Synthetic molecular feedback circuits and methods of use thereof

Abstract

Molecular feedback circuits are provided, as well as nucleic acids encoding such molecular feedback circuits, and cells genetically modified with the subject molecular feedback circuits. Methods of regulating signaling of a cellular signaling pathway using molecular feedback circuits and methods of treating a subject for a condition by administering cells containing nucleic acids encoding the molecular feedback circuits are also provided. Aspects of the molecular feedback circuits of the present disclosure include a signaling protein of the signaling pathway that includes a cryptic inactivation domain. Such circuits can include a regulatory sequence operably linked to a nucleic acid encoding a switch polypeptide that is responsive to the output of the signaling pathway and that, when expressed, triggers the inactivation domain to inactivate the signaling molecule.

Description

[Technical field]

[0001] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH This invention was made with Government support under Grant No. HR0011-16-2-0045 awarded by the Defense Advanced Research Projects Agency. The Government has certain rights in the invention.

[0002] cross reference This application claims the benefit of U.S. Provisional Application No. 62 / 789,402, filed January 7, 2019, which is incorporated herein by reference. [Background technology]

[0003] Introduction Traditionally, the desired regulation of cellular activity has been controlled by repeated inputs to a cell system provided by a user. For example, in the context of some medical treatments, the desired level of a subject's cellular output over an extended period of time is achieved by repeated cycles of administering, evaluating, re-administering, and re-evaluating a drug over the course of the treatment. Similarly, in bioproduction applications and metabolic engineering, the growth medium is repeatedly augmented, for example, by supplementing growth factors and / or removing toxic by-products, to induce the producing cells to output a product at a desired yield.

[0004] The ability of engineered cells to perform desired tasks, and methods for producing such engineered cells, have made great advances over the past few decades. For example, recent advances in synthetic biology and systems metabolic engineering technologies offer the potential for microbial cell factories to produce industrially relevant bulk and fine chemicals from renewable biomass resources in an environmentally friendly manner. Furthermore, designer cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, which can be targeted to a variety of user-defined targets, have shown great promise in the clinic and are being widely adopted and continually gaining regulatory approval.

[0005] For example, the output of such engineered cells, once administered to a subject or activated within a bioreactor, for a variety of purposes as described above, persists without additional user input, and adjustments can be made to modulate the output of the engineered cells using external input, for example, in the form of small molecules, or other stimuli or user-executed manipulations. Summary of the Invention

[0006] This disclosure includes the following [1] to

[65] . [1] A signaling protein that drives an output of a signaling pathway when activated by an input of the signaling pathway, the signaling protein comprising a latent inactivation domain but not a caged degron; and and a regulatory sequence responsive to the output operably linked to a nucleic acid sequence encoding a switch polypeptide, the expression of which triggers the inactivation domain to inactivate the signaling molecule. [2] The circuit of [1] above, wherein the input or the output, or both, include an intracellular signal. [3] The circuit described in [1] above, wherein the input or the output, or both, include an intercellular signal. [4] The circuit described in any one of [1] to [3] above, wherein the inactivation domain is a degradation domain. [5] The circuit described in [4] above, wherein the degradation domain includes a degron. [6] The circuit of [4] or [5] above, wherein the latent inactivation domain comprises a protective domain that prevents degradation of the signaling protein and is deprotected by the switch polypeptide. [7] The circuit of [6] above, wherein the switch polypeptide comprises a protease. [8] The circuit of any one of [1] to [3] above, wherein the inactivation domain comprises a first member of a binding pair. [9] The circuit of [8], wherein the switch polypeptide comprises a second member of the binding pair linked to a sequestration domain.

[10] The circuit described in [9] above, wherein the isolation domain comprises a plasma membrane targeting tag, a mitochondrial membrane targeting tag, a peroxisome targeting tag, a vacuolar targeting tag, or an actin cytoskeleton targeting tag.

[11] The circuit of [8] above, wherein the switch domain comprises a second member of the binding pair that comprises a dominant negative domain.

[12] The circuit of

[11] , wherein the latent inactivation domain comprises a competitive binding domain non-covalently bound to the first member of the binding pair.

[13] The circuit of any one of [8] to

[12] above, wherein the first and second members of the binding pair comprise first and second portions of a leucine zipper.

[14] The circuit of any one of [1] to

[13] above, wherein the signal transduction protein is a positive regulator of the signal transduction pathway.

[15] The circuit of any one of [1] to

[14] , wherein the signal transduction protein is a negative regulator of the signal transduction pathway.

[16] The circuit of any one of [1] to

[15] above, wherein the signal transduction protein is an intermediate member of the signal transduction pathway or a transcription factor.

[17] The circuit described in

[16] above, wherein the transcription factor is a synthetic transcription factor.

[18] The circuit of any one of [1] to

[17] , wherein the regulatory sequence comprises a binding site for a transcription factor of the output.

[19] The circuit of any one of

[16] to

[18] , wherein the output is expression of the transcription factor.

[20] The circuit of any one of [1] to

[15] , wherein the signaling protein is a receptor and the input is a ligand for the receptor.

[21] The circuit according to any one of [1] to

[20] above, wherein the signal transduction pathway is selected from the group consisting of AKT signal transduction pathway, Akt / PKB signal transduction pathway, AMPK signal transduction pathway, apoptosis signal transduction pathway, BMP signal transduction pathway, cAMP-dependent pathway, estrogen signal transduction pathway, Hedgehog signal transduction pathway, Hippo signal transduction pathway, immune activation pathway, immune suppression pathway, immune cell differentiation pathway, insulin signal transduction pathway, JAK-STAT signal transduction pathway, MAPK / ERK signal transduction pathway, mTOR signal transduction pathway, NF-κB signal transduction pathway, nodal signal transduction pathway, Notch signal transduction pathway, p53 signal transduction pathway, PI3K signal transduction pathway, TGF beta signal transduction pathway, TLR signal transduction pathway, TNF signal transduction pathway, VEGF signal transduction pathway, and Wnt signal transduction pathway.

[22] The circuit of any one of [1] to

[21] , further comprising a regulatory sequence operably linked to the nucleic acid sequence encoding the signal transduction protein.

[23] The circuit of

[22] above, wherein the regulatory sequence operably linked to the nucleic acid sequence encoding the signaling protein is a native promoter of the signaling protein.

[24] The circuit of any one of [1] to

[20] above, wherein the signal transduction pathway is a synthetic signal transduction pathway.

[25] The circuit described in

[24] above, wherein the receptor is a synthetic receptor.

[26] The circuit described in

[25] above, wherein the synthetic receptor is a synNotch receptor.

[27] The circuit described in

[25] above, wherein the synthetic receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[28] The circuit described in

[27] above, wherein the output is immune activation or immune suppression.

[29] One or more nucleic acid molecules encoding the molecular feedback circuit described in any one of [1] to

[28] above.

[30] A cell genetically modified to contain one or more of the nucleic acid molecules described in

[19] above.

