an EFla minimal promoter.

[0051] Also provided herein is a method of generating a variant MND promoter, the method comprising inserting one or more core regulatory element motifs into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEB Pg, cMyc, AP1_IRF4, TP53, STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1. Also provided herein is a method of generating a polynucleotide comprising a variant MND promoter, a vector comprising a variant MND promoter, a lipid particle comprising a variant MND promoter, a cell comprising a variant MND promoter, an engineered immune cell comprising a variant MND promoter, or a pharmaceutical composition comprising an engineerined immune cell comprising a variant MND promoter, in which the variant MND promoter of such methods is generated by inserting one or more core regulatory element motifs into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a core regulatoryelement motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEB Pg, cMyc, AP1_IRF4, TP53, STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0052] In some embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1JRF4, and TP53. In some of any embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACHL

[0053] In some of any embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some of any embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5.

[0054] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91.

[0055] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71.

[0056] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA and a core regulatory element motif of PATZE. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86.

[0057] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of FLU; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FEI1 comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55.vln some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of CEB Pg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1 ; and a core regulatory element motif of RUNX1.

[0058] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEB Pg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94.

[0059] In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4. In some of any embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52.

[0060] In some of any embodiments, the variant MND promoter is not naturally occurring.

[0061] Also provided herein is a method of generating a synthetic transcriptional regulatory sequence, comprising operably linking an enhancer sequence and a promoter sequence, wherein: (a) the enhancer sequence is identified by: (i) identifying, using gene expression levels, highly expressed genes from an immune cell engineered to express a recombinant receptor; (ii) identifying open chromatin regions of the genome in proximity to the highly expressed genes; and (iii) excluding transcription start site (TSS) sequences from the identified open chromatin regions, thereby identifying one or more enhancer sequences comprising the enhancer sequence; and (b) the promoter sequence is identified by: (i) identifying, using gene expression levels, highly expressed genes from an immune cell engineered to express a recombinant receptor; (ii) identifying open chromatin regions of the genome in proximity to the highly expressed genes; and (iii) identifying the identified open chromatin regions having known core promoter element configurations, thereby identifying one or more promoter sequences comprising the promoter sequence. Also provided herein is a method of generating a polynucleotide comprising a synthetic transcriptional regulatory sequence, a vector comprising a synthetic transcriptional regulatory sequence, a lipid particle comprising a synthetic transcriptional regulatory sequence, a cell comprising a synthetic transcriptional regulatory sequence, an engineered immune cell comprising a synthetic transcriptional regulatory sequence, or a pharmaceutical composition comprising an engineerined immune cell comprising a synthetic transcriptional regulatory sequence, in which the synthetic transcriptional regulatory element of such methods is generated by operably linking an enhancer sequence and a promoter sequence, wherein: (a) the enhancer sequence is identified by: (i) identifying, using gene expression levels, highly expressed genes from an immune cell engineered to express a recombinant receptor; (ii) identifying open chromatin regions of the genome in proximity to the highly expressed genes; and (iii) excluding transcription start site (TSS) sequences from the identified open chromatin regions, thereby identifying one or more enhancer sequences comprising the enhancer sequence; and (b) the promoter sequence is identified by: (i) identifying, using gene expression levels, highly expressed genes from an immune cell engineered to express a recombinant receptor; (ii) identifying open chromatin regions of the genome in proximity to the highly expressed genes; and (iii) identifying the identified open chromatin regions having known core promoter element configurations, thereby identifying one or more promoter sequences comprising the promoter sequence.

[0062] In some embodiments, the enhancer sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 104-106. In some of any embodiments, the promoter sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 107- 110.

[0063] In some of any embodiments, the enhancer sequence comprises the nucleic acid sequence of SEQ ID NO: 104 and the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 107;the enhancer sequence comprises the nucleic acid sequence of SEQ ID NO: 104 and the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 108; the enhancer sequence comprises the nucleic acid sequence of SEQ ID NO: 105 and the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 109; or the enhancer sequence comprises the nucleic acid sequence of SEQ ID NO: 106 and the promoter sequence comprises the nucleic acid sequence of SEQ ID NO: 110.

[0064] In some of any embodiments, the generated synthetic transcriptional regulatory sequence is not naturally occurring.

[0065] Also provided herein is a method of identifying a synthetic transcriptional regulatory sequence having activity in T cells, the method comprising: (a) providing a library comprising a plurality of synthetic transcriptional regulatory sequences generated by any method described herein operably linked to a transcribable sequence; (b) screening for expression of the transcribable sequence; and (c) identifying a synthetic transcriptional regulatory sequence of the plurality of synthetic transcriptional regulatory sequences having activity in a T cell. Also provided herein is a method of identifying a polynucleotide comprising a synthetic transcriptional regulatory sequence having activity in T cells, a vector comprising a synthetic transcriptional regulatory sequence having activity in T cells, a lipid particle comprising a synthetic transcriptional regulatory sequence having activity in T cells, a cell comprising a synthetic transcriptional regulatory sequence having activity in T cells, an engineered immune cell comprising a synthetic transcriptional regulatory sequence having activity in T cells, or a pharmaceutical composition comprising an engineerined immune cell comprising a synthetic transcriptional regulatory sequence having activity in T cells, in which the synthetic transcriptional regulatory sequence having activity in T cells of such methods is identified by: (a) providing a library comprising a plurality of synthetic transcriptional regulatory sequences generated by any method described herein operably linked to a transcribable sequence; (b) screening for expression of the transcribable sequence; and (c) identifying a synthetic transcriptional regulatory sequence of the plurality of synthetic transcriptional regulatory sequences having activity in a T cell.

[0066] Also provided herein is a method of identifying a variant MND promoter having activity in T cells, the method comprising: (a) providing a library comprising a plurality of variant MND promoters generated by any of the methods described herein operably linked to a transcribable sequence; (b) screening for expression of the transcribable sequence; and (c) identifying a variant MND promoter of the plurality of variant MND promoters having activity in a T cell. Also provided herein is a method of identifying a polynucleotide comprising a variant MND promoter having activity in T cells, a vector comprising a variant MND promoter having activity in T cells, a lipid particle comprising a variant MND promoter having activity in T cells, a cell comprising a variant MND promoter having activity in T cells, an engineered immune cell comprising a variant MND promoter having activity in T cells, or apharmaceutical composition comprising an engineerined immune cell comprising a variant MND promoter having activity in T cells, in which the variant MND promoter having activity in T cells of such methods is identified by: (a) providing a library comprising a plurality of variant MND promoters generated by any of the methods described herein operably linked to a transcribable sequence; (b) screening for expression of the transcribable sequence; and (c) identifying a variant MND promoter of the plurality of variant MND promoters having activity in a T cell.

[0067] In some embodiments, the synthetic transcriptional regulatory sequence having activity in the T cell has constitutive activity in the T cell. In some embodiments, the synthetic transcriptional regulatory sequence having activity in the T cell has inducible activity in the T cell. In some embodiments, the synthetic transcriptional regulatory sequence having activity in the T cell has inducible repressibility in the T cell. In some embodiments, the variant MND promoter having activity in the T cell has constitutive activity in the T cell. In some embodiments, the variant MND promoter having activity in the T cell has inducible activity in the T cell. In some embodiments, the variant MND promoter having activity in the T cell has inducible repressibility in the T cell.

[0068] In some of any embodiments, the transcribable sequence is a reporter sequence. In some of any embodiments, the transcribable sequence encodes a recombinant receptor, an antibody or antigenbinding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

[0069] In some of any embodiments, a synthetic transcriptional regulatory sequence is generated using the methods herein. In some of any embodiments, a variant MND promoter is generated using the methods herein. In some of any embodiments, a synthetic transcriptional regulatory sequence is identified using the methods herein. In some of any embodiments, a variant MND promoter is identified using the methods herein.

[0070] Also provided herein is a polynucleotide comprising any synthetic transcriptional regulatory sequence described herein or variant MND promoter described herein. Also provided herein is a polynucleotide comprising any synthetic transcriptional regulatory sequence described herein, and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

[0071] In some of any embodiments, the polynucleotide comprises any variant MND promoter described herein, and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the variant MND promoter.

[0072] In some of any embodiments, the transcribable sequence encodes a recombinant receptor, an antibody or antigen binding fragment thereof, a cytokine of variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some of any embodiments, transcription of thetranscribable sequence is under the operable control of the synthetic transcriptional regulatory sequence. In some of any embodiments, transcription of the transcribable sequence is under the operable control of the variant MND promoter. In some of any embodiments, transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence. In some of any embodiments, transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

[0073] In some of any embodiments, the transcribable sequence produces a coding RNA molecule. In some of embodiments, the coding RNA molecule encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some embodiments, the coding RNA molecule encodes a recombinant receptor that is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0074] In some of any embodiments, transcription of the transcribable sequence produces a noncoding RNA molecule. In some embodiments, the non-coding RNA molecule is an siRNA, miRNA, or shRNA molecule.

[0075] In some of any embodiments, the transcribable sequence comprises a chimeric antigen receptor (CAR), a decoy receptor, CD40L, 4-1BBL, a pro-inflammatory binder, a chemokine, or a safety switch. In some of any embodiments, the decoy receptor comprises TGFpRIIDN or a chimeric cytokine receptor. In some of any embodiments, the pro-inflammatory binder comprises an anti-PDl scFv, a CD40R agonist antibody, a CD47 antibody, or a bispecific T cell engager. In some of any embodiments, the chemokine comprises CCL19 or CXCL10.

[0076] In some of any embodiments, the transcribable sequence comprises a chimeric antigen receptor (CAR), IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-36, TNFa, FTL3L, IFNy, and / or CCL21. In some of any embodiments, the transcribable sequence comprises IL-12.

[0077] Also provided herein is a vector comprising any synthetic transcriptional regulatory sequence described herein or variant MND promoter described herein. In some of any embodiments, a vector comprises the synthetic transcriptional regulatory sequence or variant MND promoter described herein. In some of any embodiments, a vector comprises the polynucleotide described herein. In some embodiments, a vector comprises the synthetic transcriptional regulatory sequence described herein.

[0078] In some of any embodiments, the vector is an adeno-associated virus (AAV) vector. In some of any embodiments, the vector is a retroviral vector. In some of any embodiments, the vector is a lentiviral vector.

[0079] Also provided herein is a lipid particle comprising any synthetic transcriptional regulatory sequence described herein, or any variant MND promoter described herein. In some embodiments, a lipid particle comprises any polynucleotide described herein. In some embodiments, a lipid particle comprisesany synthetic transcriptional regulatory sequence described herein. In some embodiments, a lipid particle comprises any variant MND promoter described herein.

[0080] Also provided herein is a cell comprising any synthetic transcriptional regulatory sequence described herein, or any variant MND promoter described herein. In some embodiments, the cell comprises any polynucleotide described herein. In some embodiments, the cell comprises any vector described herein. In some embodiments, the cell comprises any lipid particle described herein. In some embodiments, the cell is an engineered immune cell.

[0081] Also provided herein is an engineered immune cell comprising any synthetic transcriptional regulatory sequence described herein, any variant MND promoter described herein, any polynucleotide described herein, any vector described herein, or any lipid particle described herein.

[0082] In some of any embodiments, the engineered immune cell comprises any synthetic transcriptional regulatory sequence described herein, any polynucleotide described herein, or any vector described herein. In some of any embodiments, the engineered immune cell comprises any synthetic transcriptional regulatory sequence or variqnt MND promoter described herein that is inducibly active, any polynucleotide described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly active, or any vector described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly active.

[0083] In some of any embodiments, the engineered immune cell comprises any synthetic transcriptional regulatory sequence described herein, any polynucleotide described herein, or any vector described herein. In some of any embodiments, the engineered immune cell comprises any synthetic transcriptional regulatory sequence or variqnt MND promoter described herein that is inducibly repressible, any polynucleotide described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly repressible, or any vector described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly repressible.

[0084] In some of any embodiments, the engineered immune cell expresses a recombinant receptor under the operable control of the synthetic transcriptional regulatory sequence. In some of any embodiments, the engineered immune cell expresses a recombinant receptor under the operable control of the variant MND promoter. In some of any embodiments, the recombinant receptor is a T cell receptor (TCR).

[0085] In some of any embodiments, the recombinant receptor is a CAR comprising an extracellular antigen-binding domain and an intracellular signaling domain comprising an IT AM signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a CD3 chain. In some embodiments, the CD3 chain is a CD3-zeta chain.

[0086] In some of any embodiments, binding to the recombinant receptor induces a costimulatory signal. In some of any embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of CD28 or 4- IBB.

[0087] In some of any embodiments, the engineered immune cell is an engineered lymphocyte. In some of any embodiments, the engineered immune cell is an engineered T cell.

[0088] In some of any embodiments, the synthetic transcriptional regulatory sequence and the transcribable sequence are comprised within the genome of the engineered immune cell. In some of any embodiments, the synthetic transcriptional regulatory sequence and the transcribable sequence are comprised within an intron or a transcriptional regulatory element of the genome of the engineered immune cell.

[0089] In some of any embodiments, the variant MND promoter and the transcribable sequence are comprised within the genome of the engineered immune cell. In some of any embodiments, the variant MND promoter and the transcribable sequence are comprised within an intron or a transcriptional regulatory element of the genome of the engineered immune cell.

[0090] Also provided herein is an engineered immune cell comprising any polynucleotide described herein.

[0091] In some of any embodiments, the polynucleotide is comprise in an adeno-associated virus (AAV) vector.

[0092] In some of any embodiments, the polynucleotide is comprised in the engineered immune cell by insertion or integration into the genome of the engineered immune cell. In some of any embodiments, the polynucleotide is comprised in the engineered immune cell by insertion or integration into an intron or a transcriptional regulatory element of the genome of the engineered immune cell for expression of the transcribable sequence. In some of any embodiments, the insertion or integration is by homology- directed repair (HDR).

[0093] Also provided herein is a method of producing an engineered immune cell comprising introducing into an immune cell any synthetic transcriptional regulatory sequence described herein, any variant MND promoter described herein, any polynucleotide described herein, any vector described herein, or any lipid particle described herein.

[0094] Also provided herein is a method of producing an engineered immune cell comprising introducing into an immune cell any synthetic transcriptional regulatory sequence described herein, any polynucleotide described herein, any vector described herein, or any lipid particle described herein.

[0095] Also provided herein is a method of producing an engineered immune cell comprising introducing into an immune cell any variant MND promoter described herein, any polynucleotide described herein, any vector described herein, or any lipid particle described herein.

[0096] Also provided herein is a method of producing an engineered immune cell comprising introducing into an immune cell any synthetic transcriptional regulatory sequence described herein, any polynucleotide described herein, or any vector described herein.

[0097] Also provided herein is a method of producing an engineered immune cell, comprising introducing into an immune cell any synthetic transcriptional regulatory sequence described herein, any polynucleotide described herein or any vector described herein.

[0098] In some of any of such embodiments, the synthetic transcriptional regulatory sequence or variat MND promoter is inducibly repressible. In some of any of such embodiments, the synthetic transcriptional regulatory sequence or variat MND promoter is inducibly active. In some of any of such embodiments, the synthetic transcriptional regulatory sequence or variat MND promoter is constitutively active.

[0099] In some of any embodiments, the introducing comprises introducing any synthetic transcriptional regulatory sequence described herein and any transcribable sequence described herein into the genome of the immune cell. In some of any embodiments, the introducing comprises introducing any synthetic transcriptional regulatory sequence described herein and any transcribable sequence described herein into an intron or a transcriptional regulatory element of the genome of the immune cell.

[0100] In some of any embodiments, the introducing comprises introducing any variant MND promoter described herein and any transcribable sequence described herein into the genome of the immune cell. In some of any embodiments, the introducing comprises introducing any variant MND promoter described herein and any transcribable sequence described herein into an intron or a transcriptional regulatory element of the genome of the immune cell.

[0101] Also provided herein is a method of producing an engineered immune cell comprises introducing into an immune cell any polynucleotide described herein. In some embodiments, the introducing comprises insertion or integration of any polynucleotide described herein into the genome of the immune cell. In some of any embodiments, the introducing comprises insertion or integration of any polynucleotide described herein into an intron or a transcriptional regulatory element of the genome of the immune cell for expression of the transcribable sequence. In some of any embodiments, the insertion or integration is by homology-directed repair (HDR).

[0102] In some of any embodiments, wherein the engineered immune cell expresses a recombinant receptor under the operable control of any synthetic transcriptional regulatory sequence described herein.In some of any embodiments, the engineered immune cell expresses a recombinant receptor under the operable control of any variant MND promoter described herein.

[0103] In some of any embodiments, the recombinant receptor is a T cell receptor (TCR). In some of any embodiments, the recombinant receptor is a CAR comprising an extracellular antigen-binding domain and an intracellular signaling domain comprising an IT AM signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a CD3 chain.In some embodiments, the CD3 chain is a CD3-zeta chain. In some of any embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain comprises an intracellular signaling domain of CD28 or 4-1BB.

[0104] In some of any embodiments, the engineered immune cell is an engineered lymphocyte. In some of any embodiments, the engineered immune cell is an engineered T cell. In some of any embodiments, the engineered immune cell is a human cell.

[0105] Also provided herein is an engineered immune cell is produced by any method described herein. In some of any embodiments, the engineered immune cell is a human cell.

[0106] Also provided herein is a method of expressing a transcribable sequence comprising introducing a polynucleotide described herein or a vector described herein into a cell. In some embodiments, the cell is a T cell.

[0107] Also provided herein is a method of decreasing transcription of a transcribable sequence in an immune cell comprises stimulating any engineered immune cell described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly repressible.

[0108] Also provided herein is a method of increasing transcription of a transcribable sequence in an immune cell comprises stimulating the engineered immune cell described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly active.

[0109] Also provided herein is a method of decreasing transcription of a transcribable sequence in an immune cell, comprises: (a) introducing into an immune cell any polynucleotide described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly repressible, or any vector described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly repressible; and (b) stimulating the immune cell, thereby decreasing transcription of the transcribable sequence of the polynucleotide or vector.

[0110] Also provided herein is a method of increasing transcription of a transcribable sequence in an immune cell, comprising: (a) introducing into an immune cell any polynucleotide described herein that comprises a synthetic transcriptional regulatory sequence or variat MND promoter that is inducibly active, or any vector described herein that comprises a synthetic transcriptional regulatory sequence orvariat MND promoter that is inducibly active; and (b) stimulating the immune cell, thereby increasing transcription of the transcribable sequence of the polynucleotide or vector.

