Regulated synthetic gene expression systems

#The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 15, 2020, is named 701586-095350_SL.txt and is 921,581 bytes in size.

#The technology described herein relates to regulated synthetic gene expression systems.

#Next generation cell therapies seek to create designer immune cells that can sense and respond to disease in sophisticated ways. Achieving this goal fundamentally requires engineered regulatory elements and circuitry that can be used to program human cell functions by processing complex environmental inputs and mediating precisely regulated expression of therapeutic agents. Towards this goal, synthetic transcriptional programs can interface with sense and response modules to enable new layers of regulation in cells.

#FIG. 1A-1H is a series of schematics and graphs showing the design and characterization of mammalian synTFs based on orthogonal ZF arrays. FIG. 1A is a schematic showing the design of synTFs based on orthogonal 6-unit ZF arrays. FIG. 1B is a schematic showing a synTF and reporter system. FIG. 1C is a bar graph showing that synTFs robustly activate corresponding integrated reporters, across a library of cognate synTF/DBM pairs; note that mCherry expression occurred only in the presence of a specific ZF-p65 synTF and not in the absence of a TF or in the presence of GFP-p65. FIG. 1D is a heatmap showing the co-expression of specific synTFs and reporters; note that mCherry reporter is activated by a synthetic operator and its cognate synTF. FIG. 1E-1G is a series of scatterplots showing transcriptome profiling of human cells expressing synTFs, including ZF1-p65 (FIG. 1E), ZF3-p65 (FIG. 1F), and ZF10-p65 (FIG. 1G), which reveals highly specific, orthogonal regulation of the synTFs. FIG. 1H is a bar graph showing a transcriptome profiling of synTFs compared to Gal4 and TetR. Differentially regulated transcripts=Log 2|fold change|>1; FDR<0.1 (for all 3 independent replicates).

#FIG. 2 is a schematic showing a small molecule-responsive synTF system.

#FIG. 3 is a schematic showing an exemplary synTF regulated by induced proximity.

#The following abbreviations used herein are defined as follows: synTF (synthetic transcription factor); ZF (zinc finger); HEK (human embryonic kidney 293 cells); ED (effector domain); AD (activator domain); RD (repressor domain); GOI (gene of interest); OUT (output); DBM (DNA binding motif); pUb (Ubiquitin C promoter); p65 (Transcription Factor P65); minCMV (minimal cytomegalovirus promoter); AAVS1 (adeno-associated virus integration site 1); chr19 (human chromosome 19); GFP (green fluorescent protein); TPM (transcripts per million; i.e., a normalized value of each individual RNA transcript across the total pool of RNA transcripts for the sequenced samples); ABI (ABA-insensitive); PYL (PYR1-like, protein pyrabactin resistance 1-like); ABA (abscisic acid); ERT2 (a mutated variant of the estrogen receptor ligand binding domain), 4OHT (4-hydroxyrtamoxifen); NS3 (nonstructural protein 3 of HCV); GZV or GRZ (grazoprevir); SMASh (small molecule assisted shut-off); a.u. (arbitrary units or relative emission intensity); IFNγ (interferon gamma); IL-2 (interleukin 2); IL-4 (interleukin 4); CD19 (Cluster of Differentiation 19); mCh (mCherry); pSFFV (silencing-prone spleen focus forming virus promoter); minTK (minimal promoter fragment from the HSV thymidine kinase (TK) promoter); lenti (lentivirus).

#Described herein are synTFs for use in the methods and compositions as disclosed herein, where the synTFs comprise (i) a DNA binding domain (DBD) which binds to a target nucleic acid sequence (or target DNA binding motif (DBM)), (ii) an effector domain (ED) and a regulator protein (RP), where the regulator protein controls the coupling or linkage of the DNA binding domain (DBD) with the effector domain (ED) (such coupling can also be referred to as a “mediator domain”), or controls the cellular localization of the ED, such that when the ED and DBD are attached and/or located in the nucleus, the ED can function to recruit or repress translation machinery to the promoter to regulate gene expression of a gene of interest.

#In some embodiments of any of the aspects, regulator proteins can be activated or inhibited by to a variety of inputs, non-limiting examples of which include: inducers (e.g., small molecules), light-inducible control (e.g., dimerization, assembly, localization), temperature, pH, phosphorylation, oxygen, lipid, magnetic, electric, spatial mechanisms (e.g., intracellular and/or extracellular; e.g., synthetic receptors and/or soluble factors), endogenous ligands (e.g., biomarkers), cell-cycle state, native signaling pathways, or disease and/or pathogenic states (e.g., aggregation, infection).