[31] The cell described in

[30] above, which is a eukaryotic cell.

[32] A method for treating a subject for a condition, comprising administering to the subject an effective amount of the eukaryotic cell described in

[31] above.

[33] The method according to

[32] , wherein the condition is cancer and the output of the molecular feedback circuit is immune activation.

[34] The method according to

[32] above, wherein the condition is an autoimmune disease and the output of the molecular feedback circuit is immunosuppression.

[35] The method according to

[32] , wherein the symptom is a metabolic or hormonal deficiency and the output of the molecular feedback circuit is the production and / or secretion of the metabolic or hormonal deficiency.

[36] A method for modulating signal transduction of a signal transduction pathway in a cell, comprising genetically modifying the cell with a molecular feedback loop; The molecular feedback circuit is a nucleic acid sequence encoding a signaling protein of the signaling pathway, the signaling protein comprising a cryptic inactivation domain but not a caged degron; a regulatory sequence responsive to an output of the signal transduction pathway, the regulatory sequence being operably linked to a nucleic acid sequence encoding a switch polypeptide that, when expressed, activates the latent inactivation domain; The method, wherein the activated inactivation domain inactivates the signaling protein, thereby regulating signaling of the signaling pathway.

[37] The method according to

[36] above, wherein said regulating comprises negative feedback.

[38] The method according to

[36] above, wherein said regulating comprises positive feedback.

[39] The method according to any one of

[36] to

[38] above, wherein inactivation of the signal transduction protein by the inactivation domain comprises degradation of the signal transduction protein.

[40] The method according to

[39] above, wherein the inactivation domain is a degradation domain.

[41] The method of

[39] or

[40] above, wherein the latent inactivation domain is activated by a proteolytic cleavage event mediated by the switch polypeptide.

[42] The method of any one of

[36] to

[38] above, wherein inactivation of the signaling protein by the inactivation domain comprises sequestration of the signaling protein.

[43] The method of

[42] above, wherein the inactivation domain comprises a first member of a binding pair and the switch domain comprises a second member of the binding pair linked to a sequestration domain.

[44] The method according to any one of

[36] to

[38] above, wherein inactivation of the signal transduction protein by the inactivation domain comprises dominant-negative inhibition of the signal transduction protein.

[45] The method of

[44] above, wherein the switch domain comprises a second member of the binding pair linked to a dominant negative domain.

[46] The method of

[44] or

[45] above, wherein the latent inactivation domain comprises a competitive binding domain non-covalently bound to the first member of the binding pair.

[47] The method of any one of

[43] to

[46] above, wherein the first and second members of the binding pair comprise first and second portions of a leucine zipper.

[48] ​​The method according to any one of

[36] to

[47] above, wherein the cell is an in vitro cell or an ex vivo cell.

[49] The method according to any one of

[36] to

[48] above, wherein the signal transduction pathway is a native signal transduction pathway of the cell.

[50] The method according to

[49] above, wherein the native signal transduction pathway is a native biosynthetic pathway.

[51] The method according to

[50] above, wherein the native biosynthetic pathway is a hormone production pathway.

[52] The method according to

[51] above, wherein the hormone production pathway is selected from the group consisting of an insulin production pathway, an estrogen / progesterone production pathway, an androgen production pathway, and a growth hormone production pathway.

[53] The method according to

[42] above, wherein the cell is an immune cell and the natural signaling pathway is an immune stimulatory pathway or an immune suppressive pathway.

[54] The method according to

[46] above, wherein the immune activation pathway is selected from the group consisting of a cytokine signaling pathway, a B cell receptor signaling pathway, and a T cell receptor signaling pathway.

[55] The method according to

[46] above, wherein the immunosuppressive pathway is an inhibitory immune checkpoint pathway.

[56] The method according to any one of

[36] to

[48] above, wherein the signal transduction pathway is a synthetic signal transduction pathway.

[57] The method according to

[56] above, wherein the signaling protein is a synNotch receptor and the output is release of the intracellular domain of the synNotch receptor.

[58] The method according to

[56] above, wherein the cell is an immune cell and the signal transduction pathway is a synthetic immune stimulatory pathway or a synthetic immune suppressive pathway.

[59] The method according to

[58] above, wherein the immune cells are myeloid cells or lymphoid cells.

[60] The method according to

[59] above, wherein the immune cells are lymphoid cells selected from the group consisting of T lymphocytes, B lymphocytes, and natural killer cells.

[61] The method according to any one of

[58] to

[60] above, wherein the signal transduction protein is a synthetic immune receptor.

[62] The method according to

[61] above, wherein the synthetic immune receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[63] The method according to any one of

[56] to

[62] above, wherein the output is immune activation or immune suppression.

[64] The method according to

[56] above, wherein the synthetic signaling pathway is a synthetic biosynthetic pathway.

[65] The method according to

[64] above, wherein the synthetic biosynthetic pathway is selected from the group consisting of a hormone production pathway, an opioid production pathway, an antibiotic production pathway, a chemotherapeutic drug production pathway, an artemisinic acid production pathway, a terpenoid production pathway, and a polyketide production pathway. Molecular feedback circuits are provided, as well as nucleic acids encoding such molecular feedback circuits, and cells genetically modified with the subject molecular feedback circuits. Methods of regulating signaling of a cellular signaling pathway using the molecular feedback circuits, and methods of treating a subject for a condition by administering a cell containing a nucleic acid encoding the molecular feedback circuit are also provided. Aspects of the molecular feedback circuits of the present disclosure include a signaling protein of the signaling pathway that includes a latent inactivation domain. Such circuits may include a regulatory sequence operably linked to a nucleic acid encoding a switch polypeptide that is responsive to the output of the signaling pathway and that, when expressed, triggers the inactivation domain to inactivate the signaling molecule.

[0007] In any embodiment, the signaling protein does not include a caged degron. In other words, in any embodiment, the signaling protein does not include (a) a degron, (b) a locker domain comprising five alpha helices, and (c) a latch domain comprising an alpha helix, and (i) in the absence of a key polypeptide, the degron forms a six-helix bundle with the locker domain to cage the signaling protein and prevent degradation, and (ii) in the presence of a key polypeptide, the degron is uncaged and the signaling protein is degraded, where the key polypeptide may include an alpha helix that binds to the locker domain with higher affinity than the latch domain. Such caged degron molecules are described in U.S. Provisional Application Nos. 62 / 789,418 (filed January 7, 2019), 62 / 850,336 (filed May 20, 2019), and 62 / 789,351, filed January 7, 2019, which applications are incorporated herein by reference. [Brief description of the drawings]

[0008] [Figure 1]FIG. 1 is a schematic diagram of the signaling pathways described herein.

[0009] [Diagram 2] FIG. 2 is a schematic diagram of a signal transduction pathway having a cryptic inactivation domain linked to a positive regulatory member of the signal transduction pathway of FIG. 1 as described herein.