[0111] In some of any embodiments, the introducing comprises introducing the polynucleotide into the genome of the immune cell. In some of any embodiments, the introducing comprises introducing the polynucleotide into an intron or a transcriptional regulatory element of the genome of the immune cell. In some of any embodiments, the immune cell is a lymphocyte. In some of any embodiments, the immune cell is a T cell. In some of any embodiments, the stimulating comprises inducing an IT AM-mediated signal, wherein the immune cell expresses a recombinant receptor, and the IT AM-mediated signal is induced via binding to the recombinant receptor. In some of any embodiments, the recombinant receptor is a CAR comprising an extracellular antigen-binding domain and an intracellular signaling domain comprising an IT AM signaling domain. In some of any embodiments, the stimulating occurs in vivo following administration of the immune cell to a subject having a disease or condition. In some of any embodiments, the stimulating comprises administering the immune cell to a subject having a disease or condition. In some of any embodiments, the stimulating occurs in vitro or ex vivo. In some of any embodiments, the stimulating comprises contacting the immune cell with a T cell stimulatory agent.

[0112] In some of any embodiments, the recombinant receptor binds to a target antigen that is expressed by cells associated with the disease or condition in the subject. In some of any embodiments, the disease or condition is a cancer or an autoimmune or inflammatory disease.

[0113] In some of any embodiments, the transcribable sequence encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some of any embodiments, the cytokine or variant thereof comprises IL- 12.

[0114] In some of any embodiments, any of the engineered immune cells described herein is used in treating a disease or condition in a subject. In some of any embodiments, the immune cell expresses a recombinant receptor that binds to a target antigen expressed by cells associated with the disease or condition. In some of any embodiments, the disease or condition is a cancer or an autoimmune or inflammatory disease. In some of any embodiments, the disease or condition is a cancer. In some of any embodiments, the disease or condition is an autoimmune or inflammatory disease.

[0115] In some of any embodiments, the engineered cell comprises transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence or the variant MND promoter, wherein the transcribable sequence encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some embodiments, the cytokine or variant thereof comprises IL-12. In some of anyembodiments, a pharmaceutical composition comprises the engineered immune cell described herein and a pharmaceutically acceptable carrier.Brief Description of the Drawings

[0116] FIG. 1 depicts the activity of an exemplary set of identified promoter sequences containing FOS and NFkB core regulatory element motifs separated by spacer sequences of different length (1, 4, 6, 9, or 12 nucleotides) or no spacer sequence (0 nucleotides).

[0117] FIG. 2A depicts expression data on mean fluorescent intensity (MFI) for various promoter sequences grouped into three categories based on expression of the CAR transgene following activation, as compared to control promoters. “Antigen up” corresponds to promoters exhibiting increased expression following T cell stimulation. “Ag-down” corresponds to promoters exhibiting decreased expression following T cell stimulation. “Stable” corresponds to promoters with relatively stable expression with or without T cell stimulation. A no stimulation control is also shown for each promoter.

[0118] FIG. 2B depicts expression data for various promoter sequences grouped into three categories based on expression of the CAR transgene following activation.

[0119] FIG. 3 depicts a Daudi cytolytic killing assay with various exemplary candidate promoter sequences. Cytolytic killing of target cells in the absence of CAR T cells is also shown as a control.

[0120] FIG. 4A depicts expression of the exemplary pay load IL- 12 in nonactivated CAR T cells from two different donors (Donor 1 and Donor 2) engineered to constitutively express exemplary CAR transgene and an exemplary IL- 12 transgene payload that is operably linked to either an exemplary constitutive MND promoter or exemplary inducible promoter trono 4x FOS. Controls included cells that were mock transduced and contained no knock-in transgenes (Mock) and cells that were transduced with the CAR transgene only (CAR Alone).

[0121] FIG. 4B depicts expression of the exemplary payload IL-12 in activated T cells engineered from T cells of two different donors (Donor 1 and Donor 2) to contain knock-ins of constitutively expressed exemplary CAR transgene and an exemplary IL- 12 transgene payload that is operably linked to either an exemplary constitutive MND promoter or exemplary inducible promoter trono 4x FOS. Controls included cells that were transduced with the CAR transgene only and included a spike-in (exogenously added) of either 1 pg / mL or 100 pg / mL of IL- 12.

[0122] FIGs. 5A-5B depict expression of IL- 12 in serum (top) and CAR expansion (bottom) in peripheral blood where CAR T cells prepared from Donor 1 (left) and Donor 2 (right) were transplanted into non-tumor bearing NSG mice. Cells were transplanted at an exemplary dose of 3 million cells (FIG. 5A) or 6 million cells (FIG. 5B). Cells contained only a constitutively expressed CAR transgene or 1contained both a constituvely expressed CAR transgene and an IL- 12 transgene operably linked to either a consitutively expressed MND promoter or an inducible trono 4x FOS promoter.Detailed Description

[0123] Provided herein are synthetic transcriptional regulatory sequences, including variant MND promoters, polynucleotides encoding the same, and vectors and immune cells containing the same, along with methods of generating, identifying, and using the same. Expression of transcribable sequences, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR), is often controlled by promoters like EFl alpha HTLV1R. Other transcribable sequences, such as pro-inflammatory molecules (e.g., IL-2, IL- 12, IL-15, IL-18, Flt3L, IFNy, IL-7, CCL19), are often expressed in immune cells (e.g., using leaky antigen-inducible promoters such as NF AT) to improve function. However, there are safety concerns with existing promoters regarding expression. For example, expression that is too high may cause toxicity. For instance, administration of cells engineered to express pro-inflammatory molecules (e.g., IE- 12) can result in clinic toxicities, such as liver dysfunction, fever, and hemodynamic instability. See, e.g., Zhang E, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin- 12 for the immunotherapy of metastatic melanoma. Clin Cancer Res. 2015 May 15;21(10):2278-88. doi: 10.1158 / 1078-0432.CCR-14-2085. In contrast, low expression of a therapeutic protein, e.g., a TCR or CAR, depending on the specific therapeutic protein and its target, may result in insufficient efficacy. Further, knock-in of transgenes into genomic loci that then rely on endogenous promoters for expression can prevent further fine-tuning or optimization of the expression of the transcribable sequence. Therefore, there are times when high or low expression may be needed depending on the situation. There are also times when consistent expression of a therapeutic protein may be needed. The methods and promoters described herein address these needs.

[0124] Embodiments of the provided disclosure relates to identification of regulatory sequences that include promoter sequences (also referred to herein as synthetic transcriptional regulatory sequences or variant MND promoters) that support either stable (constitutive) expression of a transcribable sequence or conditional expression of a transcribable sequence in T cells, such as expression that is able to be inducibly activated or inducibly repressed following stimulation of T cells to activate the T cells. Among the provided embodiments are promoters (also referred to herein as synthetic transcriptional regulatory sequences or variant MND promoters) identified from unique promoter libraries operably linked to a transcribable sequence in screens for expression activity of the transcribable sequence in cells (e.g., T cells). In some embodiments, the promoter libraries as described are generated by combining transcription factor motifs with varying nucleotide spacing. In some embodiments, the promoter libraries as described are generated by inserting endogenous transcription factor motifs into known promoters. Insome embodiments, the promoter libraries as described are generated by combining candidate enhancer and promoters into pairs. In some embodiments, identified promoter regulatory sequences as provided exhibit improved expression activity, such as compared to a reference promoter such as the EFl alpha HTLV1R. The improved expression activity includes, but is not limited to, increased expression of the transcribable sequence, stable or consistent expression of the transcribable sequence even following activation of the cell (e.g., T cells), inducible activation of the transcribable sequence with activation of the cell (e.g., T cell), or inducible repression of the transcribable sequence with activation of the cell (e.g., T cell). In some embodiments, the promoter driven expression increases upon T cell activation, such as is inducibly activated with T cell activation. In some embodiments, the promoter driven expression decreases upon T cell activation, such as is inducibly repressed with T cell activation. In some embodiments, the promoter is stable in that its expression is not affected by T cell activation. In some embodiments, the promoter is referred to as a synthetic transcriptional regulatory sequence. In some embodiments, the promoter is referred to as a variant MND promoter, such as in embodiments involving the modification of a parental MND promoter.

[0125] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0126] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. SYNTHETIC TRANSCRIPTIONAL REGULATORY SEQUENCES AND VARIANT MND PROMOTERS

[0127] Provided herein, in some embodiments, is a synthetic transcriptional regulatory sequence. In some embodiments, the synthetic transcriptional regulatory sequence contains one or more core regulatory element motifs and a minimal promoter, such as any of those described herein. In some embodiments, the one or more core regulatory element motif is operably linked to the minimal promoter. In some embodiments, the one or more core regulatory element motif is operably linked 5’ to the minimal promoter.

[0128] In some embodients, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif. In some embodiments, the synthetic transcriptional regulatory sequence comprises two or more core regulatory element motifs that are the same or different. In someembodiments, each core regulatory element motif of the synthetic transcriptional regulatory sequence is a core regulatory element motif of one or more transcription factors. In some embodiments, each core regulatory element motif of the synthetic transcriptional regulatory sequence is capable of being bound by one or more transcription factors.

[0129] In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

[0130] Exemplary transcribable sequences are described in Section I-C. In some embodiments, transcription of the transcribable sequence produces a coding RNA molecule. In some embodiments, the coding RNA encodes a transgene. In some embodiments, transcription of the transcribable sequence produces two or more coding RNA molecules each encoding a transgene. In some embodiments, the two or more coding RNA molecules encode the same transgene. In some embodiments, the two or more coding RNA molecules encode different transgenes.

[0131] In some embodiments, transcription of the transcribable sequence produces a non-coding RNA molecule. In some embodiments, the non-coding RNA molecule is an siRNA. In some embodiments, the non-coding RNA molecule is a miRNA. In some embodiments, the non-coding RNA molecule is an shRNA. In some embodiments, the non-coding RNA molecule is anti-sense RNA. In some embodiments, transcription of the transcribable sequence produces two or more non-coding RNA molecules. In some embodiments, the two or more non-coding RNA molecules include sequences complementary to different genes. In some embodiments, the two or more non-coding RNA molecules include sequences complementary to the same gene. In some embodiments, the two or more non-coding RNA molecules are identical.

[0132] In some embodiments, transcription of a transcribable sequence, when under the operable control of the synthetic transcriptional regulatory sequence, is modulated by immune cell stimulation, e.g., T cell stimulation, involving conditions to activate the cell (e.g., T cell). In some embodiments, the immune cell is a T cell, and the stimulation is T cell stimulation.

[0133] In some embodiments, the synthetic transcriptional regulatory sequence is constitutively active in T cells to control constitutive expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence, even in conditions in which the cell can be activated.

[0134] In some embodiments, the synthetic transcriptional regulatory element is inducibly repressible in T cells to control inducible repression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cellstimulatory agent. In other embodiments, binding of the one or more transcription factors is repressed by T cell stimulation. In some embodiments, the one or more transcription factors are not bound to the regulatory element during and / or after T cell stimulation.

[0135] In some embodiments, the synthetic transcriptional regulatory element is inducibly active in T cells to control inducible activation of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent. In some embodiments, binding of the one or more transcription factors is induced by T cell stimulation. In some embodiments, the one or more transcription factors are bound to the core regulatory element motif during and / or after T cell stimulation.

[0136] In some embodiments, the T cell stimulation includes (e.g., leads to) activation of one or more T cell signaling pathways. In some embodiments, the T cell stimulation includes activation of two or more T cell signaling pathways. In some embodiments, the T cell stimulation includes activation of the NF AT signaling pathway; i.e., the T cell stimulation includes NF AT activation. In some embodiments, the T cell stimulation includes activation of the NF-kB signaling pathway; i.e., the T cell stimulation includes NF-kB activation. In some embodiments, the T cell stimulation includes NF AT and / or NF-kB activation. In some embodiments, the T cell stimulation includes NF AT and NF-kB activation.

[0137] In some embodiments, the T cell stimulation includes activation of one or more intracellular signaling domains of one or more components of a TCR complex. In some aspects, the T cell stimulation initiates a TCR / CD3 intracellular signaling cascade in a T cell, e.g., a primary signal, e.g., an ITAM- induced signal. In some embodiments, the T cell stimulation initiates a primary signal through, e.g., via binding to, CD3.

[0138] In some embodiments, the T cell stimulation further includes (e.g., leads to) activation of one or more costimulatory molecules. In some embodiments, the T cell stimulation includes activation of one or more intracellular signaling domains of one or more costimulatory molecules, e.g., CD28, CD137 (4- 1-BB), 0X40, or ICOS. In some embodiments, the T cell stimulation initiates a costimulatory signal through, e.g., via binding to, CD28.

[0139] In some embodiments, the T cell stimulation is by (e.g., is induced by) a T cell stimulatory agent. In some embodiments, the T cell stimulation is by contacting a T cell with a T cell stimulatory agent. In some embodiments, the T cell stimulatory agent is or comprises an agent that activates a T cell receptor. In some embodiments, the T cell stimulation is by an agent that specifically binds to CD3 and / or an agent that specifically binds to CD28. In some embodiments, the T cell stimulatory agent is LPS. In some embodiments, the T cell stimulatory agent is PMA. In some embodiments, the T cell stimulatory agent is ionomycin. In some embodiments, the T cell stimulatory agent comprises PMA andionomycin. In some embodiments, the T cell stimulatory agent is purified protein derivative of tuberculin (PPD). In some embodiments, the stimulatory agent is a plant lectin (e.g., PHA, Con A, or PWM).

[0140] In some embodiments, the T cell stimulatory agent comprises an agent that specifically binds to CD3 and / or an agent that specifically binds to CD28. In some embodiments, the T cell stimulatory agent comprises an agent that specifically binds to CD3. In some embodiments, the T cell stimulatory agent comprises an anti-CD3 antibody or antibody fragment thereof. In some embodiments, the T cell stimulatory agent comprises an anti-CD3 Fab. In some embodiments, the T cell stimulatory agent comprises an agent that specifically binds to CD28. In some embodiments, the T cell stimulatory agent comprises an anti-CD28 antibody or antibody fragment thereof. In some embodiments, the antibody fragment is a F(ab’)2-fragment, a divalent single-chain Fv (scFv) fragment, a Fab fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody fragment is a Fab. In some embodiments, the T cell stimulatory agent comprises an anti-CD28 Fab. In some embodiments, the T cell stimulatory agent comprises an agent that specifically binds to CD3 and an agent that specifically binds to CD28. In some embodiments, the T cell stimulatory agent comprises an anti-CD3 antibody or antibody fragment thereof and an anti-CD28 antibody or antibody fragment thereof. In some embodiments, the T cell stimulatory agent comprises an anti-CD3 Fab and an anti-CD28 Fab.

[0141] In some embodiments, the T cell expresses a recombinant receptor, such as a chimeric antigen receptor (CAR or an engineered T cell receptor (TCR)), that contains or can complex with an ITAM-induced signaling domain and / or a costimulatory signaling domain. In some embodiments, the T cell stimulation of such recombinant receptor-expressing T cells includes (e.g., leads to) activation of one or more intracellular signals from the recombinant receptor, such as the CAR or TCR. In some embodiments, the T cell stimulation includes activation of one or more ITAM-induced intracellular signals. In some embodiments, the ITAM-induced intracelula signal provides a primary activation signal in the cell. In some embodiments, the T cell stimulation also includes activation of one or more costimulatory signals to facilitate effector functions.

[0142] In some embodiments, the T cell stimulatory agent induces a signal of a TCR, such as an engineered TCR. Thus, in some embodiments, the T cell stimulation is by a T cell stimulatory agent that induces a signal of a TCR. In some embodiments, the T cell stimulatory agent that induces a signal of a TCR is any signal that results in anti-TCR crosslinking. In some embodiments, the T cell stimulatory agent is a peptide-MHC complex (e.g., tetramer) that can be engaged by the TCR. In some embodiments, the T cell stimulatory agent that induces a signal of a TCR is an anti-CD3 antibody that engages the TCR complex, which is required for T cell activation, alone or combined with a further costimulatory signal such a using an anti-CD28 antibody.

[0143] In some embodiments, the T cell stimulatory agent induces a signal of a CAR. Thus, in some embodiments, the T cell stimulation is by a T cell stimulatory agent that induces a signal of a CAR. In some embodiments, the T cell stimulatory agent that induces a signal of a CAR binds to an extracellular domain of the CAR. In some embodiments, the T cell stimulatory agent that induces a signal of a CAR is a target antigen able to bind to an extracellular domain of the CAR. In some embodiments, the T cell stimulatory agent that induces a signal of a CAR is an anti-idiotype antibody or antigen-binding fragment thereof that is able to bind to an extracellular domain of the CAR.

[0144] In some embodiments, whether transcription of a transcribable sequence, when under the operable control of the synthetic transcriptional regulatory sequence, is induced or repressed by T cell stimulation, or is constitutively active, is determined using T cells in which the synthetic transcriptional regulatory sequence has been introduced. In some embodiments, transcription of the transcribable sequence in the assay produces a fluorescent reporter (e.g., tdTomato). In some embodiments, the T cells are stimulated using a stimulatory agent, e.g., any T cell stimulatory agent described in the preceding embodiments, e.g., PMA / ionomycin, anti-CD3 / anti-CD28 Fabs, or an anti-CAR anti-idiotype antibody. In some embodiments, expression of the fluorescent reporter is determined using flow cytometry. In some embodiments, increased expression of the fluorescent reporter following T cell stimulation using a T cell stimulatory agent is indicative of a synthetic transcriptional regulatory sequence under which transcription of the transcribable sequence is induced. In other embodiments, decreased expression of the fluorescent reporter following T cell stimulation is indicative of a synthetic transcriptional regulatory sequence under which transcription of the transcribable sequence is repressed. In other embodiments, stabilized or unchanged expression of the fluorescent reporter following T cell stimulation as compared to without T cell stimulation is indicative of a synthetic transcriptional regulatory sequence under which transcription of the transcribable sequence is constitutively active.A. Synthetic Transcriptonal Regulatory Sequences, Variant MND Promoters, and Core Regulatory Element Motifs

[0145] Provided herein, in some embodiments, are synthetic transcriptional regulatory sequences that comprise one or more core regulatory element motifs. In some embodiments, the synthetic transcriptional regulatory sequence comprises one or more core regulatory element motifs and a minimal promoter. In some embodiments, the synthetic transcriptional regulatory sequence comprises one or more core regulatory element motifs, a minimal promoter, and a transcribable sequence. In some embodiments, the synthetic transcriptional regulatory sequence comprises two or more core regulatory element motifs. In some embodiments, the two or more core regulatory element motifs are different, e.g., include at least one core regulatory element motif that differs from another core regulatory element motif. In some embodiments, the two or more core regulatory element motifs are the same.

[0146] Provided herein, in some embodiments, is a synthetic transcriptional regulatory sequence, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

[0147] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 6, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 6, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells.

[0148] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 38; and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28 and comprises a nucleic acid sequence having at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0149] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 7, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 7, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells.

[0150] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 38; and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7.

[0151] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 8, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 8, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0152] In some embodiments, the synthetic transcriptional regulatory sequence comprises a plurality of a core regulatory element motif of BLIMP (PRDM1) comprising the nucleic acid sequence of SEQ ID NO: 111 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 111, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8.

[0153] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 9, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 9, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 9, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells.

[0154] In some embodiments, the synthetic transcriptional regulatory sequence comprises a plurality of a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 38, andcomprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

[0155] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 10, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 10, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells.