[0010] [Diagram 3] FIG. 3 is a diagram showing activation of a latent inactivation domain in the signaling pathway depicted generally in FIG. 2 as described herein.

[0011] [Figure 4] FIG. 4 is a schematic diagram of a signal transduction pathway having a cryptic inactivation domain linked to a negative regulatory member of the signal transduction pathway of FIG. 1 as described herein.

[0012] [Diagram 5] FIG. 5 is a schematic diagram of a molecular feedback loop strategy using the synthetic Notch receptors described herein.

[0013] [Figure 6] FIG. 6 shows a schematic of the molecular feedback loop strategy using chimeric antigen receptors (CARs) described herein.

[0014] [Figure 7] FIG. 7 is a diagram that illustrates generally various strategies for controlling feedback in an example feedback circuit described in the present disclosure.

[0015] [Figure 8] FIG. 8 is a schematic diagram of an example of an isolation-based strategy for controlling feedback in circuits described herein.

[0016] [Figure 9]FIG. 9 is an alternative diagram of one example of an isolation-based strategy for controlling feedback in the circuits described herein.

[0017] [Figure 10] FIG. 10 illustrates a feedback control using an example isolation-based feedback circuit of the present disclosure.

[0018] [Figure 11] FIG. 11 is a schematic diagram showing leucine zipper transcription factors and dominant-negative inhibitors of leucine zipper transcription factors.

[0019] [Figure 12] FIG. 12 is a schematic diagram of an example of a contention-based strategy for controlling feedback in the circuits described herein.

[0020] [Figure 13] FIG. 13 is a diagram illustrating a feedback control using an example of a contention-based feedback circuit of the present disclosure.

[0021] [Figure 14] FIG. 14 is a schematic diagram of degronLOCKR-based feedback circuits or control of biological pathways.

[0022] [Figure 15] FIG. 15 provides a panel of mating pathway regulators tested in degronLOCKR.

[0023] [Figure 16] FIG. 16 demonstrates that the degronLOCKR module successfully implements synthetic feedback control of the conjugation pathway.

[0024] [Figure 17-1] FIG. 17 illustrates the performance characteristics of the degronLOCKR feedback module as quantified by the control of the synthesis circuit. [Figure 17-2] FIG. 17 illustrates the performance characteristics of the degronLOCKR feedback module as quantified by the control of the synthesis circuit.

[0025] [Figure 18] FIG. 18 is a diagram providing a steady-state solution in response to a positive or negative disturbance.

[0026] [Figure 19] FIG. 19 shows circuit behavior as a function of Pg for a fixed dose of E2.

[0027] [Figure 20] FIG. 20 shows circuit behavior as a function of E2 for a fixed dose of Pg.

[0028] [Figure 21] FIG. 21 illustrates the circuit behavior when constructively expressing different amounts of a key.

[0029] [Figure 22-1] FIG. 22 demonstrates that the DegronLOCKR synthesis feedback strategy is predictably tunable. [Figure 22-2] FIG. 22 demonstrates that the DegronLOCKR synthesis feedback strategy is predictably tunable.

[0030] [Diagram 23] FIG. 23 demonstrates that altering promoter strength or key length modulates feedback gain.

[0031] [Figure 24] FIG. 24 is a diagram showing how adjusting the feedback strength changes the dynamic behavior of the circuit output.

[0032] [Diagram 25]FIG. 25 illustrates combinatorial regulation of synthetic feedback in a joint pathway.

[0033] [Figure 26] FIG. 26 shows the control of protein localization using nesLOCKR.

[0034] [Figure 27] FIG. 27 shows cytosolic aggregation of nesLOCKR when Key is expressed.

[0035] [Figure 28] FIG. 28 shows a fluorescence histogram of tagBFP (left panel) and a fluorescence histogram of mCherry (right panel).

[0036] [Figure 29] FIG. 29 shows schematics without and with feedback (top panel) and representative histograms comparing output and key fluorescence of both circuits in the presence and absence of drug (bottom panel).

[0037] [Diagram 30] FIG. 30 shows a comparison of the output of different feedback mutants (left panel) with the normalized output of a circuit without feedback and a feedback circuit with mCMV-Key (right panel). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0038] definition The terms "synthetic," "chimeric," and "engineered" as used herein generally refer to an artificially derived polypeptide or a non-naturally occurring nucleic acid encoding a polypeptide. Synthetic polypeptides and / or nucleic acids may be constructed de novo from basic subunits, including, for example, single amino acids or single nucleotides, or may be derived from existing polypeptides or polynucleotides, whether naturally occurring or artificially derived, for example, by recombinant genetic techniques. Chimeric and engineered polypeptides or nucleic acids encoding polypeptides are generally constructed by combining, joining, or fusing two or more different polypeptides or nucleic acids encoding polypeptides, or polypeptide domains or nucleic acids encoding polypeptide domains. Chimeric and engineered polypeptides or nucleic acids encoding polypeptides include cases where the two or more linked polypeptide or nucleic acid "portions" are derived from different proteins (or nucleic acids encoding different proteins), as well as cases where the linked portions comprise different regions of the same protein (or nucleic acid encoding a protein), but the portions are linked in a manner that would not occur in nature.

[0039] The term "recombinant" as used herein refers to a nucleic acid molecule, e.g., a genomic polynucleotide, a cDNA polynucleotide, a viral polynucleotide, a semisynthetic polynucleotide, and / or a polynucleotide of synthetic origin that is not related to all or a portion of the polynucleotide sequence with which it is associated in nature because of its origin or manipulation. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used with respect to a host cell or virus means a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein with respect to a material (e.g., a cell, a nucleic acid, a protein, or a vector) to refer to the material having been altered by the introduction of heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0040] The term "operably linked" refers to the juxtaposition of the components so described in a relationship that allows them to function in their intended manner. For example, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may, but need not, be contiguous. For example, in some cases, a coding sequence operably linked to a promoter may be contiguous with the promoter. In some cases, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. In some cases, more than two sequences may be operably linked, including, but not limited to, two or more coding sequences operably linked to a single promoter.

[0041] A "biological sample" encompasses a variety of sample types obtained from an individual or a population of individuals and can be used in a variety of ways, including, for example, isolation of cells or biological molecules and diagnostic assays. The definition encompasses blood and other liquid samples from an organism, as well as solid tissue samples, such as biopsy specimens or tissue cultures or cells derived therefrom and their progeny. The definition also includes samples that have been manipulated in some way after their procurement, such as by mixing or pooling individual samples, treatment with reagents, solubilization, or enrichment for specific elements (e.g., cells, polynucleotides, polypeptides, etc.). The term "biological sample" encompasses clinical samples, and also includes cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples. The term "biological sample" includes urine, saliva, cerebrospinal fluid, interstitial fluid, ocular fluid, synovial fluid, blood fractions (e.g., plasma and serum), and the like. The term "biological sample" also encompasses solid tissue samples, tissue culture samples, and cell samples. Thus, a biological sample can be a cellular or acellular sample.

[0042] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the terms include, but are not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or polymers containing purine and pyrimidine bases, or polymers containing other naturally occurring, chemically or biochemically modified, non-naturally occurring, or derivatized nucleotide bases.

[0043] The terms "polypeptide," "peptide," and "protein," as used interchangeably herein, refer to polymeric forms of amino acids of any length, which may include genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides with modified peptide backbones. The terms include fusion proteins, including, but not limited to, fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, and the like.

[0044] A "vector" or "expression vector" is a replicon, such as a plasmid, phage, virus or cosmid, to which another DNA segment, or "insert," can be attached so as to bring about the replication of the attached segment in a cell.

[0045] The terms "domain" and "motif", as used interchangeably herein, refer to both structured domains having one or more specific functions, and unstructured segments of a polypeptide that are unstructured but retain one or more specific functions. For example, a structured domain may include, but is not limited to, a contiguous or non-contiguous amino acid or portion thereof in a folded polypeptide that includes a three-dimensional structure that contributes to a specific function of the polypeptide. In another example, a domain may include an unstructured segment of a polypeptide that includes two or more amino acids or portions thereof and maintains a specific function of the unfolded or disordered polypeptide. This definition also includes domains that may be disordered or unstructured, but become structured or ordered upon association with a target or binding partner. Non-limiting examples of intrinsically unstructured domains and intrinsically unstructured protein domains are described, for example, in Dyson & Wright. Nature Reviews Molecular Cell Biology 6:197-208.

[0046] As used herein, the term "affinity" refers to the equilibrium constant of reversible binding of two agents, expressed as a dissociation constant (Kd). The affinity can be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater, or more. The affinity of an antibody to a target protein can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM) or more.

[0047] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges. Nonspecific binding is approximately 10 -7 refers to binding with an affinity of less than 10 M, e.g., -6 M, 10 -5 M, 10 -4 It refers to binding with an affinity such as M.

[0048] As used herein, the terms "treatment", "treating" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in that it completely or partially prevents a disease or its symptoms, and / or it may be therapeutic, in that it partially or completely cures a disease and / or adverse effects resulting from the disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal (e.g., a human), including methods of (a) preventing the development of a disease in a subject who may be predisposed to the disease but has not yet been diagnosed with the disease, (b) inhibiting the disease (i.e., preventing its onset), and (c) relieving the disease (i.e., causing regression of the disease).

[0049] The terms "individual," "subject," "host," and "patient," used interchangeably herein, refer to mammals, including but not limited to murines (e.g., rats, mice), lagomorphs (e.g., rabbits), non-human primates, humans, dogs, cats, ungulates (e.g., horses, cows, sheep, pigs, goats), and the like.

[0050] "Therapeutically effective amount" or "effective amount" refers to the amount of one agent, or the amount of two agents combined, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. A "therapeutically effective amount" will vary depending on the agent, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0051] The terms "chimeric antigen receptor" and "CAR", as used interchangeably herein, generally, but not exclusively, refer to an artificial multi-module molecule that includes an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain, and one or more intracellular signaling domains and can trigger or inhibit the activation of immune cells. The term CAR is not strictly limited to CAR molecules, but also includes variants of CARs. CAR variants include split CARs, in which the extracellular portion (e.g., a ligand binding portion) and the intracellular portion (e.g., an intracellular signaling portion) of the CAR are present on two separate molecules. CAR variants also include on-switch CARs, which are conditionally activatable CARs, including split CARs in which the conditional heterodimerization of its two portions is pharmacologically controlled (e.g., as described in WO 2014 / 127261 and U.S. Patent Application Publication No. 2015 / 0368342, the disclosures of which are incorporated herein by reference in their entireties). CAR variants also include bispecific CARs that include a secondary CAR binding domain that can amplify or inhibit the activity of the primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs), which can be used, for example, as components of bispecific CAR systems in which binding of a secondary CAR binding domain results in inhibition of primary CAR activation.CAR molecules and their derivatives (i.e., CAR variants) can be used in a variety of applications, including, for example, those described in PCT Application No. US2014 / 016527; Fedorov et al., Sci Transl Med (2013); 5(215):215 ra 172; Glienke et al., Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151-5; Riddell et al., Cancer J (2014) 20(2):141-4; Pegram et al., Cancer J (2014) 20(2):127-33; Cheadle et al., Immunol Rev (2014) 257(1):91-106; Barrett et al., Annu Rev Med (2014) 65:333-47; Sadelain et al., Cancer J (2014) 257(1):91-106. Discov (2013) 3(4): 388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19-4-1BB-CD3ζ CAR expressed by lentivirus-loaded CTL019 (Tisagenlecleucel-T) CAR-T cells marketed by Novartis (Basel, Switzerland). The terms "chimeric antigen receptor" and "CAR" also include SUPRA CAR and PNE CAR (see, e.g., Cho et al., Cell 2018 173: 1426-1438 and Rodgers et al., Proc. Acad. Sci. 2016 113: E 459-468).

[0052] The terms "T cell receptor" and "TCR" are used interchangeably and generally refer to a molecule found on the surface of a T cell or T lymphocyte that is responsible for recognizing fragments of antigens as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complex is a disulfide-linked membrane-anchored heterodimeric protein consisting of highly variable alpha (α) and beta (β) chains that are usually expressed as part of a complex with a CD3 chain molecule. Most naturally occurring TCRs exist in a heterodimeric αβ or heterodimeric γδ form. The complete endogenous TCR complex in the heterodimeric αβ form contains eight chains: an alpha chain (referred to herein as TCRα or TCR alpha), a beta chain (referred to herein as TCRβ or TCR beta), a delta chain, a gamma chain, two epsilon chains, and two zeta chains. In some instances, TCRs are generally referred to with reference to only the TCR alpha and beta chains, however, as an assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains, one of skill in the art will readily appreciate that reference to a TCR present at a cell membrane can include reference to a fully or partially assembled TCR complex, as appropriate.

[0053] Recombinant or engineered individual TCR chains and TCR complexes have been developed. Reference to the use of TCRs in a therapeutic context may refer to individual recombinant TCR chains. Thus, engineered TCRs may include individual modified TCR alpha or beta chains, as well as single chain TCRs comprising modified and / or unmodified TCR alpha and beta chains linked into a single polypeptide by a linking polypeptide.

[0054] The terms "synthetic Notch receptor," "synNotch," and "synNotch receptor," as used interchangeably herein, refer to a recombinant, chimeric binding-triggered transcriptional switch that includes at least an extracellular binding domain, a portion of a Notch receptor that includes at least one proteolytic cleavage site, and an intracellular domain that provides a signaling function. SynNotch polypeptides, elements thereof, and methods of using same are described in U.S. Pat. Nos. 9,834,608 and 9,670,281, as well as Toda et al., Science (2018) 361(6398):156-16; Roybal & Lim, Annu Rev Immunol. (2017) 35:229-253; Lim & June Cell. (2017) 168(4):724-740; Roybal et al., Cell. (2016) 167(2):419-432.e 16; Roybal et al., Cell. (2016) 164(4):770-9; and Morsut et al., Cell. (2016) 164(4):780-91; the disclosures of which are incorporated herein by reference in their entireties.