[0156] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 32; and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10.

[0157] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 11, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11, optionally wherein the synthetic transcriptional regulatory sequence is inducibly repressible in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 11, optionally wherein the synthetic transcriptional regulatory sequence is inducibly repressible in T cells.

[0158] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 12, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 12, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0159] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13, optionally wherein the synthetic transcriptional regulatory sequence is inducibly repressible in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 13, optionally wherein the synthetic transcriptional regulatory sequence is inducibly repressible in T cells.

[0160] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14, optionally wherein the synthetic transcriptional regulatory sequence is inducibly repressible in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 14, optionally wherein the synthetic transcriptional regulatory sequence is inducibly repressible in T cells.

[0161] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 15, optionally wherein the synthetic transcriptional regulatory sequence is inducibly active in T cells.

[0162] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 16, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 16, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0163] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 17, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0164] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 18, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 18.

[0165] In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 104 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 104, and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 107 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 107, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 18.

[0166] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 19, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 19.

[0167] In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 104 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 104, and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 108 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 108, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 19.

[0168] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 20, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequencecomprises the nucleic acid sequence of SEQ ID NO: 20, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0169] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of ZKSCAN comprising the nucleic acid sequence of SEQ ID NO: 112 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 112, and a core regulatory element motif of EFla comprising the nucleic acid sequence of SEQ ID NO: 113 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 113, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20.

[0170] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 21, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 21, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 21, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0171] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 22, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 22, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0172] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 23, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 23, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 23, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0173] In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 105 or a nucleic acid sequencehaving at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 105, and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 109 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 109, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 23.

[0174] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 24, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 24, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 24, optionally wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells.

[0175] In some embodiments, the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 106 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 106, and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 110 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 110, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 24.

[0176] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 101, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 101.

[0177] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 38; and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101.

[0178] In some embodiments, the synthetic transcriptional regulatory sequence comprises a core regulatory element motif. In some embodiments, the synthetic transcriptional regulatory sequence comprises two or more core regulatory element motifs that are different. In some embodiments, the synthetic transcriptional regulatory sequence comprises two of more core regulatory element motifs that are the same. In some embodiments, the core regulatory element motif, or each of the two or more core regulatory element motifs, comprises a sequence of a regulatory element motif of a transcription factor.

[0179] Also provided herein, in some embodiments, is a synthetic transcriptional regulatory sequence, comprising a first core regulatory element motif and a second core regulatory element motif, wherein the first core regulatory element motif and the second core regulatory element motif each independently comprise a core regulatory element motif selected from the group consisting of FOS, NFkB, and FLU; and wherein the first core regulatory element motif and a second core regulatory element motif are separated by a spacer sequence.

[0180] In some embodiments, the first core regulatory element motif and the second core regulatory element motif are different. In some embodiments, the first core regulatory element motif and the second core regulatory element motif each bind to a different transcription factor. In some embodiments, the first core regulatory element motif and the second core regulatory element motif comprise different sequences.

[0181] In some embodiments, the first core regulatory element motif is a core regulatory element motif of FOS, and the second core regulatory element motif is a core regulatory element motif of NFkB. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6; and a first core regulatory element motif of FOS.

[0182] In some embodiments, the first core regulatory element motif is a core regulatory element motif of NFkB, and the second core regulatory element motif is a core regulatory element motif of FEI1. In some embodiments, the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10; and a first core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 32.

[0183] In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 103, or a nucleic acid sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 103. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 103.

[0184] Also provided herein, in some embodiments, is a variant MND promoter, comprising one or more core regulatory element motifs inserted into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KEF10, KEF3, NFAT:AP1, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEB Pg, cMyc, AP1JRF4, TP53, STAT1, IRF9, KEF3, ETV6, FOXJ2, KEF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NF YA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCE6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0185] In some embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FEI1, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KEF10, KEF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1JRF4, and TP53. In some embodiments, the one or more core regulatory motifs independently comprise an activator core regulatory motif selected from the group consisting of NFkB, FEI1, ATF4, E2F6, EOMES, SRF, FOSE1, FOS, KEF10, KEF3, NFAT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53.

[0186] In some embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of STAT1, IRF9, KEF3, ETV6, FOXJ2, KEF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCE6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1. In some embodiments, the one or more core regulatory motifs independently comprise a repressor core regulatory motif selected from the group consisting of STAT1, IRF9, KEF3, ETV6, FOXJ2, KEF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCE6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0187] In some embodiments, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98, or anucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some embodiment, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some embodiments, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some embodiments, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98.

[0188] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 28, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 28.

[0189] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 30, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 30. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 31, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 31. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 32, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 33, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 34, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 35, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 35. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 36, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 37, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 37. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 38, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 38. In some embodiments,the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 39, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 39.

[0190] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 40, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 40. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 41, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 41. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 42, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 42. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 43, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 43. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 44, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 44. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 45, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 45. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 46, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 46. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 47, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 47. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 48, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 48. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 49, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 49.

[0191] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 50, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 50. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 51, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 51. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQID NO: 52, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 53, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 53. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 54, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 54. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 55, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 56, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 56. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 57, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 57. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 58, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 58. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 59, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 59.

[0192] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 60, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 60. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 61, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 61. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 62, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 62. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 63, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 63. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 64, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 64. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 65, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 65. In some embodiments, the one or more coreregulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 66, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 66. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 67, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 67. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 68, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 68. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 69, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 69.

[0193] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 70, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 70. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 71, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 71. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 72, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 72. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 73, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 73. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 74, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 74. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 75, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 75. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 76, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 76. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 77, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 77. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 78, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 78. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 79, or anucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 79.

[0194] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 80, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 80. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 81, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 81. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 82, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 82. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 83, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 83. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 84, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 84. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 85, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 85. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 86, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 87, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 87. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 88, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 88. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 89, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 89.

[0195] In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 90, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 90. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 91, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 91. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 92, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleicacid sequence of SEQ ID NO: 92. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 93, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 93. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 94, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 94. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 95, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 95. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 96, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 96. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 97, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 97. In some embodiments, the one or more core regulatory motifs comprises the nucleic acid sequence of SEQ ID NO: 98, or a nucleic acid sequence having at least 85%, 90%, or 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 98.

[0196] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91. In some embodiments, one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

[0197] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0198] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA and a core regulatory element motif of PATZE. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA and a core regulatory element motif of PATZE; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

[0199] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0200] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of FLU; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatory element motif of FLU; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT ; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

[0201] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of CEB Pg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1; and a core regulatory element motif of RUNX1. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motifof NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEB Pg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatory element motif of CEB Pg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1 ; and a core regulatory element motif of RUNX1 ; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEB Pg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16.

[0202] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52; and the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

[0203] In some embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5.

[0204] In some embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17. In some embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17.

[0205] In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the variant MND promoter comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

[0206] In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14. In some embodiments, the variant MND promoter comprises thenucleic acid sequence of SEQ ID NO: 15. In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 17.

[0207] Also provided herein, in some embodiments, is a synthetic transcriptional regulatory sequence, comprising two or more core regulatory element motifs, wherein each of the two or more core regulatory element motifs is the same and is a core regulatory element motif of PRDM1 or FOS; wherein each of the two or more core regulatory element motifs are separated from one another by a spacer sequence.

[0208] In some embodiments, the two or more core regulatory element motifs comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 core regulatory element motifs that are each the same. In some embodiments, the two or more core regulatory element motifs comprises at least 2 core regulatory element motifs that are each the same. In some embodiments, the two or more core regulatory element motifs comprises at least 4 core regulatory element motifs that are each the same. In some embodiments, the two or more core regulatory element motifs comprises 2 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 3 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 4 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 5 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 6 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 7 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 8 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 9 core regulatory element motifs that are the same. In some embodiments, the two or more core regulatory element motifs comprises 10 core regulatory element motifs that are the same.

[0209] In some embodiments, the first core regulatory element motif and / or the second core regulatory element motif is a core regulatory element motif of a transcription factor. In some embodiments, each of the two or more core regulatory element motifs is a regulatory element of a transcription factor. In some embodiments, the binding and / or activity of the transcription factor is modulated by T cell stimulation. In some embodiments, the first core regulatory element motif and / or the second core regulatory element motif are each independently a regulatory element of any of the following transcription factors: NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KEF10, KEF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4,IRF4, CEB Pg, cMyc, AP1JRF4, TP53, STAT1, IRF9, KEF3, ETV6, F0XJ2, KEF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NF YA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1. In some embodiments, each of the two or more core regulatory element motifs is a regulatory element of any of the following transcription factors: NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KEF10, KEF3, NFAT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEB Pg, cMyc, AP1JRF4, TP53, STAT1, IRF9, KEF3, ETV6, FOXJ2, KEF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCE6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0210] In some embodiments, the first core regulatory element motif and / or the second core regulatory element motif are independently selected from the group consisting of core regulatory element motifs of NFkB, FEU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KEF10, KEF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1JRF4, TP53, STAT1, IRF9, KEF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCE6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1. In some embodiments, the first core regulatory element motif and / or the second core regulatory element motif are activator motifs independently selected from the group consisting of core regulatory element motifs of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSE1, FOS, KEF10, KEF3, NFAT:AP1, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53. In some embodiments, the first core regulatory element motif and / or the second core regulatory element motif are repressor motifs independently selected from the group consisting of core regulatory element motifs of STAT1, IRF9, KEF3, ETV6, FOXJ2, KEF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCE6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0211] In some embodiments, each of the two or more core regulatory element motifs is selected from the group consisting of core regulatory element motifs of NFkB, FEU, ATF4, E2F6, EOMES, SRF, FOSE1, FOS, KEF10, KEF3, NFAT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5,SMAD3, BATF_JUNB_IRF4, IRF4, CEB Pg, cMyc, AP1JRF4, TP53, STAT1, IRF9, KLF3, ETV6, F0XJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1. In some embodiments, each of the two or more core regulatory element motifs is an activator motif selected from the group consisting of core regulatory element motifs of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT:AP1, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53. In some embodiments, each of the two or more core regulatory element motifs is a repressor motif selected from the group consisting of core regulatory element motifs of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0212] In some embodiments, each of the one or more core regulatory element motifs inserted into the parental MND promoter is independently selected from the group consisting of core regulatory element motifs of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT:AP1, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, TP53, STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACHL In some embodiments, the one or more core regulatory element motifs inserted into the parental MND promoter comprises one or more core regulatory element motifs that are activator motifs selected from the group consisting of core regulatory element motifs of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53. In some embodiments, the one or more core regulatory element motifs inserted into the parental MND promoter comprises one or more core regulatory element motifs that are repressor motifs selected from the group consisting of core regulatory element motifs of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACHL

[0213] In some of any of such embodiments, the synthetic transcriptional regulatory sequence comprises one or more core regulatory element motifs each independently comprising a nucleic acidsequence selected from the group consisting of SEQ ID NOs: 28-98 and 104-113, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98 and 104-113. In some of any of such embodiments, the synthetic transcriptional regulatory sequence comprises one or more core regulatory element motifs each independently comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98 and 104-113.

[0214] In some embodiments, a spacer sequence separates each of two or more core regulatory element motifs. In some embodiments, the spacer sequence has a length of at least 1 nucleotide and up to 20, 19, 18, 17, 16, 15, 14, 13, or 12 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 12 nucleotides, 1 to 10 nucleotides, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 4 to 12 nucleotides, 4 to 10 nucleotides, 4 to 9 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides, or 4 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 11 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 8 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 11 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 8 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 11 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 8 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 3 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 11 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 8 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 6 nucleotides.

[0215] In some embodiments, the spacer sequence has a length of 1 nucleotide. In some embodiments, the spacer sequence has a length of 2 nucleotides. In some embodiments, the spacer sequence has a length of 3 nucleotides. In some embodiments, the spacer sequence has a length of 4 nucleotides. In some embodiments, the spacer sequence has a length of 5 nucleotides. In some embodiments, the spacer sequence has a length of 6 nucleotides. In some embodiments, the spacer sequence has a length of 7 nucleotides. In some embodiments, the spacer sequence has a length of 8 nucleotides. In some embodiments, the spacer sequence has a length of 9 nucleotides. In some embodiments, the spacer sequence has a length of 10 nucleotides. In some embodiments, the spacer sequence has a length of 11 nucleotides. In some embodiments, the spacer sequence has a length of 12 nucleotides.

[0216] In some embodiments, the spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 99 or 100, or a nucleic acid sequence of or of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99 or 100. In some embodiments, the spacer sequence comprises a nucleic acid sequence of or of at least 4, 6, or 9 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99 or 100. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 4 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99 or 100. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 6 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99 or 100. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 9 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99 or 100. In some embodiments, the spacer sequence comprises a nucleotide of T, G, or A.

[0217] In some embodiments, the spacer sequence comprises a nucleic acid sequence of or of at least 4, 6, or 9 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 4 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 6 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 9 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the spacer sequence comprises a nucleic acid sequence of or of at least 4, 6, or 9 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 100. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 4 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 100. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 6 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 100. In some embodiments, the spacersequence comprises a nucleic acid sequence of 9 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 100.

[0218] In some embodiments, the spacer sequence comprises a nucleic acid sequence of 4 to 6 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the spacer sequence comprises a nucleic acid sequence of 4 to 6 consecutive nucleotides within the nucleic acid sequence of SEQ ID NO: 100.

[0219] In some embodiments, the spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 99 or 100. In some embodiments, the spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 99. In some embodiments, the spacer sequence comprises the nucleic acid sequence of SEQ ID NO: 100.

[0220] In some embodiments, the spacer sequences comprises the nucleic acid sequence of SEQ ID NO: 102.1. Exemplary Synthetic Transcriptional Regulatory Sequences, Variant MND Promoters, and Core Regulatory Element Motifs For Inducing Transcription

[0221] In some embodiments, transcription of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence is induced by T cell stimulation. In some embodiments, the synthetic transcriptional regulatory sequence is a variant MND promoter. In some embodiments, binding of a transcription factor to the synthetic transcriptional regulatory sequence is modulated by T cell stimulation such that the synthetic transcriptional regulatory element is inducibly active in T cells to control inducible activation of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent, thereby inducing increased transcription of the transcribable sequence. In some embodiments, it may be desirable to induce transcription of a transcribable sequence following T cell stimulation, such as when transcription of the transcribable sequence has the potential for inducing toxicity when constitutively expressed. Moreover, in some embodiments, it may be desirable to induce transcription of a potent agent, such as a potent cytokine, following T cell stimulation so that increased expression only occurs in the desired environment where the engineered immune cell is active, such as a tumor environment.

[0222] Accordingly, in some embodiments, provided herein is a synthetic transcriptional regulatory element that is inducibly active in T cells to control inducible activation of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent.

[0223] In some embodiments, the synthetic transcriptional regulatory element that is inducibly active in T cells comprises a core regulatory element motif of FOS and / or NFkB.

[0224] In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101.

[0225] In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 6. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 7. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 9. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 10. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 15. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 101.

[0226] In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises the nucleic acid of SEQ ID NO: 6. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises the nucleic acid of SEQ ID NO: 7. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises the nucleic acid of SEQ ID NO: 9. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly active in T cells comprises the nucleic acid of SEQ ID NO: 10. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid of SEQ ID NO: 15. In some embodiments, the synthetic transcriptional regulatory sequence comprises the nucleic acid of SEQ ID NO: 101.

[0227] In some embodiments, the synthetic transcriptional regulatory element that is inducibly active in T cells exhibits an increase of at least 20%, 25%, 30%, 35%, 40%, 45%, or 50% expression after activation of the T cells with the T cell stimulatory agent, e.g., as compared to without being activated with the T cell stimulatory agent. In some embodiments, the increase in expression is determined from RNA expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the increase in expression is determined from protein expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the increase in expression is measured by reverse transcriptase polymerase chain reaction (RT-PCR) and / or real time PCR (qPCR). In some embodiments, the increase in expression is determined using flow cytometry for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the increase in expression is determined based on a reporter assay for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the reporter assay detects a fluorescent protein; an enzyme, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT); or a light-emitting reporter gene.2. Exemplary Synthetic Trancriptional Regulatory Sequences, Variant MND Promoters, and Core Regulatory Element Motifs For Repressing Transcription

[0228] In some embodiments, transcription of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence is repressed by T cell stimulation. In some embodiments, the synthetic transcriptional regulatory sequence is a variant MND promoter. In some embodiments, the binding of a transcription factor to the synthetic transcriptional regulatory sequence is modulated by T cell stimulation such that the synthetic transcriptional regulatory element is inducibly repressible in T cells to control inducible repression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent, thereby repressing transcription of the transcribable sequence. In some embodiments, repressing or downregulating expression following T cell stimulation may be beneficial for, e.g., preventing T cell exhaustion.

[0229] Accordingly, in some embodiments, provided herein is a synthetic transcriptional regulatory element that is inducibly repressible in T cells to control inducible repression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent.

[0230] In some embodiments, the synthetic transcriptional regulatory element that is inducibly repressible in T cells comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14.

[0231] In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly repressible in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 11. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly repressible in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 13. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly repressible in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 14.

[0232] In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly repressible in T cells comprises the nucleic acid of SEQ ID NO: 11. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly repressible in T cells comprises the nucleic acid of SEQ ID NO: 13. In some embodiments, the synthetic transcriptional regulatory sequence that is inducibly repressible in T cells comprises the nucleic acid of SEQ ID NO: 14.

[0233] In some embodiments, the synthetic transcriptional regulatory element that is inducibly repressible in T cells exhibits a decrease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% expression after activation of the T cells with the T cell stimulatory agent, e.g., as compared to without being activated with the T cell stimulatory agent. In some embodiments, the decrease in expression is determined from RNA expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the decrease in expression is determined from protein expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the decrease in expression is measured by reverse transcriptase polymerase chain reaction (RT-PCR) and / or real time PCR (qPCR). In some embodiments, the decrease in expression is determined using flow cytometry for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the decrease in expression is determined based on a reporter assay for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the reporter assay detects a fluorescent protein; an enzyme, such as luciferase, the lacZ gene from E. coli,alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT); or a light-emitting reporter gene.3. Exemplary Synthetic Transcriptional Regulatory Sequences, Variant MND Promoters, and Core Regulatory Element Motifs For Constitutive Expression

[0234] In some embodiments, transcription of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence is constitutively active in T cells. In some embodiments, the synthetic transcriptional regulatory sequence is a variant MND promoter. In some embodiments, the synthetic transcriptional regulatory sequence is constitutively active regardless of whether or not there is activation of a T cell containing the transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence with a T cell stimulatory agent. In some embodiments, stable or constitutive expression of a transcribable sequence, such as a CAR or TCR, may allow for stable and predictable expression of the transcribable sequence, thereby allowing for maintaining a balance between efficacy and safety (low toxicity).

[0235] Accordingly, in some embodiments, provided herein is a synthetic transcriptional regulatory element that is constitutively active in T cells to control constitutive expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence.

[0236] In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of one or more of PRDM1, ZKSCAN1, NOP56, B2, PPP2R5C, and RN7SL2. In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of PRDM1. In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of ZKSCAN 1. In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of NOP56. In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of B2. In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of PPP2R5C. In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a core regulatory element motif of RN7SL2.

[0237] In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103.

[0238] In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 8. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 12. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 16. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 17. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 18. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 19. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 20. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 21. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 22. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 23. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutively active in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 24. In some embodiments, the synthetic transcriptional regulatory sequence that is constitutivelyactive in T cells comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid of SEQ ID NO: 103.

[0239] In some embodiments, the synthetic transcriptional regulatory element that is constitutively active in T cells exhibits a decrease of less than 10%, 9%, 8%, 7%, 6%, or 5% after activation of the T cells with the T cell stimulatory agent or an increase of less than 10%, 9%, 8%, 7%, 6%, or 5% after activation of the T cells with the T cell stimulatory agent, e.g., as compared to without being activated with the T cell stimulatory agent.

[0240] In some embodiments, the increase in expression is determined from RNA expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the increase in expression is determined from protein expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the increase in expression is measured by reverse transcriptase polymerase chain reaction (RT-PCR) and / or real time PCR (qPCR). In some embodiments, the increase in expression is determined using flow cytometry for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the increase in expression is determined based on a reporter assay for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the reporter assay detects a fluorescent protein; an enzyme, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT); or a light-emitting reporter gene.

[0241] In some embodiments, the decrease in expression is determined from RNA expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the decrease in expression is determined from protein expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the decrease in expression is measured by reverse transcriptase polymerase chain reaction (RT-PCR) and / or real time PCR (qPCR). In some embodiments, the decrease in expression is determined using flow cytometry for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the decrease in expression is determined based on a reporter assay for expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, the reporter assay detects a fluorescent protein; an enzyme, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT); or a light-emitting reporter gene.B. Minimal Promoters

[0242] In some embodiments, the synthetic transcriptional regulatory element comprises a minimal promoter or variant thereof. Minimal promoters suitable for inclusion in the synthetic transcriptional regulatory elements provided herein have sequences sufficient to specify a transcription start site, but that otherwise weakly activate transcription. For instance, suitable minimal promoters and variants thereof include those that do not recruit RNA polymerase or transcription factors strongly. In some embodiments, the one or more core regulatory element motifs and spacer sequences, such as any described above, is / are operably linked to the minimal promoter or variant thereof. In some embodiments, the one or more core regulatory element motifs and spacer sequences, such as any described above, is / are operably linked 5’ to the minimal promoter or variant thereof.

[0243] In some embodiments, the minimal promoter or variant thereof is a minimal promoter variant. In some embodiments, the minimal promoter variant is an SCP2 minimal promoter. In some embodiments, the minimal promoter or variant thereof is an EFla minimal promoter.C. Transcribable Sequences

[0244] In some embodiments, the synthetic transcriptional regulatory sequence is operably linked to a transcribable sequence to control or modulate expression of the transcribable sequence. In some embodiments, transcription of a transcribable sequence is inducible active by T cell stimulation when under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, transcription of a transcribable sequence is inducibly repressible by T cell stimulation when under the operable control of the synthetic transcriptional regulatory sequence. In some embodiments, transcription of a transcribable sequence is constitutively active when under the operable control of the synthetic transcriptional regulatory sequence.

[0245] In some embodiments, transcription of the transcribable sequence produces a coding RNA molecule. Exemplary coding RNA molecules are described in Section V-A-l and Section V-A-2. In some embodiments, the coding RNA encodes a transgene. In some embodiments, transcription of the transcribable sequence produces two or more coding RNA molecules each encoding a transgene. In some embodiments, the two or more coding RNA molecules encode the same transgene. In some embodiments, the two or more coding RNA molecules encode different transgenes.

[0246] In some embodiments, the transcribable sequence produces a coding RNA molecule encoding a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

[0247] In some embodiments, transcription of the transcribable sequence produces a non-coding RNA molecule. Exemplary non-coding RNA molecule are described in Section I- A throughC-1. In some embodiments, the non-coding RNA molecule is an siRNA. In some embodiments, the non-coding RNAmolecule is a miRNA. In some embodiments, the non-coding RNA molecule is an shRNA. In some embodiments, the non-coding RNA molecule is anti-sense RNA. In some embodiments, transcription of the transcribable sequence produces two or more non-coding RNA molecules. In some embodiments, the two or more non-coding RNA molecules include sequences complementary to different genes. In some embodiments, the two or more non-coding RNA molecules include sequences complementary to the same gene. In some embodiments, the two or more non-coding RNA molecules are identical.

[0248] In some embodiments, transcription of a transcribable sequence, when under the operable control of the synthetic transcriptional regulatory sequence, is induced by T cell stimulation. In other embodiments, transcription of a transcribable sequence, when under the operable control of the synthetic transcriptional regulatory sequence, is repressed by T cell stimulation. In some embodiments, transcription of a transcribable sequence, when under the operable control of the synthetic transcriptional regulatory sequence, is constitutively active with or without T cell stimulation.1. Exemplary Transcription Products For Induction by T Cell Stimulation

[0249] In some embodiments, transcription of the transcribable sequence is induced by T cell stimulation when under the operable control of the synthetic transcriptional regulatory sequence or variant MND promoter. In some embodiments, the synthetic transcriptional regulatory sequence or variant MND promoter includes any of those described in Section I-A-l. In some embodiments, he transcribable sequence can encode, or transcription of the transcribable sequence can produce, any molecule for which expression or upregulation of the molecule during T cell stimulation is desired, such as when transcription of the transcribable sequence has the potential for inducing toxicity when constitutively expressed and, thus, induction of expression following activation of the engineered immune cell would be advantageous for avoiding toxicity. In some embodiments, transcription of the transcribable sequence is transiently upregulated by T cell stimulation (e.g., is upregulated until T cell stimulation is terminated, or is upregulated until shortly after T cell stimulation). In some embodiments, transcription of the transcribable sequence exhibits sustained upregulation following T cell stimulation (i.e., is upregulated even after T cell stimulation is terminated).

[0250] In some embodiments, transcription of the transcribable sequence produces a coding RNA molecule. In some embodiments, the coding RNA molecule encodes a transgene. In some embodiments, the coding RNA molecule encode multiple transgenes.

[0251] In some embodiments, the coding RNA molecule encodes a fluorescent reporter protein (e.g., tdTomato). In some embodiments, the coding RNA molecule encodes a tumor-targeting molecule. In some embodiments, the coding RNA molecule encodes an anticancer agent. In some embodiments, the coding RNA molecule encodes an immune system-targeting molecule. In some embodiments, the coding RNA molecule encodes an oncolytic peptide. In some embodiments, the coding RNA molecule encodes aSMAC mimetic peptide. In some embodiments, the coding RNA molecule encodes an apoptosisinducing agent (e.g., TNF-related apoptosis-inducing ligand).

[0252] In some embodiments, the coding RNA molecule is an element that enhances therapeutic responses, such as therapeutic responses within a tumor microenviroment, when expressed on a CAR T cell. See, e.g., Tang L, et al. Arming CAR-T cells with cytokines and more: Innovations in the fourthgeneration CAR-T development. Mol Ther. 2023;31(l l):3146-3162. doi:10.1016 / j.ymthe.2023.09.021; Yeku 00, Brentjens RJ. Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy. Biochem Soc Trans. 2016 Apr 15;44(2):412-8. doi: 10.1042 / BST20150291; Hawkins ER, D'Souza RR, Klampatsa A. Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy. Biologies. 2021 Apr 14;15:95-105. doi: 10.2147 / BTT.S291768; and Carcopino C, Erdogan E, Henrich M, Kobold S. Armoring chimeric antigen receptor (CAR) T cells as micropharmacies for cancer therapy. Immunooncol Technol. 2024;24: 100739. Published 2024 Sep 25. doi:10.1016 / j.iotech.2024.100739, each of which is hereby incorporated in its entirety for all purposes. In some embodiments, the coding RNA molecule encodes a chimeric antigen receptor (CAR), IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-36, TNFa, FTL3L, IFNy, and / or CCL21.

[0253] In some embodiments, the coding RNA molecule encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR), such as any of those described in Section V-A-l. In some embodiments, the recombinant receptor is a T cell receptor (TCR), such as any of those described in Section V-A-2.

[0254] In some embodiments, the coding RNA molecule encodes a cytokine (e.g., IL-12) or variant thereof. In some embodiments, the cytokine or variant thereof is a pro-inflammatory cytokine. In some embodiments, the cytokine or variant thereof results in systematic toxicity at constant or high doses. For instance, IL- 12 is toxic when driven using promoters such as NF AT. Accordingly, use of synthetic transcriptional regulatory sequences described herein that allow inducible activation of otherwise systematically toxic transcriable sequences can increase the safety and efficacy of CAR T cells. See, e.g., Zhang E, et al. Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin- 12 for the immunotherapy of metastatic melanoma. Clin Cancer Res. 2015 May 15;21(10):2278-88. doi: 10.1158 / 1078-0432.CCR-14-2085, which is hereby incorporated in its entirety for all purposes. In some embodiments, the cytokine or variant thereof comprises IE-2, IL-7, IL-12, IL- 15, IL-18, IL-21, IL-23, IL-36, tumor necrosis factor alpha (TNFa), Fms-related tyrosine kinase 3 ligand (FET3EG), type II interferon (IFNy), and Chemokine (C-C motif) ligand 21 (CCE21). In someembodiments, the cytokine or variant thereof comprises IL-12. In some embodiments, the cytokine or variant thereof comprises the sequence set forth in SEQ ID NO: 114, or a sequence sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 114. In some embodiments, the cytokine or variant thereof comprises the sequence set forth in SEQ ID NO: 114. In some of these embodiments, transcription of the transcribable sequence is transiently upregulated by T cell stimulation.

[0255] In some embodiments, transcription of the transcribable sequence produces a non-coding RNA molecule. In some embodiments, the non-coding RNA molecule downregulates or knocks out expression of another molecule during T cell stimulation. In some embodiments, the non-coding RNA molecule contains a sequence complementary to a portion of a gene encoding the molecule to be downregulated or knocked out during T cell stimulation. Any molecule for which downregulation or knockout of the molecule during T cell stimulation is desired can be targeted. In some embodiments, the non-coding RNA molecule downregulates (e.g., transiently downregulates) a regulator of T cell exhaustion and / or effector function. In some embodiments, the non-coding RNA molecule downregulates (e.g., transiently downregulates) a tumor suppressor gene.

[0256] In some embodiments, the non-coding RNA molecule is an siRNA. In some embodiments, the non-coding RNA molecule is a miRNA. In some embodiments, the non-coding RNA molecule is an shRNA. In some embodiments, the non-coding RNA molecule is anti-sense RNA.2. Exemplary Transcription Products For Repression by T Cell Stimulation

[0257] In some embodiments, transcription of the transcribable sequence is repressed by T cell stimulation when under the operable control of the synthetic transcriptional regulatory sequence or variant MND promoter. In some embodiments, the synthetic transcriptional regulatory sequence or variant MND promoter includes any of those described in Section I-A-2. In some embodiments, the transcribable sequence can encode, or transcription of the transcribable sequence can produce, any molecule for which repression or downregulation of the molecule during T cell stimulation is desired. For example, repression may be desired such as to prevent T cell exhaustion or to control memory phenotype imprinting for better persistence of T cells. In another example, the inducible repression of may avoid knocking out a gene in a T cell, such as to allow context-dependent tuning to avoid the downsides of permanently knocking out certain genes. For example, the inducible repression of trafficking inhibitors (e.g., molecules inhibiting genes that assist with cell trafficking) can enhance CAR T cell migration to solid tumors without permanently knocking out these genes that assist with cell trafficking. In some embodiments, the transcribable sequence is transcribed and expressed prior to T cell stimulation. In some embodiments, transcription of the transcribable sequence is transiently downregulated by T cell stimulation (e.g., is downregulated until T cell stimulation is terminated, or is downregulated until shortlyafter T cell stimulation). In some embodiments, transcription of the transcribable sequence exhibits sustained downregulated following T cell stimulation (i.e., is downregulated even after T cell stimulation is terminated).

[0258] In some embodiments, transcription of the transcribable sequence produces a coding RNA molecule. In some embodiments, the coding RNA molecule encodes a transgene. In some embodiments, the coding RNA molecule encode multiple transgenes.

[0259] In some embodiments, the coding RNA molecule encodes a chimeric antigen receptor (CAR) or a chimeric cytokine receptor.

[0260] In some embodiments, the coding RNA molecule encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR), such as any of those described in Section V-A-l. In some embodiments, the recombinant receptor is a T cell receptor (TCR), such as any of those described in Section V-A-2.

[0261] In some embodiments, the CAR is expressed prior to T cell stimulation via transcription of the transcribable sequence, and CAR expression is downregulated following T cell stimulation. In some embodiments, the TCR is expressed prior to T cell stimulation via transcription of the transcribable sequence, and TCR expression is downregulated following T cell stimulation.

[0262] In some embodiments, transcription of the transcribable sequence produces a non-coding RNA molecule. In some embodiments, the non-coding RNA molecule downregulates or knocks out expression of another molecule during cell migration, such as CAR T cell migration to solid tumors. In some embodiments, the non-coding RNA molecule contains a sequence complementary to a portion of a gene encoding the molecule to be downregulated or knocked out during cell migration. Any molecule for which downregulation or knockout of the molecule during cell migration is desired can be targeted. In some embodiments, the non-coding RNA molecule downregulates (e.g., transiently downregulates) a regulator of cell trafficking.3. Exemplary Transcription Products for Constitutive Expression

[0263] In somee embodiments, transcription of the transcribable sequence is constitutively active in T cells when under the operable control of the synthetic transcriptional regulatory sequence or variant MND promoter. In some embodiments, the synthetic transcriptional regulatory sequence or variant MND promoter is constitutively active with or without T cell stimulation. In some embodiments, the synthetic transcriptional regulatory sequence or variant MND promoter that is constitutively active does not substantially increase or decrease its activity following T cell stimulation. In some embodiments, thesynthetic transcriptional regulatory sequence or variant MND promoter includes any of those described in Section I-A-3. In some embodiments, the transcribable sequence can encode, or transcription of the transcribable sequence can produce, any molecule for which constitutive (or stable) expression of the molecule before, during, and after T cell stimulation is desired, such as for stable and predictable expression of the transcribable sequence, thereby allowing for maintaining a balance between efficacy and safety (low toxicity). In some embodiments, the transcribable sequence is transcribed and expressed prior to T cell stimulation and exhibits sustained expression following T cell stimulation (i.e., is stabily expressed regardless of whether T cell stimulation occurs).

[0264] In some embodiments, the provided synthetic transcriptional regulatory sequences or variant MND promoters are constitutively active to different degrees to confer varying levels of expression of a transcribable sequence. Accordingly, a specific synthetic transcriptional regulatory sequence or variant MND promoter that is constitutively active may be selected to fine-tune the level of expression based on the desired expression level of a transcribable sequence (e.g., high or low expression of the transcribable sequence).

[0265] In some embodiments, transcription of the transcribable sequence produces a coding RNA molecule. In some embodiments, the coding RNA molecule encodes a transgene. In some embodiments, the coding RNA molecule encode multiple transgenes.

[0266] In some embodiments, the coding RNA molecule is an element that enhances therapeutic responses, such as therapeutic responses within a tumor microenviroment, when expressed on a CAR T cell. See, e.g., Tang L, et al. Arming CAR-T cells with cytokines and more: Innovations in the fourthgeneration CAR-T development. Mol Ther. 2023;31(l l):3146-3162. doi:10.1016 / j.ymthe.2023.09.021; Yeku 00, Brentjens RJ. Armored CAR T-cells: utilizing cytokines and pro-inflammatory ligands to enhance CAR T-cell anti-tumour efficacy. Biochem Soc Trans. 2016 Apr 15;44(2):412-8. doi: 10.1042 / BST20150291; Hawkins ER, D'Souza RR, Klampatsa A. Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy. Biologies. 2021 Apr 14;15:95-105. doi: 10.2147 / BTT.S291768; Carcopino C, Erdogan E, Henrich M, Kobold S. Armoring chimeric antigen receptor (CAR) T cells as micropharmacies for cancer therapy. Immunooncol Technol. 2024;24: 100739. Published 2024 Sep 25. doi:10.1016 / j.iotech.2024.100739; and Tri Minh Tran, et al. Armored TGFpRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFpi: Journal for ImmunoTherapy of Cancer 2024;12:e008261, each of which is hereby incorporated in its entirety for all purposes. In some embodiments, the coding RNA molecule encodes a chimeric antigen receptor (CAR), a decoy receptor, CD40L, 4-1BBL, a pro-inflammatory binder, a chemokine, or a safety switch. In some embodiments, the decoy receptor comprises TGFpRIIDN or a chimeric cytokine receptor. In some embodiments, the pro-inflammatory bindercomprises an anti-PDl scFv, a CD40R agonist antibody, a CD47 antibody, or a bispecific T cell engager. In some embodiments, the chemokine comprises CCL19 or CXCL10.

[0267] In some embodiments, the coding RNA molecule encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof. In some embodiments, the recombinant receptor is a chimeric antigen receptor (CAR), such as any of those described in Section V-A-l. In some embodiments, the recombinant receptor is a T cell receptor (TCR), such as any of those described in Section V-A-2.

[0268] In some embodiments, the CAR is expressed prior, during, and after T cell stimulation via transcription of the transcribable sequence. In some embodiments, the TCR is expressed prior to, during, and after T cell stimulation via transcription of the transcribable sequence.II. METHODS OF GENERATING SYNTHETIC TRANSCRIPTIONAL REGULATORY SEQUENCES AND VARIANT MND PROMOTERS

[0269] Provided herein are methods of generating synthetic transcriptional regulatory sequences and variant MND promoters. The methods described herein are useful for generating synthetic transcriptional regulatory sequences and variant MND promoters that can be screened for certain desirable characteristics, such as inducible activation of expression of a transcribable sequence upon T cell stimulation, inducible repression of a transcribable sequence upon T cell stimulation, or constitutive expression of a transcribable sequence regardless of T cell stimulation. For instance, in some embodiments, it may be desirable to induce transcription of a transcribable sequence following T cell stimulation, such as when transcription of the transcribable sequence has the potential for inducing toxicity when constitutively expressed, thereby limting induced expression to when it is aligned with stimulation / activation of the engineered immune cell. Alternatively, in some embodiments, it may be desirable to repress or downregulate transcription of a transcribable sequence following T cell stimulation, such as when repressing or downregulating such expression can prevent or delay T cell exhaustion. Further, in some embodiments, it may be desirable to maintain stable or constitutive transcription of a transcribable sequence, because it can allow for stable and predictable expression of the transcribable sequence, thereby allowing for maintaining a balance between efficacy and safety (low toxicity).