[0055] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0056] Where a range of values ​​is presented, it is understood that each intervening value between the upper and lower limits of that range, to the tenth of the unit of the lower limit, and any other declared or intervening values ​​within the declared range, unless the context clearly dictates otherwise, are encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, which are also encompassed within the invention, subject to any specifically excluded limit in the declared range. Where a declared range includes one or both of its limits, ranges excluding either or both of those included limits are also included in the invention.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any method and material similar or equivalent to those described herein can also be used to carry out or test the present invention, the preferred method and material are described herein. All publications mentioned herein are incorporated by reference herein to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0058] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to a "circuit" includes a plurality of such circuits, a reference to a "nucleic acid" includes a reference to one or more nucleic acids and equivalents thereof known to those of skill in the art, and so forth. It is further recognized that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate basis for using exclusive language such as "solely," "only," or "negative" limitations in relation to the recitation of claim elements.

[0059] It is understood that certain features of the invention that are described for clarity in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the invention that are described for brevity in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments related to the present invention are specifically embraced by the present invention and are disclosed herein as if each and every combination were individually and expressly disclosed herein. Moreover, all subcombinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein as if each and every such subcombination were individually and expressly disclosed herein.

[0060] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the publication dates provided may be different from the actual publication dates which may need to be independently confirmed.

[0061] Detailed Description Molecular feedback circuits are provided, as well as nucleic acids encoding such molecular feedback circuits, and cells genetically modified with the subject molecular feedback circuits. Methods of regulating signaling of a cellular signaling pathway using the molecular feedback circuits, and methods of treating a subject for a condition by administering a cell containing a nucleic acid encoding the molecular feedback circuit are also provided. Aspects of the molecular feedback circuits of the present disclosure include a signaling protein of the signaling pathway that includes a latent inactivation domain. Such circuits may include a regulatory sequence operably linked to a nucleic acid encoding a switch polypeptide that is responsive to the output of the signaling pathway and that, when expressed, triggers the inactivation domain to inactivate the signaling molecule.

[0062] molecular circuit The molecular circuits of the present disclosure may optionally be encoded, in whole or in part, by a nucleic acid sequence. Such circuits may optionally be present and / or comprised in an expression vector and / or expression cassette. The subject nucleic acids of the circuits of the present invention may optionally be contained within a vector, including, for example, viral and non-viral vectors. Such circuits may optionally be present within a cell (e.g., immune cells, stem cells) or may be introduced into a cell by various means, including, for example, the use of viral vectors. Cells may optionally be genetically modified to contain and / or encode the subject circuits, and such modifications may be substantially permanent (e.g., integrating) and / or transient, as desired.

[0063] The disclosed circuits, whose components are modular, may include signaling proteins that include latent inactivation domains. As used herein, the term "signaling protein" generally refers to a protein of a signaling pathway, including natural and synthetic signaling pathways, as described in more detail below. Any convenient and suitable signaling protein of any convenient signaling pathway may be used. In general, signaling proteins include proteins that can be activated by an input of the signaling pathway with which the signaling protein associates. A signaling pathway may generate an output that depends on, or at least is influenced by, the function of the signaling protein. Such an output may be a direct or indirect result of the response of the signaling protein to the input. Useful signaling proteins include members from any convenient and suitable point of a signaling pathway, including input-receiving members, intermediate members, and output-producing members.

[0064] As used herein, "input receptor member" generally refers to the first component of a signaling pathway that receives an input to initiate signaling along the signaling pathway. Examples of input receptor members include, but are not limited to, extracellular receptors (e.g., G protein-coupled receptors, protein kinases, integrins, Toll-like receptors, ligand-gated ion channels, etc.) and intracellular receptors (e.g., nuclear receptors, cytoplasmic receptors, etc.). In some cases, the input receptor member may be a protein that directly binds to an input of a signaling pathway, such as a ligand input of a signaling pathway. In some cases, a signaling protein that includes a potential inactivation domain in a circuit of the present disclosure may be an input receptor member. In some cases, a signaling protein that includes a potential inactivation domain in a circuit of the present disclosure may not be an input receptor member, but may be, for example, an intermediate member or an output-producing member.

[0065] As used herein, "intermediate member" generally refers to a component of a signaling pathway that is required for or at least involved in signal transduction, but does not directly receive the initial input of the signaling pathway, or directly generates or causes the final output of the signaling pathway. Examples of intermediate members of a signaling pathway include, but are not limited to, enzymes, binding partners, protein complex subunits, scaffolding proteins, transport proteins, coactivators, corepressors, and the like. In some cases, a signaling protein that includes a potential inactivation domain in a circuit of the present disclosure may be an intermediate member. In some cases, a signaling protein that includes a potential inactivation domain in a circuit of the present disclosure may not be an intermediate member, and may be, for example, an input-receiving member or an output-producing member.

[0066] As used herein, "output-producing member" generally refers to a component of a signaling pathway that directly produces or otherwise causes the output of the signaling pathway. Examples of output-producing members of a signaling pathway include, but are not limited to, DNA-binding proteins, such as transcription factors, enzymes, and the like. In some cases, a signaling protein that includes a potential inactivation domain in a circuit of the present disclosure may be an output-producing member. In some cases, a signaling protein that includes a potential inactivation domain in a circuit of the present disclosure may not be an output-producing member, and may be, for example, an input-receiving member or an intermediate member.

[0067] A schematic example of a signaling pathway is shown in Figure 1. As shown, the signaling pathway includes an input 100 that activates an input receptor member 101 of the pathway. Activation of the input receptor member 101 positively regulates a first intermediate member 102 of the pathway, which in turn positively regulates a second intermediate member 103 of the pathway. In the illustrated pathway, the second intermediate member 103 is negatively regulated by a third intermediate member 104. In the absence of inhibition by the third intermediate member 104, the second intermediate member 103 positively regulates an output producing member 105 of the pathway. Thus, in the event of activation by the second intermediate member 103, the output producing member 105 is active and binds to a regulatory region 107 operably linked to a sequence 106 that encodes an output of the signaling pathway.

[0068] Useful signaling proteins include those cases where the signaling protein can be a regulator of one or more signaling pathways to which it is associated, where the signaling protein can be a negative regulator of a signaling pathway or a positive regulator of a signaling pathway. Thus, the molecular feedback circuits of the present disclosure include positive feedback circuits and negative feedback circuits.