[0270] The following methods provide exemplary methods for generating synthetic transcriptional regulatory sequences and variant MND promoters that can be selected for any of these desirable characteristics.A. Variants of Transcription Factor Motif Designs

[0271] Provided herein, in some embodiments, are methods of generating synthetic transcriptional regulatory sequences based on variations of transcription factor motif designs. Without being bound by any theory, it is believed that spacing alters reporter strength by changing motif rotation, therefore affecting promoter signaling strength. In some embodiments, the motif designs are generated by combining two or more different core regulatory element motifs and altering the spacing between them via one or more spacer sequences. In some embodiments, the synthetic transcriptional regulatory sequences are generated by combining two or more of the same core regulatory element motif and altering space between them via one or more spacer sequences. In some embodiments, the synthetic transcriptional regulatory sequences are designed with any of the core regulatory element motifs described herein, along with a minimal promoter, an optimized minimal promoter (SCP2), or an EFla core promoter. In some embodiments, synthetic transcriptional regulatory sequences are designed in which motifs are first clustered and combined into pairs. In some embodiments, promoter sequences are designed with alternate spacing between the motifs.

[0272] Accordingly, provided herein is a method of generating a synthetic transcriptional regulatory sequence, the method comprising operably linking a first core regulatory element motif and a second core regulatory element motif via a spacer sequence, wherein the first core regulatory element motif and the second core regulatory element motif each independently comprise a core regulatory element motif selected from the group consisting of FOS, NFkB, and FLU.

[0273] In some embodiments, the first core regulatory element motif and the second core regulatory element motif are different. In some embodiments, the first core regulatory element motif is a core regulatory element motif of FOS, and the second core regulatory element motif is a core regulatory element motif of NFkB. In some embodiments, the first core regulatory element motif is a core regulatory element motif of NFkB, and the second core regulatory element motif is a core regulatory element motif of FLU. In some embodiments, the first core regulatory element motif is a core regulatory element motif of FOS, and the second core regulatory element motif is a core regulatory element motif of FLU.

[0274] Also provided herein is a method of generating a synthetic transcriptional regulatory sequence, the method comprising operably linking two or more core regulatory element motifs from one another via a spacer sequence, wherein each of the two or more core regulatory element motifs is the same and is a core regulatory element motif of PRDM1 or FOS. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of PRDM1. In some embodiments, each of the two or more core regulatory element motifs is a core regulatory element motif of FOS. In some embodiments, the spacer sequence has a length of at least 1 nucleotide and up to 20, 19,18, 17, 16, 15, 14, 13, or 12 nucleotides. In some embodiments, the spacer sequence has a length of up to 20 nucleotides. In some embodiments, the spacer sequence has a length of up to 19 nucleotides. In some embodiments, the spacer sequence has a length of up to 18 nucleotides. In some embodiments, the spacer sequence has a length of up to 17 nucleotides. In some embodiments, the spacer sequence has a length of up to 16 nucleotides. In some embodiments, the spacer sequence has a length of up to 15 nucleotides. In some embodiments, the spacer sequence has a length of up to 14 nucleotides. In some embodiments, the spacer sequence has a length of up to 13 nucleotides. In some embodiments, the spacer sequence has a length of up to 12 nucleotides.

[0275] In some embodiments, the spacer sequence has a length of 1 to 12 nucleotides, 1 to 10 nucleotides, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 4 to 12 nucleotides, 4 to 10 nucleotides, 4 to 9 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides, or 4 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 8 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 1 to 5 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 2 to 5 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 12 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 10 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 9 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 8 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 7 nucleotides. In some embodiments, the spacer sequence has a length of 4 to 6 nucleotides. In some embodiments, the spacer sequence has a length of 1 nucleotide. In some embodiments, the spacer sequence has a length of 2 nucleotides. In some embodiments, the spacer sequence has a length of 3 nucleotides. In some embodiments, the spacer sequence has a length of 4 nucleotides. In some embodiments, the spacer sequence has a length of 5 nucleotides. In some embodiments, the spacer sequence has a length of 6 nucleotides. In some embodiments, the spacer sequence has a length of 7 nucleotides. In some embodiments, the spacer sequence has a length of 8 nucleotides. In some embodiments, the spacer sequence has a length of 9 nucleotides. In some embodiments, the spacersequence has a length of 10 nucleotides. In some embodiments, the spacer sequence has a length of 11 nucleotides. In some embodiments, the spacer sequence has a length of 12 nucleotides.

[0276] In some embodiments, the method further comprises operably linking a minimal promoter or variant thereof to the first core regulatory element motif and / or the second core regulatory element motif. In some embodiments, the method further comprises operably linking a minimal promoter or variant thereof to at least one of the two or more core regulatory element motifs. In some embodiments, the minimal promoter or variant thereof is a minimal promoter variant. In some embodiments, the minimal promoter variant is an SCP2 minimal promoter. In some embodiments, the minimal promoter or variant thereof is an EFla (EFla) minimal promoter.

[0277] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-10, 20, and 101, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-10, 20, and 101. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-10, 20, and 101.

[0278] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 6, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 6.

[0279] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 7, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 7.

[0280] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 8, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 8.

[0281] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 9, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 9. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 9.

[0282] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 10, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 10.

[0283] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 20, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 20.

[0284] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 101, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 101.

[0285] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method is not naturally occurring.B. Variants of MND Promoter

[0286] Also provided herein are methods of generating variant MND promoters, which can be used to control expression of a transcribable sequence. In some embodiments, the variant MND promoters are generated by inserting or introducing one or more core regulatory element motifs into a parental MND promoter. Without being bound by any theory, it is believed that varying key characteristics of an existing MND promoter (e.g., a particular core regulatory element motif postitioning, density, and / or spacing) may improve expression of a transcribable sequence.

[0287] Accordingly, provided herein, in some embodiments, is a method of generating a variant MND promoter, the method comprising inserting one or more core regulatory element motifs into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, TP53, STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

[0288] In some embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53. In some embodiments, the one or more core regulatory motifs independently comprise an activator core regulatory motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NF AT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NF AT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53.

[0289] In some embodiments, one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACHL In some embodiments, the one or more core regulatory motifs independently comprise a repressor core regulatory motif selected from the group consisting of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACHL

[0290] In some embodiments, each of the one or more core regulatory motifs independently comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98. In some embodiments, each of the one or more core regulatory motifsindependently comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28- 98. In some embodiments, the one or more core regulatory motifs are any of the core regulatory motifs described herein, e.g., in Section I or II.

[0291] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91. In some embodiments, one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

[0292] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0293] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA and a core regulatory element motif of PATZE. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA and a core regulatory element motif of PATZE; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

[0294] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of FLU; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatory element motif of FLU; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT ; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

[0295] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB; a core regulatory element motif of CEB Pg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1; and a core regulatory element motif of RUNX1. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEB Pg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatory element motif of CEB Pg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1 ; and a core regulatory element motif of RUNX1 ; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEB Pg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a coreregulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16.

[0296] In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17. In some embodiments, the one or more core regulatory element motifs comprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52; and the variant MND promoter generated by this method comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

[0297] In some embodiments, promoter sequences were generated by random insertion of endogenous transcription factor motifs. In some embodiments, the parental MND promoter (SEQ ID NO: 5) is altered by insertion of endogenous transcription sites and motifs. In some embodiments, the transcription factor motifs were selected from ATAC-seq data, regulatory activity assessments via T-cell reporter assays, and literature.

[0298] In some embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5. In some embodiments, the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5.

[0299] In some embodiments, the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17. In someembodiments, the variant MND promoter that is generated by this method comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17.

[0300] In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 11, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:11. In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 11.

[0301] In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 12, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:12. In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 12.

[0302] In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:13. In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 13.

[0303] In some embodiments, the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO:14. In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 14.

[0304] In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 15.

[0305] In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 16, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16. In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 16.

[0306] In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17. 1 In some embodiments, the variant MND promoter that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 17.

[0307] In some embodiments, the variant MND promoter that is generated by this method is not naturally occurring.C. Endogenous Enhancer-Promoter Combinations

[0308] Also provided herein are methods of generating synthetic transcriptional regulatory sequences by linking an enhancer sequence with a promoter sequence, which can be used to control expression of a transcribable sequence.

[0309] Accordingly, provided herein, in some embodiments, is a method of generating a synthetic transcriptional regulatory sequence, comprising operably linking an enhancer sequence and a promoter sequence, wherein: (a) the the enhancer sequence is identified by: i) identifying, using gene expression levels, highly expressed genes from an immune cell engineered to express a recombinant receptor; (ii) identifying open chromatin regions of the genome in proximity to the highly expressed genes; (iii) excluding transcription start site (TSS) sequences from the identified open chromatin regions, thereby identifying one or more enhancer sequences comprising the enhancer sequence; and (b) the promoter sequence is identified by: (i) identifying, using gene expression levels, highly expressed genes from an immune cell engineered to express a recombinant receptor; (ii) identifying open chromatin regions of the genome in proximity to the highly expressed genes; and (iii) identifying the identified open chromatin regions having known core promoter element configurations, thereby identifying one or more promoter sequences comprising the promoter sequence.

[0310] In some embodiments, synthetic transcriptional regulatory sequences are generated from endogenous enhancer-promoter combinations. Without being bound by any theory, it is believed that combining enhancers and promoters into novel regulatory units may improve expression of transcribable sequences. In some embodiments, the candidate enhancers and promoters are selected from RNA-seq data. In some embodiments, candidate enhancers are intersected with annotated CD4 and CD8 enhancers using software. In some embodiments, the candidate enhancers and promoters are selected from expression profile clusters. In some embodiments, enhancer / promoter pairs are generated using different combinations of the selected enhancers and promoters.

[0311] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 22-24, and 103 or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs: 18, 19, 22-24, and 103. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises a nucleic acid sequence having at least 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 22-24, and 103.

[0312] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 22-24, and 103. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 19. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 22. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 23. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 24. In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method comprises the nucleic acid sequence of SEQ ID NO: 103.

[0313] In some embodiments, the synthetic transcriptional regulatory sequence that is generated by this method by this method is not naturally occurring.III. METHODS OF IDENTIFYING SYNTHETIC TRANSCRIPTIONAL REGULATORY SEQUENCES

[0314] Also provided herein, in some embodiments, is a method of identifying synthetic transcriptional regulatory sequences having activity in T cells, the method comprising: (a) providing a library comprising a plurality of synthetic transcriptional regulatory sequences generated by any of the methods described herein, e.g., in Sections II-A and II-C, operably linked to a transcribable sequence; (b) screening for expression of the transcribable sequence; and (c) identifying a synthetic transcriptional regulatory sequence of the plurality of synthetic transcriptional regulatory sequences having activity in a T cell.

[0315] In some embodiments, the synthetic transcriptional regulatory sequence having activity in the T cell has constitutive activity in the T cell. In some embodiments, the constitutive activity in a T cell is based on the transcribable sequence exhibiting a decrease of less than 10%, 9%, 8%, 7%, 6%, or 5% after activation of the T cell with a T cell stimulatory agent or an increase of less than 10%, 9%, 8%, 7%, 6%, or 5% after activation of the T cell with a T cell stimulatory agent, e.g., as compared to without being activated with the T cell stimulatory agent.

[0316] In some embodiments, the synthetic transcriptional regulatory sequence having activity in the T cell has inducible activity in the T cell. In some embodiments, the inducible activity in a T cell is based on the transcribable sequence exhibiting an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% expression after activation of the T cell with a T cell stimulatory agent, e.g., as compared to without being activated with a T cell stimulatory agent.

[0317] In some embodiments, the synthetic transcriptional regulatory sequence having activity in the T cell has inducible repressibility in the T cell. In some embodiments, the inducible repressibility in a T cell is based on the transcribable sequence exhibiting a decrease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% expression after activation of the T cells with a T cell stimulatory agent, e.g., as compared to without being activated with a T cell stimulatory agent.

[0318] Also provided herein, in some embodiments, is a method of identifying a variant MND promoter having activity in T cells, the method comprising: (a) providing a library comprising a plurality of variant MND promoters generated by any of the methods described herein, e.g., in Section II-B, operably linked to a transcribable sequence; (b) screening for expression of the transcribable sequence; and (c) identifying a variant MND promoter of the plurality of variant MND promoters having activity in a T cell.

[0319] In some embodiments, the variant MND promoter having activity in the T cell has constitutive activity in the T cell. In some embodiments, the constitutive activity in a T cell is based on the transcribable sequence exhibiting a decrease of less than 10%, 9%, 8%, 7%, 6%, or 5% after activation of the T cell with a T cell stimulatory agent or an increase of less than 10%, 9%, 8%, 7%, 6%, or 5% after activation of the T cell with a T cell stimulatory agent, e.g., as compared to without being activated with the T cell stimulatory agent.

[0320] In some embodiments, the variant MND promoter having activity in the T cell has inducible activity in the T cell. In some embodiments, the inducible activity in a T cell is based on the transcribable sequence exhibiting an increase of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% expression after activation of the T cell with a T cell stimulatory agent, e.g., as compared to without being activated with a T cell stimulatory agent.

[0321] In some embodiments, the variant MND promoter having activity in the T cell has inducible repressibility in the T cell. In some embodiments, the inducible repressibility in a T cell is based on the transcribable sequence exhibiting a decrease of at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% expression after activation of the T cells with a T cell stimulatory agent, e.g., as compared to without being activated with a T cell stimulatory agent.

[0322] In some embodiments, the transcribable sequence is a reporter sequence. In some embodiments, the reporter sequence encodes a fluorescent protein protein, such as green fluorescentprotein (GFP), enhanced green fluorescent protein (EGFP), such as super-fold GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), blue green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), and yellow fluorescent protein (YFP), and variants thereof, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of the fluorescent proteins. In some embodiments, the reporter sequence encodes an enzyme, such as luciferase, the lacZ gene from E. coli, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), or chloramphenicol acetyltransferase (CAT). In some embodiments, the reporter sequence encodes a light-emitting reporter gene. In some embodiments, the light-emitting reporter gene is luciferase, P-galactosidase, chloramphenicol acetyltransferase (CAT), P-glucuronidase (GUS), or variants thereof.IV. POLYNUCLEOTIDES, VECTORS, AND LIPID PARTICLES

[0323] Also provided herein in some embodiments are polynucleotides that include any of the synthetic transcriptional regulatory sequences provided herein (e.g., any as described in Section I, or any generated by methods as described in Section II, or any identified by the methods described in Section III) as well as a transcribable sequence (e.g., any as described in Section I-C). In some embodiments, nucleotides of the synthetic transcriptional regulatory sequenceand transcribable sequence are operably linked such that transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

[0324] Also provided herein is a polynucleotide comprising any synthetic transcriptional regulatory sequence described herein and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

[0325] Also provided herein is a polynucleotide comprising any variant MND promoter described herein and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the variant MND promoter.

[0326] In some embodiments, transcription of the transcribable sequence produces a coding RNA molecule. In some embodiments, the coding RNA molecule encodes a transgene. In some embodiments, the coding RNA molecule encodes an engineered recombinant molecule. In some embodiments, the engineered recombinant molecule is a chimeric antigen receptor (CAR). In some embodiments, the engineered recombinant molecule is a T cell receptor (TCR). Exemplary CARs and TCRs are described in Section V-A.

[0327] In some embodimets, transcription of the transcribable sequence produces a non-coding RNA molecule. In some embodiments, the non-coding RNA molecule is an siRNA. In someembodiments, the non-coding RNA molecule is a miRNA. In some embodiments, the non-coding RNA molecule is an shRNA.

[0328] Also provided herein in some embodiments are vectors that include any of the synthetic transcriptional regulatory sequences provided herein (e.g., any as described in Section I, or any generated by methods as described in Section II, or any identified by the methods described in Section III). Also provided herein in some embodiments are vectors that include any of the synthetic transcriptional regulatory sequences provided herein (e.g., any as described in Section I, or any generated by methods as described in Section II, or any identified by the methods described in Section III) as well as a transcribable sequence (e.g., any as described in Section I-C). Also provided herein in some embodiments are vectors that include any of the polynucleotides provided herein (e.g., any as described in Section IV). Also provided herein is a vector comprising any synthetic transcriptional regulatory sequence described herein or any variant MND promoter described herein. Also provided herein is a vector comprising any synthetic transcriptional regulatory sequence described herein. Also provided herein is a vector comprising any variant MND promoter described herein.

[0329] In some embodiments, the vector is derived from simian virus 40 (SV40), adenoviruses, or adeno-associated virus (AAV). In some embodiments, the vector is an AAV vector. In some embodiments, the vector is a retroviral vector, such as a lentiviral vector or a gamma-retroviral vector (see, e.g., Koste et al. (2014) Gene Therapy 2014 Apr 3. doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. 2011 November 29(11): 550-557).

[0330] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), e.g., a retroviral vector derived from the Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), or spleen focus forming virus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. The retroviruses typically are amphotropic, meaning that they are capable of infecting host cells of several species, including humans. In one embodiment, the transcribable sequence replaces the retroviral gag, pol and / or env sequences. A number of illustrative retroviral systems have been described (e.g., U.S. Pat. Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, A. D. (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109.

[0331] The viral vector genome is typically constructed in a plasmid form that can be transfected into a packaging or producer cell line. In any of such examples, the transcribable sequence is inserted orlocated in a region of the viral vector, such as generally in a non-essential region of the viral genome. In some embodiments, the transcribable sequence is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication defective.

[0332] Any of a variety of known methods can be used to produce retroviral particles whose genome contains an RNA copy of the viral vector genome. In some embodiments, at least two components are involved in making a virus-based gene delivery system: first, packaging plasmids, encompassing the structural proteins as well as the enzymes necessary to generate a viral vector particle, and second, the viral vector itself, i.e., the genetic material to be transferred. Biosafety safeguards can be introduced in the design of one or both of these components.

[0333] In some embodiments, the packaging plasmid can contain all retroviral, such as HIV-1, proteins other than envelope proteins (Naldini et al., 1998). In other embodiments, viral vectors can lack additional viral genes, such as those that are associated with virulence, e.g. vpr, vif, vpu and nef, and / or Tat, a primary transactivator of HIV. In some embodiments, lentiviral vectors, such as HIV-based lentiviral vectors, comprise only three genes of the parental virus: gag, pol and rev, which reduces or eliminates the possibility of reconstitution of a wild-type virus through recombination.

[0334] In some embodiments, the viral vector genome is introduced into a packaging cell line that contains all the components necessary to package viral genomic RNA, transcribed from the viral vector genome, into viral particles. Alternatively, the viral vector genome may comprise one or more genes encoding viral components in addition to the one or more sequences, e.g., transcribable sequences, of interest. In some aspects, in order to prevent replication of the genome in the target cell, however, endogenous viral genes required for replication are removed and provided separately in the packaging cell line.

[0335] In some embodiments, a packaging cell line is transfected with one or more plasmid vectors containing the components necessary to generate the particles. In some embodiments, a packaging cell line is transfected with a plasmid containing the viral vector genome, including the LTRs, the cis-acting packaging sequence and the transcribable sequence; and one or more helper plasmids encoding the virus enzymatic and / or structural components, such as Gag, pol and / or rev. In some embodiments, multiple vectors are utilized to separate the various genetic components that generate the retroviral vector particles. In some such embodiments, providing separate vectors to the packaging cell reduces the chance of recombination events that might otherwise generate replication competent viruses. In some embodiments, a single plasmid vector having all of the retroviral components can be used.

[0336] In some embodiments, the retroviral vector particle, such as lentiviral vector particle, is pseudotyped to increase the transduction efficiency of host cells. For example, a retroviral vector particle, such as a lentiviral vector particle, in some embodiments is pseudotyped with a VSV-Gglycoprotein, which provides a broad cell host range extending the cell types that can be transduced. In some embodiments, a packaging cell line is transfected with a plasmid or polynucleotide encoding a nonnative envelope glycoprotein, such as to include xenotropic, polytropic or amphotropic envelopes, such as Sindbis virus envelope, GALV or VSV-G.

[0337] In some embodiments, the packaging cell line provides the components, including viral regulatory and structural proteins, that are required in trans for the packaging of the viral genomic RNA into lenti viral vector particles. In some embodiments, the packaging cell line may be any cell line that is capable of expressing lentiviral proteins and producing functional lentiviral vector particles. In some aspects, suitable packaging cell lines include 293 (ATCC CCL X), 293T, HeLA (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430) cells.

[0338] In some embodiments, the packaging cell line stably expresses the viral protein(s). For example, in some aspects, a packaging cell line containing the gag, pol, rev and / or other structural genes but without the LTR and packaging components can be constructed. In some embodiments, a packaging cell line can be transiently transfected with nucleic acid molecules encoding one or more viral proteins along with the viral vector genome containing a nucleic acid molecule encoding a heterologous protein, and / or a nucleic acid encoding an envelope glycoprotein.

[0339] In some embodiments, the viral vectors and the packaging and / or helper plasmids are introduced via transfection or infection into the packaging cell line. The packaging cell line produces viral vector particles that contain the viral vector genome. Methods for transfection or infection are well known. Non-limiting examples include calcium phosphate, DEAE-dextran and lipofection methods, electroporation and microinjection.

[0340] When a recombinant plasmid and the retroviral LTR and packaging sequences are introduced into a special cell line (e.g., by calcium phosphate precipitation for example), the packaging sequences may permit the RNA transcript of the recombinant plasmid to be packaged into viral particles, which then may be secreted into the culture media. The media containing the recombinant retroviruses in some embodiments is then collected, optionally concentrated, and used for gene transfer. For example, in some aspects, after cotransfection of the packaging plasmids and the transfer vector to the packaging cell line, the viral vector particles are recovered from the culture media and titered by standard methods used by those of skill in the art.

[0341] In some embodiments, a retroviral vector, such as a lentiviral vector, can be produced in a packaging cell line, such as an exemplary HEK 293T cell line, by introduction of plasmids to allow generation of lentiviral particles. In some embodiments, a packaging cell is transfected and / or contains a polynucleotide encoding gag and pol, and a polynucleotide encoding a recombinant receptor, such as anantigen receptor, for example, a CAR. In some embodiments, the packaging cell line is optionally and / or additionally transfected with and / or contains a polynucleotide encoding a rev protein. In some embodiments, the packaging cell line is optionally and / or additionally transfected with and / or contains a polynucleotide encoding a non-native envelope glycoprotein, such as VSV-G. In some such embodiments, approximately two days after transfection of cells, e.g. HEK 293T cells, the cell supernatant contains recombinant lenti viral vectors, which can be recovered and titered.

[0342] In some embodiments, any synthetic transcriptional regulatory sequence, any variant MND promoter, or any polynucleotide described herein can be encompassed within a nanoparticle, such as a lipid particle, for delivery. In some embodiments, an exemplary delivery vehicle is a nanoparticle, e.g., a liposome or other suitable sub-micron sized delivery system.

[0343] Accordingly, also provided herein is a lipid particle comprising any synthetic transcriptional regulatory sequence, any variant MND promoter, or any polynucleotide described herein.

[0344] In some embodiments, the use of lipid formulations is contemplated for the introduction of the nucleic acids into a cell. The lipid particle may be a nucleic acid-lipid particle, which may be formed from a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle. The nucleic acid may be encapsulated in the lipid portion of the particle, thereby protecting it from enzymatic degradation. A stable nucleic acid-lipid particle can be a particle made from lipids (e.g., a cationic lipid, a non-cationic lipid, and optionally a conjugated lipid that prevents aggregation of the particle), wherein the nucleic acid is fully encapsulated within the lipid.

[0345] In some embodiments, the lipid particles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the lipid particles are substantially non-toxic. In some embodiments, nucleic acids, when present in the lipid particles of the present disclosure, can be resistant in aqueous solution to degradation with a nuclease.

[0346] In some embodiments, a lipid particle provides a nucleic acid with full encapsulation, partial encapsulation, or both. In some embodiments, the nucleic acid is fully encapsulated in the lipid particle to form a nucleic acid-lipid particle.

[0347] In some embodiments, a conjugated lipid inhibits aggregation of lipid particles, including, polyethylene glycol (PEG)-lipid conjugates such as, e.g., PEG coupled to dialkyloxypropyls e.g., PEG- DAA conjugates), PEG coupled to diacylglycerols (e.g., PEG-DAG conjugates), PEG coupled tocholesterol, PEG coupled to phosphatidylethanolamines, and PEG conjugated to ceramides, cationic PEG lipids, polyoxazoline (POZ)-lipid conjugates (e.g., POZ-DAA conjugates; polyamide oligomers (e.g., ATTA-lipid conjugates), and mixtures thereof. In some embodiments, PEG or POZ can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG or the POZ to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In some embodiments, non-ester containing linker moieties, such as amides or carbamates, are used.

[0348] In some embodiments, an amphipathic lipid can have a hydrophobic portion that orients into a hydrophobic phase, and a hydrophilic portion orients toward the aqueous phase. In some embodiments, hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. In some embodiments, hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0349] Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and (3-acyloxyacids, are also within the group designated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

[0350] In some embodiments, a neutral lipid exists either in an uncharged or neutral zwitterionic form at a selected pH. In some embodiments, at physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

[0351] In some embodiments, a non-cationic lipid may be any amphipathic lipid as well as any other neutral lipid or anionic lipid.

[0352] In some embodiments, an anionic lipid is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols,palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

[0353] In some embodiments, a hydrophobic lipid has apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups optionally substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Suitable examples include, but are not limited to, diacylglycerol, dialkylglycerol, N — N-dialkylamino, l,2-diacyloxy-3- aminopropane, and l,2-dialkyl-3-aminopropane.

[0354] In some embodiments, the lipid particle comprises (i) any of the synthetic transcriptional regulatory sequences described herein, any of the variant MND promoters described herein, or any of the polynucleotides described herein, (ii) a cationic lipid, and (iii) a non-cationic lipid. In some embodiments, the lipid particle further comprises a conjugated lipid that prevents aggregation of the particle.

[0355] In some embodiments, the nucleic acid-lipid particle comprises: (a) a nucleic acid (e.g., any of the synthetic transcriptional regulatory sequences described herein, any of the variant MND promoters described herein, or any of the polynucleotides described herein); (b) a cationic lipid comprising from about 50 mol % to about 65 mol % of the total lipid present in the particle; (c) a non-cationic lipid comprising from about 25 mol % to about 45 mol % of the total lipid present in the particle; and (d) a conjugated lipid that inhibits aggregation of particles comprising from about 5 mol % to about 10 mol % of the total lipid present in the particle.

[0356] In some embodiments, the lipid particle comprises: (a) a nucleic acid (e.g., any of the synthetic transcriptional regulatory sequences described herein, any of the variant MND promoters described herein, or any of the polynucleotides described herein); (b) a cationic lipid; (c) a mixture of a phospholipid and cholesterol or a derivative thereof; and (d) a PEG-lipid conjugate.V. ENGINEERED IMMUNE CELLS AND METHODS FOR PRODUCING SAME

[0357] Also provided herein in some embodiments are engineered immune cells, e.g., engineered T cells, that contain any of the synthetic transcriptional regulatory sequences, variant MND promoters, polynucleotides, or vectors described herein. In some embodiments, provided herein is an engineered T cell comprising any of the synthetic transcriptional regulatory sequences, variant MND promoters, polynucleotides, or vectors described herein. In some embodiments, the engineered T cell expresses a chimeric antigen receptor (CAR). In some embodiments, the engineered T cell expresses an engineered T cell receptor (TCR). In some embodiments, provided herein is an engineered T cell that expresses a CAR under the operable control of any synthetic transcriptional regulatory sequences or variant MND promoter described herein. In some embodiments, provided herein is an engineered T cell that expressesa TCR under the operable control of any synthetic transcriptional regulatory sequences or variant MND promoter described herein.

[0358] Also provided herein in some embodiments are methods of producing an engineered immune cell, e.g., an engineered T cell, said methods comprising introducing into an immune cell any synthetic transcriptional regulatory sequence described herein, any variant MND promoter described herein, any polynucleotide described herein, or any vector described herein.

[0359] In some embodiments, the engineered immune cell is an engineered lymphocyte. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is an engineered T cell.

[0360] Accordingly, also provided herein is a method of producing an engineered T cell, comprising introducing into a T cell any synthetic transcriptional regulatory sequence described herein, any variant MND promoter described herein, any polynucleotide described herein, or any vector described herein.

[0361] In some embodiments, the T cell expresses a chimeric antigen receptor (CAR). In some embodiments, the T cell has been engineered to express a CAR. In some embodiments, the T cell expresses a CAR under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein. In some embodiments, the T cell expresses a T cell receptor (TCR). In some embodiments, the T cell expresses a recombinant TCR. In some embodiments, the T cell expresses a TCR under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein.

[0362] In some embodiments, the T cell has been engineered to express a CAR. In some embodiments, the T cell has been engineered to express a CAR under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein. In some embodiments, the T cell has been engineered to express a TCR. In some embodiments, the T cell has been engineered to express a TCR under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein. In some embodiments, the methods further include introducing into the T cell a polyucleotide encoding a CAR. In some embodiments, the methods further include introducing into the T cell a polyucleotide encoding a TCR.

[0363] Also provided herein is an engineered immune cell, e.g., T cell, produced by any of the methods described herein.A. Recombinant Receptors

[0364] In some embodiments, the immune cells, e.g., T cells, express recombinant receptors, such as antigen receptors including functional non-TCR antigen receptors, e.g., chimeric antigen receptors (CARs), and other antigen-binding receptors such as transgenic or engineered T cell receptors (TCRs)under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein. In some embodiments, a synthetic transcriptional regulatory sequence is operably linked to a nucleic acid sequence encoding a recombinant receptor, e.g., CAR or TCR, to control or modulate expression of the recombinant receptor, e.g., CAR or TCR. In some embodiments, the engineered immune cell expresses a recombinant receptor, and wherein binding to the recombinant receptor induces an IT AM-mediated signal.1. Chimeric Antigen Receptors (CARs)

[0365] In some embodiments, engineered immune cells, such as T cells, employed in the provided embodiments express a CAR with specificity for a particular antigen (or marker or ligand), such as an antigen expressed on the surface of a particular cell type. In some embodiments, the engineered immune cells, e.g., T cells, express the CAR under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein. Accordingly, in some embodiments, a nucleic acid sequence encoding the CAR is under the operable control of any synthetic transcriptional regulatory sequence or variant MND promoter described herein. In some embodiments, the antigen is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of a disease or condition, e.g., a tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0366] In particular embodiments, the CAR comprises an intracellular signaling region which comprises a cytoplasmic signaling domain or region (also interchangeably called an intracellular signaling domain or region), such as a cytoplasmic (intracellular) region capable of inducing a primary activation signal in a T cell, for example, a cytoplasmic signaling domain or region of a T cell receptor (TCR) component (e.g., a cytoplasmic signaling domain or region of a zeta chain of a CD3-zeta (CD3Q chain or a functional variant or signaling portion thereof) and / or that comprises an immunoreceptor tyrosine-based activation motif (IT AM).

[0367] Accordingly, in some embodiments, the recombinant receptor is a CAR comprising an extracellular antigen-binding domain and an intracellular signaling domain comprising an IT AM signaling domain. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain of a CD3 chain. In some embodiments, the CD3 chain is a CD3-zeta chain. In some embodiments, inding to the recombinant receptor induces a costimulatory signal.

[0368] In some embodiments, the chimeric antigen receptor further comprises an extracellular ligand-binding domain that specifically binds to a ligand (e.g., antigen). In some embodiments, the chimeric receptor is a CAR that comprises an extracellular antigen-recognition domain that specificallybinds to an antigen. In some embodiments, the ligand, such as an antigen, is a protein expressed on the surface of cells.

[0369] In some embodiments, the CAR is a TCR-like CAR and the antigen is a processed peptide antigen, such as a peptide antigen of an intracellular protein, which, like a TCR, is recognized on the cell surface in the context of a major histocompatibility complex (MHC) molecule. Generally, a CAR comprising an antibody or antigen-binding fragment that exhibits TCR-like specificity directed against peptide-MHC complexes also may be referred to as a TCR-like CAR. In some embodiments, the extracellular antigen binding domain specific for an MHC -peptide complex of a TCR-like CAR is linked to one or more intracellular signaling components, in some aspects via linkers and / or transmembrane domain(s). In some embodiments, such molecules can typically mimic or approximate a signal through a natural antigen receptor, such as a TCR, and, optionally, a signal through such a receptor in combination with a costimulatory receptor.

[0370] In some embodiments, transcription of a CAR, when under the operable control of the synthetic transcriptional regulatory sequence or the variant MND promoter, is repressed by T cell stimulation. In some embodiments, transcription of a CAR, when under the operable control of the synthetic transcriptional regulatory sequence or the variant MND promoter, is constitutively active with or without T cell stimulation.

[0371] In some embodiments, the CAR is constructed with a specificity for a particular antigen (or marker or ligand), such as an antigen expressed in a particular cell type to be targeted by adoptive therapy, e.g., a cancer marker, and / or an antigen intended to induce a dampening response, such as an antigen expressed on a normal or non-diseased cell type. Thus, the CAR typically includes in its extracellular portion one or more antigen binding molecules, such as one or more antigen-binding fragment, domain, or portion, or one or more antibody variable domains, and / or antibody molecules. In some embodiments, the CAR includes an antigen-binding portion or portions of an antibody molecule, such as a single-chain antibody fragment (scFv) derived from the variable heavy (VH) and variable light (VL) chains of a monoclonal antibody (mAb), or a single domain antibody (sdAb), such as sdFv, nanobody, VHH and VNAR. In some embodiments, an antigen-binding fragment comprises antibody variable regions joined by a flexible linker.

[0372] Among the antigen binding domains included in the CARs are antibody fragments. An “antibody fragment” or “antigen-binding fragment” refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabodies; linear antibodies; heavy chain variable (VH) regions, single-chain antibody molecules such as scFvs and single-domain antibodies comprising only the VH region; and multispecific antibodies formedfrom antibody fragments. In particular embodiments, the antibodies are single-chain antibody fragments comprising a heavy chain variable (VH) region and / or a light chain variable (VL) region, such as scFvs.

[0373] In certain embodiments, multispecific binding molecules, e.g., multispecific chimeric receptors, such as multispecific CARs, can contain any of the multispecific antibodies, including, e.g. bispecific antibodies, multispecific single-chain antibodies, e.g., diabodies, triabodies, and tetrabodies, tandem di-scFvs, and tandem tri-scFvs.

[0374] Single-domain antibodies (sdAbs) are antibody fragments comprising all or a portion of the heavy chain variable region or all or a portion of the light chain variable region of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody.

[0375] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells. In some embodiments, the antibodies are recombinantly-produced fragments, such as fragments comprising arrangements that do not occur naturally, such as those with two or more antibody regions or chains joined by synthetic linkers, e.g., peptide linkers, and / or that are may not be produced by enzyme digestion of a naturally-occurring intact antibody. In some aspects, the antibody fragments are scFvs.

[0376] In some embodiments, the antibody or antigen-binding fragment thereof is a single-chain antibody fragment, such as a single chain variable fragment (scFv) or a diabody or a single domain antibody (sdAb). In some embodiments, the antibody or antigen-binding fragment is a single domain antibody comprising only the VH region. In some embodiments, the antibody or antigen binding fragment is an scFv comprising a heavy chain variable (VH) region and a light chain variable (VL) region.

[0377] In some embodiments, the antigen targeted by the receptor is a polypeptide. In some embodiments, it is a carbohydrate or other molecule. In some embodiments, the antigen is selectively expressed or overexpressed on cells of the disease or condition, e.g., the tumor or pathogenic cells, as compared to normal or non-targeted cells or tissues. In other embodiments, the antigen is expressed on normal cells and / or is expressed on the engineered cells.

[0378] In certain embodiments, the antigen is or includes avP6 integrin (avb6 integrin), B cell activating factor receptor (BAFF-R), B cell maturation antigen (BCMA), B7-H3, B7-H6, carbonic anhydrase 9 (CA9, also known as CAIX or G250), a cancer-testis antigen, cancer / testis antigen IB (CTAG, also known as NY-ESO-1 and LAGE-2), carcinoembryonic antigen (CEA), a cyclin, cyclin A2, C-C Motif Chemokine Ligand 1 (CCL-1), CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44, CD44v6, CD44v7 / 8, CD70, CD123, CD133, CD138, CD171, chondroitin sulfate proteoglycan 4 (CSPG4), delta-like ligand 3 (DLL3), epidermal growth factor protein (EGFR), truncated epidermal growth factor protein (tEGFR), type III epidermal growth factor receptor mutation (EGFR vIII), epithelial glycoprotein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), ephrinB2, ephrine receptor A2(EPHa2), estrogen receptor, Fc receptor like 5 (FCRL5; also known as Fc receptor homolog 5 or FCRH5), fetal acetylcholine receptor (fetal AchR), a folate binding protein (FBP), folate receptor alpha, ganglioside GD2, O-acetylated GD2 (0GD2), ganglioside GD3, glycoprotein 100 (gplOO), glypican-3 (GPC3), G Protein Coupled Receptor 5D (GPCR5D), Her2 / neu (receptor tyrosine kinase erb-B2), Her3 (erb-B3), Her4 (erb-B4), erbB dimers, Human high molecular weight-melanoma-associated antigen (HMW-MAA), hepatitis B surface antigen, Human leukocyte antigen Al (HFA-A1), Human leukocyte antigen A2 (HFA-A2), IF-22 receptor alpha(IF-22Ra), IF-13 receptor alpha 2 (IF-13Ra2), kinase insert domain receptor (kdr), kappa light chain, Fl cell adhesion molecule (Fl-CAM), CE7 epitope of Fl- CAM, Feucine Rich Repeat Containing 8 Family Member A (FRRC8A), Fewis Y, Melanoma-associated antigen (MAGE)-Al, MAGE- A3, MAGE-A6, MAGE-A10, mesothelin (MSLN), c-Met, murine cytomegalovirus (CMV), mucin 1 (MUC1), MUC16, natural killer group 2 member D (NKG2D) ligands, melan A (MART-1), neural cell adhesion molecule (NCAM), oncofetal antigen, Preferentially expressed antigen of melanoma (PRAME), progesterone receptor, a prostate specific antigen, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1), survivin, Trophoblast glycoprotein (TPBG also known as 5T4), tumor-associated glycoprotein 72 (TAG72), Tyrosinase related protein 1 (TRP1, also known as TYRP1 or gp75), Tyrosinase related protein 2 (TRP2, also known as dopachrome tautomerase, dopachrome delta- isomerase or DCT), vascular endothelial growth factor receptor (VEGFR), vascular endothelial growth factor receptor 2 (VEGFR2), Wilms Tumor 1 (WT-1), a pathogen-specific or pathogen-expressed antigen, or an antigen associated with a universal tag, and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens. Antigens targeted by the receptors in some embodiments include antigens associated with a B cell malignancy, such as any of a number of known B cell marker. In some embodiments, the antigen is or includes CD20, CD19, CD22, ROR1, CD45, CD21, CD5, CD33, Igkappa, Iglambda, CD79a, CD79b or CD30.