[0069] For example, in some cases, the signaling proteins used in the circuits of the present disclosure may drive the output of a signaling pathway when activated. Thus, triggering the latent inactivation domain to inactivate the signaling protein may negatively regulate the output of the signaling pathway, resulting in a negative feedback. In some cases, the signaling proteins used in the circuits of the present disclosure may inhibit the output of a signaling pathway when activated. Thus, triggering the latent inactivation domain to inactivate the signaling protein may positively regulate the output of the signaling pathway, resulting in a positive feedback.

[0070] Figure 2 shows the signaling pathway shown in Figure 1 in which the first intermediate signaling member 102, which positively regulates the pathway, has been modified to include a latent inactivation domain 200. Thus, when the latent inactivation domain 200 remains latent, signaling through the signaling pathway proceeds from the input 100, through the input accepting member 101, to the first intermediate signaling member 102, which positively regulates a downstream element of the pathway, such that, in the absence of inhibition by a third intermediate member (not shown), the output producing member 105 drives the output of the signaling pathway, shown as expression of a product encoded by sequence 106.

[0071] 3, the expressed switch polypeptide 300 activates the latent inactivation domain 200, resulting in the inactivation of the first intermediate member 102, resulting in a lack of positive signaling from the first intermediate member 102 to the second intermediate member 103 and subsequent points in the pathway. Thus, output from sequence 106 is not produced or is reduced.

[0072] 4, latent inactivation domain 400 is bound to inhibitory third intermediate member 104. Thus, when latent inactivation domain 400 remains latent, the presence of third intermediate member 104 negatively regulates the second intermediate member, thereby suppressing expression and production of the product encoded by output sequence 106. Correspondingly, when latent inactivation domain 400 is activated by expressed switch polypeptide 401, third intermediate member 104 is inactivated, thereby preventing negative regulation by third intermediate member 104 and positively regulating pathway 100 to promote production of output, i.e., increased expression of at least the product encoded by sequence 106.

[0073] When integrated with a switch polypeptide whose expression is driven by the output of a signaling pathway, coupling the latent inactivation domain to a signaling protein of the pathway can result in positive or negative feedback, as desired. For example, coupling the latent inactivation domain to a positive signaling regulator results in negative feedback on the pathway, whereas coupling the latent inactivation domain to a negative regulator results in positive feedback on the pathway. In some cases, negative pathway feedback can be used to attenuate a response, while in other cases, positive pathway feedback can be used to amplify a response or to create hypersensitivity. As will be readily apparent, the feedback circuits of the present disclosure are highly modular, and thus the circuits described herein can be easily modified and / or applied to essentially any convenient and suitable signaling pathway (including, for example, signaling pathways with measurable output via a promoter), as desired.

[0074] Feedback control allows for robust and stable execution of physical processes by removing disturbances. Implementation of feedback control may generally include (1) the ability to measure or "sense" the output of a process, (2) a controller that generates a correction signal based on a comparison of the output measurement to a desired output or "set point," and (3) a method to input or "actuate" the correction signal to the process being controlled. Provided herein are engineered circuits that utilize latent inactivation domain-based protein switches triggered by an expressed switch polypeptide to generate feedback control of a biological system. Specifically, three modules similar to those described above are included: (1) a sensing promoter that is activated by the output of the process of interest, (2) a switch peptide produced by the sensing promoter that activates the degradation of (3), and (3) a signaling protein (i.e., transcription factor, kinase, etc.) fused to the latent inactivation domain. Each of these modules can be independently adjusted via simple manipulations as desired to achieve the desired feedback control of the process.

[0075] Signaling proteins that may be used in the circuits of the present disclosure include signaling proteins that are endogenous elements of a signaling pathway, as well as heterologous or synthetic elements of a signaling pathway. Such endogenous, heterologous, and / or synthetic elements of a signaling pathway may be modified to include a latent inactivation domain, as described in more detail below, for use in the circuits of the present disclosure. By "endogenous element of a signaling pathway" is generally meant an element of a signaling pathway that occurs naturally within a cell.

[0076] "Heterologous element of a signaling pathway" generally refers to an element that functions in a signaling pathway but is derived from a cell or signaling pathway other than the cell or signaling pathway used in the subject circuit. A heterologous element may be derived from another signaling pathway than the signaling pathway of the subject circuit. A heterologous element may be derived from a different type of cell and / or a different organism than the cell and / or organism of the signaling pathway that is regulated in the subject circuit. For example, in some cases, an element of a signaling pathway from a first organism (e.g., a mouse) may be used in a corresponding signaling pathway in a second organism (e.g., a human).

[0077] "Synthetic element of a signaling pathway" generally refers to an element that functions in a signaling pathway but is not naturally occurring. Non-naturally occurring elements include recombinant elements, including, for example, analogs, mimetics, fusions, mutants, truncations, fragments, etc. Non-limiting examples of synthetic elements of a signaling pathway include synthetic receptors, synthetic enzymes, synthetic coactivators, synthetic co-repressors, synthetic binding partners, synthetic scaffolding proteins, synthetic transcription factors, etc.

[0078] The circuits of the disclosure can employ one or more regulatory sequences, the control of which can depend on an element of a signaling pathway with which the signaling protein is associated. For example, in some cases, the circuits of the disclosure can include a regulatory sequence that is responsive to the output of a signaling pathway. The regulatory sequence can be operably linked to one or more nucleic acid sequences encoding one or more elements of the subject circuits. For example, the regulatory sequence can be operably linked to a nucleic acid sequence encoding a switch polypeptide.

[0079] In some cases, the circuit may include a regulatory sequence operably linked to a nucleic acid sequence encoding a signaling protein. The regulatory sequence operably linked to a sequence encoding a signaling protein of the subject circuit may vary and may include endogenous regulatory sequences and heterologous regulatory sequences, including, but not limited to, a native promoter, a native enhancer, a heterologous promoter, a heterologous enhancer, a synthetic regulatory sequence, and the like. The regulatory sequence operably linked to a sequence encoding a signaling protein may be constitutive or inducible, as desired. In some cases, the regulatory sequence operably linked to a nucleic acid sequence encoding a signaling protein is the native promoter of the signaling protein.

[0080] In some cases, the regulatory sequence can include one or more (e.g., 1 or more, 2 or more, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 2 to 6, 3 to 6, 4 to 6, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) binding sites for the output transcription factor (including, e.g., where the transcription factor is an endogenous, heterologous, or synthetic transcription factor that functions in a signal transduction pathway).

[0081] The regulatory sequences of the disclosed circuits may be controlled by or otherwise responsive to the output of a signaling pathway. For example, in some cases, the output of a signaling pathway that the subject circuits are configured to affect may induce expression of a coding sequence via a regulatory sequence operably linked to the coding sequence. By connecting the regulation of sequences encoding components of the disclosed circuits to the output of a signaling pathway, the disclosed circuits may provide feedback responsive to the output.