[0379] In some embodiments, the antigen is or includes a pathogen-specific or pathogen-expressed antigen. In some embodiments, the antigen is a viral antigen (such as a viral antigen from HIV, HCV, HBV, etc.), bacterial antigens, and / or parasitic antigens.

[0380] Exemplary CAR T cell therapies for use in accordance with the methods provided herein are known in the art. CAR T cell therapies suitable for use in accordance with the methods provided herein include any described in Marofi et al., Stem Cell Res Ther 12: 81 (2021); Townsend et al., J Exp Clin Cancer Res 37: 163 (2018); Ma et al., Int J Biol Sci 15(12): 2548-2560 (2019); Zhao and Cao, Front Immunol 10: 2250 (2019); and Han et al., J Cancer 12(2): 326-334 (2021), the contents of each of which are incorporated by reference herein in their entirety.

[0381] Exemplary antigen receptors, including CARs, and methods for engineering and introducing such receptors into cells, include those described, for example, in international patent application publication numbers W0200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, W02013 / 123061, WO2016 / 0046724, WO2016 / 014789, WO2016 / 090320, WO2016 / 094304, W02017 / 025038, WO2017 / 173256, U.S. patent application publication numbers US2002131960, US2013287748, US20130149337, U.S. Patent Nos. 6,451,995,7.446.190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762,7.446.191, 8,324,353, 8,479,118, and 9,765,342, and European patent application number EP2537416, and / or those described by Sadelain et al., Cancer Discov., 3(4): 388-398 (2013); Davila et al., PLoS ONE 8(4): e61338 (2013); Turtle et al., Curr. Opin. Immunol., 24(5): 633-39 (2012); Wu et al., Cancer, 18(2): 160-75 (2012), the contents of each of which are incorporated by reference in their entirety. In some aspects, the antigen receptors include a CAR as described in U.S. Patent No. 7,446,190, and those described in International Patent Application Publication No. WO / 2014055668 Al, the contents of each of which are incorporated by reference in their entirety. Examples of the CARs include CARs as disclosed in any of the aforementioned publications, such as WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No. 7,446,190, US Patent No. 8,389,282, Kochenderfer et al., Nature Reviews Clinical Oncology, 10, 267-276 (2013); Wang et al., J. Immunother. 35(9): 689-701 (2012); and Brentjens et al., Sci Transl Med. 5(177) (2013), the contents of each of which are incorporated by reference in their entirety. See also WO2014031687, US 8,339,645, US 7,446,179, US 2013 / 0149337, U.S. Patent No. 7,446,190, and US Patent No. 8,389,282, the contents of each of which are incorporated by reference in their entirety.

[0382] In some embodiments, the CAR is an anti-BCMA CAR that is specific for BCMA, e.g., human BCMA. Chimeric antigen receptors containing anti-BCMA antibodies, including mouse antihuman BCMA antibodies and human anti-human BCMA antibodies, and cells expressing such chimeric receptors have been previously described. See Carpenter et al., Clin Cancer Res., 2013, 19(8):2048- 2060, US 9,765,342, WO 2016 / 090320, W02016090327, W02010104949A2, WO2016 / 0046724, WO2016 / 014789, WO2016 / 094304, W02017 / 025038, and WO2017173256, the contents of each of which are incorporated by reference in their entirety.

[0383] In some embodiments, the CAR is an anti-CD19 CAR that is specific for CD19, e.g., human CD 19. In some embodiments, the antibody or an antigen-binding fragment e.g. scFv or VH domain) specifically recognizes an antigen, such as CD19. In some embodiments, the antibody or antigen-binding fragment is derived from, or is a variant of, antibodies or antigen-binding fragment that specifically binds to CD19.

[0384] In some embodiments, the antigen is CD 19. In some embodiments, the scFv contains a VH and a VL derived from an antibody or an antibody fragment specific to CD19. In some embodiments, the antibody or antibody fragment that binds CD 19 is a mouse derived antibody such as FMC63 and SJ25C1. In some embodiments, exemplary antibody or antibody fragment include those described in U.S. Patent Publication No. WO 2014 / 031687, US 2016 / 0152723 and WO 2016 / 033570, the contents of each of which are incorporated by reference in their entirety.[0...

Claims

ClaimsWHAT IS CLAIMED:

1. A synthetic transcriptional regulatory sequence, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

2. The synthetic transcriptional regulatory sequence of claim 1 , wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

3. The synthetic transcriptional regulatory sequence of claim 1 or claim 2, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

4. The synthetic transcriptional regulatory sequence of any one of claims 1-3, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-24, 101, and 103.

5. The synthetic transcriptional regulatory sequence of claim 1, wherein the synthetic transcriptional regulatory element is inducibly active in T cells to control inducible activation of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent.

6. The synthetic transcriptional regulatory sequence of claim 1 or claim 5, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101.

7. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101.

8. The synthetic transcriptional regulatory sequence of any one of claims 1 and 5-7, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101.

9. The synthetic transcriptional regulatory sequence of any one of claims 1 and 5-8, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 7, 9, 10, 15, and 101.

10. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

11. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of NFkB comprising the nucleic acid sequencesof SEQ ID NO: 28 and a core regulatory element motif of NFkB comprising the nucleic acid sequencesof SEQ ID NO: 30, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 7.

12. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a plurality of a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and comprises a nucleicacid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 9.

13. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 10.

14. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 55, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

15. The synthetic transcriptional regulatory sequence of any one of claims 1, 5, and 6, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 38, and a core regulatory element motif of NFkB comprising the nucleic acid sequence of SEQ ID NO: 28, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 101.

16. The synthetic transcriptional regulatory sequence of any one of claims 5-15, wherein the transcribable sequence comprises a chimeric antigen receptor (CAR), IL-2, IL-7, IL-12, IL-15, IL-18, IL- 21, IL-23, IL-36, TNFa, FTL3L, IFNy, and / or CCL21.

17. The synthetic transcriptional regulatory sequence of any one of claims 5-16, wherein the transcribable sequence comprises IL- 12.

18. The synthetic transcriptional regulatory sequence of claim 1, wherein the synthetic transcriptional regulatory sequence is constitutively active in T cells to control constitutive expression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence.

19. The synthetic transcriptional regulatory sequence of claim 1 or claim 18, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103.

20. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103.

21. The synthetic transcriptional regulatory sequence of any one of claims 1 and 18-20, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103.

22. The synthetic transcriptional regulatory sequence of any one of claims 1 and 18-21, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 12, 16-24, and 103.

23. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 111, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 8.

24. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 71, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

25. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 94, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16.

26. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 52, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

27. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 104 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 107, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 18.

28. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 104 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 108, and comprises a nucleic acid sequence having at least 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 19.

29. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif of ZKSCAN comprising the nucleic acid sequence of SEQ ID NO: 112 and a core regulatory element motif of EFla comprising the nucleic acid sequence of SEQ ID NO: 113, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 20.

30. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 21.

31. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 22.

32. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 105 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 109, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 23.

33. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises an enhancer sequence comprising the nucleic acid sequence of SEQ ID NO: 106 and a promoter sequence comprising the nucleic acid sequence of SEQ ID NO: 110, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 24.

34. The synthetic transcriptional regulatory sequence of any one of claims 1, 18, and 19, wherein the synthetic transcriptional regulatory sequence comprises the nucleic acid sequence of SEQ ID NO: 103, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 103.

35. The synthetic transcriptional regulatory sequence of any one of claims 18-34, wherein the transcribable sequence comprises a chimeric antigen receptor (CAR), a decoy receptor, CD40L, 4- 1BBL, a pro-inflammatory binder, a chemokine, or a safety switch.

36. The synthetic transcriptional regulatory sequence of claim 35, wherein the decoy receptor comprises TGFPRIIDN or a chimeric cytokine receptor.

37. The synthetic transcriptional regulatory sequence of claim 35, wherein the pro- inflammatory binder comprises an anti-PDl scFv, a CD40R agonist antibody, a CD47 antibody, or a bispecific T cell engager.

38. The synthetic transcriptional regulatory sequence of claim 35, wherein the chemokine comprises CCL19 or CXCL10.

39. The synthetic transcriptional regulatory sequence of claim 1, wherein the synthetic transcriptional regulatory element is inducibly repressible in T cells to control inducible repression of a transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence after activation of the T cells with a T cell stimulatory agent.

40. The synthetic transcriptional regulatory sequence of claim 1 or claim 39, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14.

41. The synthetic transcriptional regulatory sequence of any one of claims 1, 39, and 40, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 90%sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14.

42. The synthetic transcriptional regulatory sequence of any one of claims 1 and 39-41, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14.

43. The synthetic transcriptional regulatory sequence of any one of claims 1 and 39-42, wherein the synthetic transcriptional regulatory sequence comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11, 13, and 14.

44. The synthetic transcriptional regulatory sequence of any one of claims 1, 39, and 40, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 91, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

45. The synthetic transcriptional regulatory sequence of any one of claims 1, 39, and 40, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 86, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

46. The synthetic transcriptional regulatory sequence of any one of claims 1, 39, and 40, wherein the synthetic transcriptional regulatory sequence comprises a core regulatory element motif comprising the nucleic acid sequence of SEQ ID NO: 71, and comprises a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

47. The synthetic transcriptional regulatory sequence of any one of claims 39-46, wherein the transcribable sequence comprises a chimeric antigen receptor (CAR) or a chimeric cytokine receptor.

48. The synthetic transcriptional regulatory sequence of any one of claims 5-15, 18-34, and 39-46, wherein the transcribable sequence encodes an antibody or antigen-binding fragment thereof, an interleukin or variant thereof, or a cytokine or variant thereof.

49. The synthetic transcriptional regulatory sequence of any one of claims 5-15, 18-34, and 39-46, wherein the transcribable sequence encodes a recombinant receptor, an antibody or antigen binding fragment thereof, a cytokine of variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

50. A synthetic transcriptional regulatory sequence, comprising a first core regulatory element motif and a second core regulatory element motif, wherein the first core regulatory element motif and the second core regulatory element motif each independently comprise a core regulatory element motif selected from the group consisting of FOS, NFkB, FLU, EFla, and ZKSCAN1; and wherein the first core regulatory element motif and a second core regulatory element motif are separated by a spacer sequence.

51. The synthetic transcriptional regulatory sequence of claim 50, wherein the first core regulatory element motif and the second core regulatory element motif are different.

52. The synthetic transcriptional regulatory sequence of claim 50 or claim 51 , wherein the first core regulatory element motif is a core regulatory element motif of FOS, and the second core regulatory element motif is a core regulatory element motif of NFkB.

53. The synthetic transcriptional regulatory sequence of any one of claims 50-52, wherein the core regulatory element motif of FOS comprises the nucleic acid sequence of SEQ ID NO: 38 and the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28.

54. The synthetic transcriptional regulatory sequence of claim 50 or claim 51 , wherein the first core regulatory element motif is a core regulatory element motif of NFkB, and the second core regulatory element motif is a core regulatory element motif of FLU.

55. The synthetic transcriptional regulatory sequence of any one of claims 50, 51, and 54, wherein the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28, and the core regulatory element motif of FLU comprises the nucleic acid sequence of SEQ ID NO: 32.

56. The synthetic transcriptional regulatory sequence of claim 50 or claim 51, wherein the first core regulatory element motif is a core regulatory element motif of ZKSCAN, and the second core regulatory element motif is a core regulatory element motif of EFla.

57. The synthetic transcriptional regulatory sequence of any one of claims 50, 51, and 56, wherein the core regulatory element motif of ZKSCAN comprises the nucleic acid sequence of SEQ ID NO: 112, the core regulatory element motif of EFla comprises the nucleic acid sequence of SEQ ID NO: 113.

58. The synthetic transcriptional regulatory sequence of claim 50 or claim 51, wherein the first core regulatory element motif is a first core regulatory element motif of NFkB, and the second core regulatory element motif is a second core regulatory element motif of NFkB, wherein the first core regulatory element motif and the second core regulatory element motif are different.

59. The synthetic transcriptional regulatory sequence of any one of claims 50, 51, and 58, wherein the first core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28, the second core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 30.

60. A synthetic transcriptional regulatory sequence, comprising two or more core regulatory element motifs, wherein each of the two or more core regulatory element motifs is the same and is a core regulatory element motif selected from the group consisting of PRDM1, FOS, and NFkB; wherein each of the two or more core regulatory element motifs are separated from one another by a spacer sequence.

61. The synthetic transcriptional regulatory sequence of claim 60, wherein each of the two or more core regulatory element motifs is a core regulatory element motif of BEIMP l(PRDMl).

62. The synthetic transcriptional regulatory sequence of claim 61, wherein the core regulatory element motif of BEIMP l(PRDMl) comprises the nucleic acid sequence of SEQ ID NO: 111.

63. The synthetic transcriptional regulatory sequence of claim 60, wherein each of the two or more core regulatory element motifs is a core regulatory element motif of FOS.

64. The synthetic transcriptional regulatory sequence of claim 63, wherein the core regulatory element motif of FOS comprises the nucleic acid sequence of SEQ ID NO: 38.

65. The synthetic transcriptional regulatory sequence of claim 60, wherein each of the two or more core regulatory element motifs is a core regulatory element motif of NFkB.

66. The synthetic transcriptional regulatory sequence of claim 65, wherein the core regulatory element motif of NFkB comprises the nucleic acid sequence of SEQ ID NO: 28 or 30.

67. The synthetic transcriptional regulatory sequence of any one of claims 50-66, wherein the spacer sequence has a length of at least 1 nucleotide and up to 20, 19, 18, 17, 16, 15, 14, 13, or 12 nucleotides.

68. The synthetic transcriptional regulatory sequence of any one of claims 50-67, wherein the spacer sequence has a length of 1 to 12 nucleotides, 1 to 10 nucleotides, 1 to 9 nucleotides, 1 to 8 nucleotides, 1 to 7 nucleotides, 1 to 6 nucleotides, 1 to 5 nucleotides, 2 to 12 nucleotides, 2 to 10 nucleotides, 2 to 9 nucleotides, 2 to 8 nucleotides, 2 to 7 nucleotides, 2 to 6 nucleotides, 2 to 5 nucleotides, 4 to 12 nucleotides, 4 to 10 nucleotides, 4 to 9 nucleotides, 4 to 8 nucleotides, 4 to 7 nucleotides, or 4 to 6 nucleotides.

69. The synthetic transcriptional regulatory sequence of any one of claims 50-68, wherein the spacer sequence has a length of 1 to 12 nucleotides.

70. The synthetic transcriptional regulatory sequence of any one of claims 50-68, wherein the spacer sequence has a length of 4 to 6 nucleotides.

71. The synthetic transcriptional regulatory sequence of any one of claims 50-70, further comprising a minimal promoter or variant thereof.

72. The synthetic transcriptional regulatory sequence of claim 71, wherein the minimal promoter or variant thereof is a minimal promoter variant.

73. The synthetic transcriptional regulatory sequence of claim 72, wherein the minimal promoter variant is an SCP2 minimal promoter.

74. The synthetic transcriptional regulatory sequence of claim 71, wherein the minimal promoter or variant thereof is an EFla minimal promoter.

75. The synthetic transcriptional regulatory sequence of any one of claims 5-17 and 39-74, wherein the T cell stimulatory agent is or comprises an agent that activates a T cell receptor.

76. The synthetic transcriptional regulatory sequence of claim 75, wherein the T cell receptor comprises an immunoreceptor tyrosine-based activation motif (IT AM).

77. The synthetic transcriptional regulatory sequence of any one of claims 1-76, wherein the synthetic transcriptional regulatory sequence is not naturally occurring.

78. A variant MND promoter, comprising one or more core regulatory element motifs inserted into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT: API, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1JRF4, TP53, STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

79. The variant MND promoter of claim 78, wherein one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of NFkB, FLU, ATF4, E2F6, EOMES, SRF, FOSL1, FOS, KLF10, KLF3, NFAT:AP1, INSMI, ETV6, RARA, NFAT5, ZBTB3, ARID3A, YY2, ZBTB33, MAFK, SP4, HSF1, Canonical ATF4, Canonical NFAT, Canonical FOS, Canonical STAT5, SMAD3, BATF_JUNB_IRF4, IRF4, CEBPg, cMyc, AP1_IRF4, and TP53.

80. The variant MND promoter of claim 78 or claim 79, wherein one or more of the one or more core regulatory motifs independently comprises a core regulatory element motif selected from the group consisting of STAT1, IRF9, KLF3, ETV6, FOXJ2, KLF9, Canonical STAT1, Canonical STAT3, USF2, E2F6, MGA, JUNB, ZNF652, NFYA, ZNF528, CREB1, REST, PATZE, ZBTB35, BCL6B, HIC1, ZEB_EB, PRDM1, ZBTB17, RUNX1, RUNX3, IKZF1, ZBTB7A, and BACH1.

81. A variant MND promoter, comprising one or more core regulatory element motifs inserted into a parental MND promoter, wherein each of the one or more core regulatory motifs independently comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28- 98, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98.

82. The variant MND promoter of claim 78 or claim 81, wherein each of the one or more core regulatory motifs independently comprises a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98.

83. The variant MND promoter of claim 78 or claim 81, wherein each of the one or more core regulatory motifs independently comprises a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 28-98.

84. The variant MND promoter of any one of claims 78 and 81-83, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES; a core regulatory element motif of STAT1; and a core regulatory element motif of ZEB_EB.

85. The variant MND promoter of any one of claims 78 and 81-84, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of EOMES comprising the nucleic acid sequence of SEQ ID NO: 35; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 67; a core regulatory element motif of STAT1 comprising the nucleic acid sequence of SEQ ID NO: 73; and a core regulatory element motif of ZEB_EB comprising the nucleic acid sequence of SEQ ID NO: 91.