[0082] Useful signaling pathway outputs for use in the circuits of the present disclosure can be diverse and include essentially any output that can be configured to directly or indirectly affect expression via regulatory sequences. Non-limiting examples of useful signaling pathway outputs include, but are not limited to, transcription factor activity (e.g., activation, repression, etc.), transcription factor expression, transcription factor translocation, enzyme activity (e.g., activation, repression, etc.), enzyme expression, signaling molecule production, signaling molecule secretion, cell activation (including, but not limited to, activation of natural cellular programs, such as, for example, immune activation, immune suppression, proliferation, etc.), and the like.

[0083] A signaling pathway can be regulated (e.g., activated, inhibited, etc.) by one or more inputs. Signaling pathway inputs can be diverse and can include signaling pathway endogenous (e.g., natural) inputs and heterologous (e.g., engineered or synthetic) signaling pathway inputs. Just as signaling pathways and signaling pathway outputs can be natural or synthetic, signaling pathway inputs can likewise be natural or synthetic.

[0084] Native signaling pathways can often be regulated by the native or natural receptors of the pathway. Non-limiting examples of native signaling pathways include, but are not limited to, for example, AKT signaling pathway, Akt / PKB signaling pathway, AMPK signaling pathway, apoptosis signaling pathway, BMP signaling pathway, cAMP-dependent pathway, estrogen signaling pathway, hedgehog signaling pathway, hippo signaling pathway, immune activation pathway, immune suppression pathway, immune cell differentiation pathway, insulin signaling pathway, JAK-STAT signaling pathway, MAPK / ERK signaling pathway, mTOR signaling pathway, NF-κB signaling pathway, nodal signaling pathway, notch signaling pathway, p53 signaling pathway, PI3K signaling pathway, TGF beta signaling pathway, TLR signaling pathway, TNF signaling pathway, VEGF signaling pathway, Wnt signaling pathway, etc.

[0085] Non-limiting examples of synthetic signaling pathways include, but are not limited to, pathways controlled by synthetic or engineered receptors (e.g., but not limited to, CARs, engineered TCRs, synNotch, etc.) Signaling pathways are described in more detail below.

[0086] Schematic examples of the process of regulating synthetic synNotch signaling pathways and synthetic CAR signaling pathways using the circuits of the present disclosure are shown in Figures 5 and 6, respectively. As shown in Figure 5, a synNotch receptor 500 having an antigen binding domain 501, a proteolytically cleavable Notch domain 502, and an intracellular signaling domain including a synthetic transcription factor (synTF) portion 503 and a latent inactivation domain 504 is triggered by an antigen input 505 to release the intracellular signaling domain. Release of the intracellular signaling domain of synNotch after antigen binding induces expression of a desired output 506 controlled by the synTF. In the illustrated embodiment, the synTF output also controls expression 507 of a switch polypeptide 508. Thus, when the released intracellular signaling domain of synNotch induces expression of a switch polypeptide, the switch polypeptide 508 activates the latent inactivation domain 504 to inactivate the synTF-containing intracellular signaling domain of the synNotch receptor. As a result, a controlled custom output is generated by providing negative feedback through the synthetic synNotch signaling pathway.

[0087] As shown in FIG. 6, the CAR 600 is triggered by an antigen input 601 that binds to the antigen binding domain 602 to induce an internal signaling cascade leading to immune cell activation, for example, via immune stimulatory signaling through the CD3z domain 603 of the CAR 600. The CAR 600 also includes an associated latent inactivation domain 604, and the cell includes a regulatory sequence operably linked to a sequence 605 that encodes a switch polypeptide that responds to an element of the internal signaling cascade. Thus, activation of the internal signaling cascade induces expression of a desired output 606 (such as, for example, immune cell activation and / or expression of a desired immune factor controlled by the CAR). However, in the illustrated embodiment, the CAR output also controls expression of the switch polypeptide 607. Thus, when the signaling cascade induces expression of the switch polypeptide 607, the switch polypeptide 607 activates the latent inactivation domain 604, inactivating the CAR. As a result, T cell activation is controlled by providing negative feedback via the synthetic CAR signaling pathway.

[0088] As will be apparent, these examples of using synthetic signaling pathways are not intended to be limiting.

[0089] Latent inactivation domains and switch polypeptides As summarized above, the signaling proteins used in the circuits of the present disclosure may include a latent inactivation domain. The latent inactivation domains included in the subject signaling proteins of the circuits of the present invention may vary widely and may be attached or otherwise incorporated into the signaling proteins as desired. Any convenient method of attaching or incorporating the latent inactivation domain into the subject signaling proteins may be used, including, but not limited to, where the latent inactivation domain is attached via a linker.

[0090] The latent inactivation domain remains latent in the absence of the switch polypeptide, meaning that the inactivation domain does not inactivate the signaling protein with which it is associated. In the presence of the switch polypeptide, the latent inactivation domain is activated, which subsequently inactivates the signaling protein with which it is associated. Strategies for inactivation are varied, including, but not limited to, inducible degradation, inducible localization, protein splitting, and the like. The switch polypeptides can be accordingly varied, including, but not limited to, polypeptides that induce the degradation of the latent inactivation domain, polypeptides that induce the localization of the latent inactivation domain, polypeptides that induce the splitting of the latent inactivation domain, and the like.

[0091] As summarized above, latent inactivation domains include inducible degradation domains. As used herein, an "inducible degradation domain" generally refers to a domain that can regulate (e.g., enhance, increase, etc.) the degradation of a polypeptide (such as a signaling protein) to which it is bound or associated upon expression of a switch polypeptide. An inducible degradation domain generally does not regulate (e.g., enhance, increase, etc.) the degradation of a subject signaling protein when the corresponding switch polypeptide is not expressed. Thus, a degradation domain is generally latent until it is activated by expression of a switch polypeptide to induce the degradation of a polypeptide to which it is bound or associated. Degradation of a polypeptide, such as a signaling protein to which the degradation domain is bound or associated, inactivates the polypeptide.

[0092] The composition of the latent inactivation domain, including the inducible degradation domain, is as varied as the corresponding switch polypeptide. In some cases, the degradation domain can include a degron. In some cases, the inducible degradation domain can include a protection domain that prevents degradation of a polypeptide (e.g., a signaling protein) bound to the degradation domain. A degradation domain that is protected by a protection domain can be deprotected by a switch polypeptide, thereby activating the degradation domain and triggering degradation of a bound or otherwise associated polypeptide.

[0093] For example, in some embodiments, a degradation domain bound to a signaling protein can be protected by a protection domain that includes a proteolytic cleavage site cleavable by a protease contained in a switch polypeptide. In the absence of the switch polypeptide, the protease is not present and the protection domain prevents the degradation domain from triggering degradation of the signaling protein. In the presence of the switch polypeptide, the protease cleaves the proteolytic cleavage site, deprotecting the degradation domain and triggering degradation of the signaling protein.