86. The variant MND promoter of any one of claims 78 and 81-83, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2.

87. The variant MND promoter of any one of claims 78, 81-83, and 86, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of FOXJ2 comprising the nucleic acid sequence of SEQ ID NO: 71.

88. The variant MND promoter of any one of claims 78 and 81-83, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA and a core regulatory element motif of PATZE.

89. The variant MND promoter of any one of claims 78, 81-83, and 88, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of NFYA comprising the nucleic acid sequence of SEQ ID NO: 82; and a core regulatory element motif of PATZE comprising the nucleic acid sequence of SEQ ID NO: 86.

90. The variant MND promoter of any one of claims 78 and 81-83, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatory element motif of FLU ; a core regulatory element motif of ATF4; a core regulatory element motif of FOS; a core regulatory element motif of YY2; a core regulatory element motif of ZBTB33; and a core regulatory element motif of canonical NF AT.

91. The variant MND promoter of any one of claims 78, 81-83, and 90, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 28; a core regulatory element motif of FLU comprising the nucleic acid sequence of SEQ ID NO: 32; a core regulatory element motif of ATF4 comprising the nucleic acid sequence of SEQ ID NO: 33; a core regulatory element motif of FOS comprising the nucleic acid sequence of SEQ ID NO: 39; a core regulatory element motif of YY2 comprising the nucleic acid sequence of SEQ ID NO: 49; a core regulatory element motif of ZBTB33 comprising the nucleic acid sequence of SEQ ID NO: 50; and a core regulatory element motif of canonical NF AT comprising the nucleic acid sequence of SEQ ID NO: 55.

92. The variant MND promoter of any one of claims 78 and 81-83, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB ; a core regulatoryelement motif of CEB Pg; a core regulatory element motif of JUNB; a core regulatory element motif of ZNF528; a core regulatory element motif of ZBTB35; a core regulatory element motif of PRDM1 ; and a core regulatory element motif of RUNX1.

93. The variant MND promoter of any one of claims 78, 81-83, and 92, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of NFKB comprising the nucleic acid sequence of SEQ ID NO: 29; a core regulatory element motif of CEBPg comprising the nucleic acid sequence of SEQ ID NO: 62; a core regulatory element motif of JUNB comprising the nucleic acid sequence of SEQ ID NO: 79 or 80; a core regulatory element motif of ZNF528 comprising the nucleic acid sequence of SEQ ID NO: 83; a core regulatory element motif of ZBTB35 comprising the nucleic acid sequence of SEQ ID NO: 87; a core regulatory element motif of PRDM1 comprising the nucleic acid sequence of SEQ ID NO: 92; and a core regulatory element motif of RUNX1 comprising the nucleic acid sequence of SEQ ID NO: 94.

94. The variant MND promoter of any one of claims 78 and 81-83, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of SP4.

95. The variant MND promoter of any one of claims 78, 81-83, and 94, wherein the one or more core regulatory element motifs comprises a core regulatory element motif of SP4 comprising the nucleic acid sequence of SEQ ID NO: 52.

96. The variant MND promoter of any one of claims 78-95, wherein the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

97. The variant MND promoter of any one of claims 78-95, wherein the parental MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

98. The variant MND promoter of any one of claims 78-95, wherein the parental MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

99. The variant MND promoter of any one of claims 78-98, wherein the parental MND promoter comprises the nucleic acid sequence of SEQ ID NO: 5.

100. The variant MND promoter of any one of claims 78-99, wherein the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17.

101. The variant MND promoter of any one of claims 78-100, wherein the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 90% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17.

102. The variant MND promoter of any one of claims 78-100, wherein the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17, or a nucleic acid sequence having at least 95% sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17.

103. The variant MND promoter of any one of claims 78-100, wherein the variant MND promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 11-17.

104. The variant MND promoter of any one of claims 78, 81-85, and 96-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 11, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

105. The variant MND promoter of any one of claims 78, 81-85, 96-100, and 104, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

106. The variant MND promoter of any one of claims 78, 81-85, 96-100, and 104, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

107. The variant MND promoter of any one of claims 78, 81-85, 96-100, and 104, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 11.

108. The variant MND promoter of any one of claims 78, 81-85, 86, 87, and 96-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 12, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

109. The variant MND promoter of any one of claims 78, 81-83, 86, 87, 96-100, and 108, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

110. The variant MND promoter of any one of claims 78, 81-83, 86, 87, 96-100, and 108, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

111. The variant MND promoter of any one of claims 78, 81-83, 86, 87, 96-100, and 108, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 12.

112. The variant MND promoter of any one of claims 78, 81-83, 88, 89, and 96-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 13, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

113. The variant MND promoter of any one of claims 78, 81-83, 88, 89, 96-100, and 112, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

114. The variant MND promoter of any one of claims 78, 81-83, 88, 89, 96-100, and 112, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

115. The variant MND promoter of any one of claims 78, 81-83, 88, 89, 96-100, and 112, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 13.

116. The variant MND promoter of any one of claims 78, 81-83, 86, 87, and 96-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

117. The variant MND promoter of any one of claims 78, 81-83, 86, 87, 96-100, and 116, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

118. The variant MND promoter of any one of claims 78, 81-83, 86, 87, 96-100, and 116, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

119. The variant MND promoter of any one of claims 78, 81-83, 86, 87, 96-100, and 116, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 14.

120. The variant MND promoter of any one of claims 78, 81-83, 90, 91, and 96-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 15, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

121. The variant MND promoter of any one of claims 78, 81-83, 90, 91, 96-100, and 120, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

122. The variant MND promoter of any one of claims 78, 81-83, 90, 91, 96-100, and 120, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 15.

123. The variant MND promoter of any one of claims 78, 81-83, 90, 91, 96-100, and 120, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 15.

124. The variant MND promoter of any one of claims 78, 81-83, 92, 93, and 96-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 16, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 16.

125. The variant MND promoter of any one of claims 78, 81-83, 92, 93, 96-100, and 124, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 16.

126. The variant MND promoter of any one of claims 78, 81-83, 92, 93, 96-100, and 124, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 16.

127. The variant MND promoter of any one of claims 78, 81-83, 92, 93, 96-100, and 124, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 16.

128. The variant MND promoter of any one of claims 78, 81-83, and 94-100, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 17, or a nucleic acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

129. The variant MND promoter of any one of claims 78, 81-83, 94-100, and 128, wherein the variant MND promoter comprises a nucleic acid sequence having at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

130. The variant MND promoter of any one of claims 78, 81-83, 94-100, and 128, wherein the variant MND promoter comprises a nucleic acid sequence having at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 17.

131. The variant MND promoter of any one of claims 78, 81-83, 94-100, and 128, wherein the variant MND promoter comprises the nucleic acid sequence of SEQ ID NO: 17.

132. The variant MND promoter of any one of claims 78-131, wherein the variant MND promoter is not naturally occurring.

133. A polynucleotide comprising the synthetic transcriptional regulatory sequence of any one of claims 1-77, and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

134. A polynucleotide comprising the variant MND promoter of any one of claims 78-132, and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the variant MND promoter.

135. The polynucleotide of claim 133 or claim 134, wherein the transcribable sequence encodes a recombinant receptor, an antibody or antigen binding fragment thereof, a cytokine of variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

136. A polynucleotide comprising the synthetic transcriptional regulatory sequence of any one of claims 39-46 and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

137. The polynucleotide of claim 136, wherein the transcribable sequence comprises a chimeric antigen receptor (CAR) or a chimeric cytokine receptor.

138. A polynucleotide comprising the synthetic transcriptional regulatory sequence of any one of claims 18-34 and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

139. The polynucleotide of claim 138, wherein the transcribable sequence comprises a chimeric antigen receptor (CAR), a decoy receptor, CD40L, 4-1BBL, a pro-inflammatory binder, a chemokine, or a safety switch.

140. The polynucleotide of claim 139, wherein the decoy receptor comprises TGFpRIIDN or a chimeric cytokine receptor.

141. The polynucleotide of claim 139, wherein the pro-inflammatory binder comprises an anti-PDl scFv, a CD40R agonist antibody, a CD47 antibody, or a bispecific T cell engager.

142. The polynucleotide of claim 139, wherein the chemokine comprises CCL19 or CXCL10.

143. A polynucleotide comprising the synthetic transcriptional regulatory sequence of any one of claims 5-15 and a transcribable sequence, wherein transcription of the transcribable sequence is under the operable control of the synthetic transcriptional regulatory sequence.

144. The polynucleotide of claim 143, wherein the transcribable sequence comprises a chimeric antigen receptor (CAR), IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IL-36, TNFa, FTL3L, IFNy, and / or CCL21.

145. The polynucleotide of claim 143 or claim 144, wherein the transcribable sequence comprises IL- 12.

146. The polynucleotide of claim 136 or claim 143, wherein transcription of the transcribable sequence produces a coding RNA molecule.

147. The polynucleotide of claim 146, wherein the coding RNA molecule encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

148. The polynucleotide of claim 147, wherein the coding RNA molecule encodes a recombinant receptor that is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

149. The polynucleotide of claim 136 or claim 143, wherein transcription of the transcribable sequence produces a non-coding RNA molecule.

150. The polynucleotide of claim 149, the non-coding RNA molecule is an siRNA, miRNA, or shRNA molecule.

151. A vector comprising the synthetic transcriptional regulatory sequence of any one of claims 1-77, or the variant MND promoter of any one of claims 78-132.

152. A vector comprising the polynucleotide of any one of claims 133-150.

153. A vector comprising the synthetic transcriptional regulatory sequence of any one of claims 18-38, 48, and 49.

154. A vector comprising the synthetic transcriptional regulatory sequence of any one of claims 39-49.

155. A vector comprising the polynucleotide of any one of claims 136, 137, and 146-150.

156. A vector comprising the synthetic transcriptional regulatory sequence of any one of claims 5-17, 48, and 49.

157. A vector comprising the polynucleotide of any one of claims 143-150.

158. The vector of any one of claims 151-157, wherein the vector is an adeno-associated virus (AAV) vector.

159. The vector of any one of claims 151-157, wherein the vector is a retroviral vector.

160. The vector of any one of claims 151-157 and 159, wherein the vector is a lentiviral vector.

161. A lipid particle comprising the synthetic transcriptional regulatory sequence of any one of claims 1-77, or the variant MND promoter of any one of claims 78-132.

162. A lipid particle comprising the polynucleotide of any one of claims 133-150.

163. A lipid particle comprising the synthetic transcriptional regulatory sequence of any one of claims 39-49.

164. A lipid particle comprising the polynucleotide of any one of claims 136, 137, and 146- 150.

165. A lipid particle comprising the synthetic transcriptional regulatory sequence of any one of claims 5-17, 48, and 49.

166. A lipid particle comprising the polynucleotide of any one of claims 143-150.

167. A cell comprising the synthetic transcriptional regulatory sequence of any one of claims 1-77, the variant MND promoter of any one of claims 78-132, the polynucleotide of any one of claims 133-150, the vector of any one of claims 151-160, or the lipid particle of any one of claims 161-166.

168. A cell comprising the synthetic transcriptional regulatory sequence of any one of claims 18-38, 48, and 49, the polynucleotide of any one of claims 138-142, or the vector of any one of claims 156-160.

169. A cell comprising the synthetic transcriptional regulatory sequence of any one of claims 39-49, the polynucleotide of any one of claims 136, 137, and 146-150, or the vector of any one of claims 155 and 158-160.

170. A cell comprising the synthetic transcriptional regulatory sequence of any one of claims 5-17, 48, and 49, the polynucleotide of any one of claims 143-150, or the vector of any one of claims 156-160.

171. The cell of any one of claims 167-170, wherein the cell is an engineered immune cell.

172. An engineered immune cell comprising the synthetic transcriptional regulatory sequence of any one of claims 1-77, the variant MND promoter of any one of claims 78-132, the polynucleotide of any one of claims 133-150, the vector of any one of claims 151-160, or the lipid particle of any one of claims 161-166.

173. An engineered immune cell comprising the synthetic transcriptional regulatory sequence of any one of claims 18-38, 48, and 49, the polynucleotide of any one of claims 138-142, or the vector of any one of claims 156-160.

174. An engineered immune cell comprising the synthetic transcriptional regulatory sequence of any one of claims 39-49, the polynucleotide of any one of claims 136, 137, and 146-150, or the vector of any one of claims 154, 155 and 158-160.

175. An engineered immune cell comprising the synthetic transcriptional regulatory sequence of any one of claims 5-17 48, and 49„ the polynucleotide of any one of claims 143-150, or the vector of any one of claims 156-160.

176. The engineered immune cell of any one of claims 171-175, wherein the engineered immune cell expresses a recombinant receptor under the operable control of the synthetic transcriptional regulatory sequence.

177. The engineered immune cell of any one of claims 171-175, wherein the engineered immune cell expresses a recombinant receptor under the operable control of the variant MND promoter.

178. The engineered immune cell of claim 176 or claim 177, wherein the recombinant receptor is a T cell receptor (TCR).

179. The engineered immune cell of claim 176 or claim 177, wherein the recombinant receptor is a CAR comprising an extracellular antigen-binding domain and an intracellular signaling domain comprising an IT AM signaling domain.

180. The engineered immune cell of claim 179, wherein the intracellular signaling domain comprises an intracellular signaling domain of a CD3 chain.

181. The engineered immune cell of claim 180, wherein the CD3 chain is a CD3-zeta chain.

182. The engineered immune cell of any one of claims 176-181, wherein binding to the recombinant receptor induces a costimulatory signal.

183. The engineered immune cell of any one of claims 180-182, wherein the intracellular signaling domain comprises a costimulatory signaling domain.

184. The engineered immune cell of claim 183, wherein the costimulatory signaling domain comprises an intracellular signaling domain of CD28 or 4- IBB.

185. The engineered immune cell of any one of claims 171-184, wherein the engineered immune cell is an engineered lymphocyte.

186. The engineered immune cell of any one of claims 171-185, wherein the engineered immune cell is an engineered T cell.

187. The engineered immune cell of any one of claims 171-186, wherein the synthetic transcriptional regulatory sequence and the transcribable sequence are comprised within the genome of the engineered immune cell.

188. The engineered immune cell of any one of claims 171-187, wherein the synthetic transcriptional regulatory sequence and the transcribable sequence are comprised within an intron or a transcriptional regulatory element of the genome of the engineered immune cell.

189. The engineered immune cell of any one of claims 171-186, wherein the variant MND promoter and the transcribable sequence are comprised within the genome of the engineered immune cell.

190. The engineered immune cell of any one of claims 171-186 and 189, wherein the variant MND promoter and the transcribable sequence are comprised within an intron or a transcriptional regulatory element of the genome of the engineered immune cell.

191. An engineered immune cell comprising the polynucleotide of any one of claims 133-150.

192. The engineered immune cell of claim 191, wherein the polynucleotide is comprised in an adeno-associated virus (AAV) vector.

193. The engineered immune cell of claim 191, wherein the polynucleotide is comprised in the engineered immune cell by insertion or integration into the genome of the engineered immune cell.

194. The engineered immune cell of claim 191 or claim 193, wherein the polynucleotide is comprised in the engineered immune cell by insertion or integration into an intron or a transcriptional regulatory element of the genome of the engineered immune cell for expression of the transcribable sequence.

195. The engineered immune cell of claim 193 or claim 194, wherein the insertion or integration is by homology-directed repair (HDR).

196. A method of decreasing transcription of a transcribable sequence in an immune cell, comprising stimulating the engineered immune cell of any one of claims 174 and 176-190.

197. A method of increasing transcription of a transcribable sequence in an immune cell, comprising stimulating the engineered immune cell of any one of claims 175-190.

198. A method of decreasing transcription of a transcribable sequence in an immune cell, comprising:(a) introducing into an immune cell the polynucleotide of any one of claims 136, 137, and 146- 150, or the vector of any one of claims 154, 155, and 158-160; and(b) stimulating the immune cell, thereby decreasing transcription of the transcribable sequence of the polynucleotide or vector.

199. A method of increasing transcription of a transcribable sequence in an immune cell, comprising:(a) introducing into an immune cell the polynucleotide of any one of claims 134-150, or the vector of any one of claims 156-160; and(b) stimulating the immune cell, thereby increasing transcription of the transcribable sequence of the polynucleotide or vector.

200. The method of claim 198 or claim 199, wherein the introducing comprises introducing the polynucleotide into the genome of the immune cell.

201. The method of any one of claims 198-200, wherein the introducing comprises introducing the polynucleotide into an intron or a transcriptional regulatory element of the genome of the immune cell.

202. The method of any one of claims 196-201, wherein the immune cell is a lymphocyte.

203. The method of any one of claims 196-202, wherein the immune cell is a T cell.

204. The method of any one of claims 196-203, wherein the stimulating comprises inducing an IT AM-mediated signal, wherein the immune cell expresses a recombinant receptor, and the IT AM- mediated signal is induced via binding to the recombinant receptor.

205. The method of claim 204, wherein the recombinant receptor is a CAR comprising an extracellular antigen-binding domain and an intracellular signaling domain comprising an IT AM signaling domain.

206. The method of any one of claims 196-205, wherein the stimulating occurs in vivo following administration of the immune cell to a subject having a disease or condition.

207. The method of any one of claims 196-206, wherein the stimulating comprises administering the immune cell to a subject having a disease or condition.

208. The method of any one of claims 196-205, wherein the stimulating occurs in vitro or ex vivo.

209. The method of claim 208, wherein the stimulating comprises contacting the immune cell with a T cell stimulatory agent.

210. The method of claim 206 or claim 207, wherein the recombinant receptor binds to a target antigen that is expressed by cells associated with the disease or condition in the subject.

211. The method of any one of claims 206, 207, and 210, wherein the disease or condition is a cancer or an autoimmune or inflammatory disease.

212. The method of any one of claims 196-211, wherein the transcribable sequence encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

213. The method of claim 212, wherein the cytokine or variant thereof comprises IL-12.

214. An engineered immune cell of any one of claims 172-195, for use in treating a disease or condition in a subject.

215. The engineered immune cell for use of claim 214, wherein the immune cell expresses a recombinant receptor that binds to a target antigen expressed by cells associated with the disease or condition.

216. The engineered immune cell for use of claim 214 or claim 215, wherein the disease or condition is a cancer or an autoimmune or inflammatory disease.

217. The engineered immune cell of any one of claims 214-216, wherein the disease or condition is a cancer.

218. The engineered immune cell of any one of claims 214-216, wherein the disease or condition is an autoimmune or inflammatory disease.

219. The engineered immune cell of any one of claims 214-218, wherein the engineered cell comprises transcribable sequence under the operable control of the synthetic transcriptional regulatory sequence or the variant MND promoter, wherein the transcribable sequence encodes a recombinant receptor, an antibody or antigen-binding fragment thereof, a cytokine or variant thereof, a chemokine or variant thereof, or an interleukin or variant thereof.

220. The engineered immune cell of claim 219, wherein the cytokine or variant thereof comprises IL- 12.

221. A pharmaceutical composition comprising the engineered immune cell of any one of claims 172-195 and a pharmaceutically acceptable carrier.

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