[0094] In some embodiments, the switch polypeptide can include a protease, such as a tobacco etch virus (TEV) protease, which cleaves either the N- or C-terminal sequence present on the inducible inactivation domain bound to the signaling protein. Cleavage by the protease exposes either the N- or C-terminal degron, which then triggers degradation of the signaling protein. Thus, in the absence of the TEV protease-containing switch polypeptide, the degron remains hidden and does not induce degradation of the signaling protein. In some cases, components and configurations that can be used in the protease-inducible degradation domain system can include those used in the CHOMP (Circuit of Hacked Orthogonal Modular Proteases) system described in Gao et al., Science. 2018;361(6408):1252-1258, the disclosure of which is incorporated herein by reference in its entirety. As will be readily understood, in some cases, other degrons and / or other encoded proteases can be substituted for those explicitly described.

[0095] In some embodiments, induced degradation can be achieved by ligating a degradation sequence to the target signaling protein. For example, in some cases, a degradation sequence can be inducibly ligated to a target signaling molecule using two peptides that bind to each other when both peptides are present. The binding of the peptides to each other can be covalent in some cases. In some embodiments, a tag peptide that binds to a tag binding domain can be incorporated into a target signaling protein, and a switch protein can be configured to include a tag binding domain with a bound degradation sequence, such as a constitutive degron. Upon expression of such a switch protein, the tag and the tag binding domain bind to each other, thereby binding the degradation sequence to the target signaling protein and inducing degradation of the signaling protein. As will be readily understood, in some cases, the tag and the tag binding domain can be swapped, i.e., a tag binding domain that binds to a tag peptide can be incorporated into a target signaling protein, and a switch protein can be configured to include a tag peptide with a bound degradation sequence.

[0096] Useful examples of peptides that bind to each other that can be used in these and similar embodiments include SpyCatcher and SpyTag. The terms "SpyTag" and "SpyCatcher" ("Spy" refers to the bacterium Streptococcus pyogenes) refer to a convenient protein coupling tool that can bind two polypeptides with higher efficiency than typical protein-protein interactions. The SpyTag / SpyCatcher system generates an irreversible peptide ligation and can be used to bind, label or immobilize proteins. SpyTag is a genetically encoded peptide that forms a spontaneous amide bond when bound to its genetically encoded partner, SpyCatcher. SpyTag reacts with SpyCatcher under a wide range of conditions and the product after the reaction is extremely stable. SpyCatcher and SpyTag are described in Zakeri et al., (Proc Natl Acad Sci USA 2012;109(12):E 690-7) and WO 2017 / 112784, the disclosures of which are incorporated by reference in their entireties.

[0097] PROTACs are also useful examples of protein coupling strategies that can be adapted to inducibly ligate or otherwise associate a degradation signal with a target signaling protein in the disclosed circuit. Proteolysis targeting chimeras (PROTACs) are double-headed macromolecules that contain a first domain that binds to a target protein and a second domain that can engage an E3 ubiquitin ligase. Methods of using PROTACs for targeted degradation that may be compatible for use with the circuits and methods described herein include, but are not limited to, those described in, for example, Raina & Crews, J Biol Chem. (2010) 285(15): 11057-60; Raina & Crews, Curr Opin Chem Biol. (2017) 39: 46-53; Coleman & Crews, Annual Review of Cancer Biology (2018) 2: 41-58; Bondeson et al., Cell Chemical Biology (2018) 25(1): 78-87; Neklesa et al., Pharmacology & Therapeutics. (2017) 174: 138-144; the disclosures of which are incorporated herein by reference in their entireties. Ubiquibodies and peptide PROTACs are described, for example, in Ludwicki et al., ACS Central Science 2019 5:852-866; Portnoff et al., J. Biol. Chem. 2014 289:7844-7855; Fan et al., Nature Neuroscience 2014 17:471 480; and Hines et al., Proc. Natl. Acad. Sci. 2013 110:8942-8947.

[0098] As will be readily appreciated, the strategies and components for inducibly ligating degradation signals to target signaling proteins in the circuits of the present disclosure are not limited to those specifically described herein, and in some cases other protein coupling systems can be adapted for use in the described circuits.

[0099] In some embodiments, the induced degradation strategy used in the disclosed circuits may include phosphorylation of a degron contained in a target signaling protein. For example, a degron that can be phosphorylated by a kinase may be incorporated into the target signaling protein, and the switch polypeptide may include a kinase. Thus, upon expression of the switch polypeptide, the kinase phosphorylates the degron, triggering degradation of the target signaling protein. Correspondingly, when the switch polypeptide is not expressed, the kinase is not present, the degron is not phosphorylated, and the target signaling protein is not degraded.

[0100] Useful examples of phosphorylation-based inducible degradation strategies that can be adapted for use in the disclosed circuits include, but are not limited to, modular phosphodegron strategies, such as those described in Gordley et al., PNAS (2016) 113(47):13528-13533, the disclosure of which is incorporated herein by reference in its entirety. For example, a modular phosphodegron can include an extension region of the Tec1 transcription factor that is phosphorylated by Fus3 mitogen-activated protein kinase (MAPK) and contains an additional polylysine region that inducibly binds to the yeast Cdc4 E3 ubiquitin ligase complex and promotes ubiquitination. A phosphodegron can include a mutationally optimized Cdc-binding region that results in rapid degradation of the bound polypeptide upon MAPK activation. Thus, a target signaling protein can be configured to include a modular phosphodegron and a switch polypeptide can be configured to include a MAPK. Thus, upon expression of the switch polypeptide, the phosphodegron is phosphorylated by MAPK and subsequently ubiquitinated, resulting in degradation of the target signaling protein. In the absence of the switch polypeptide, the phosphodegron is not phosphorylated and the target signaling protein is not degraded.

[0101] As will be readily understood, the strategies and components for phosphorylation-dependent inducible degradation of target signaling proteins in the circuits of the present disclosure are not limited to those specifically described herein, and in some cases, other phosphorylation-dependent systems can be adapted for use in the described circuits.

[0102] In some embodiments, the inducible degradation strategy used in the disclosed circuits may include an orthogonal proteasome system. For example, a switch polypeptide may be configured to include a proteasome that is heterologous to the cell type in which the switch polypeptide is expressed (i.e., a heterologous proteasome), and a targeted signaling protein may be configured to include a degradation tag specific for the heterologous proteasome. Thus, the proteasome and the degradation tag may constitute an orthogonal pair such that the tagged signaling protein is only degraded by the heterologous proteasome (i.e., the tag does not induce degradation of the targeted signaling protein by the host-derived (endogenous) proteolytic machinery).

[0103] For example, various orthogonal proteasome / tag pairs can be used depending on the cell modified to contain the circuit. For example, in eukaryotic cells, the orthogonal proteasome / tag pair can include a prokaryotic proteasome and a prokaryotic degradation tag that signals degradation by the prokaryotic proteasome. In prokaryotic cells, the orthogonal proteasome / tag pair can include a eukaryotic proteasome and a eukaryotic degradation tag that signals degradation by the eukaryotic proteasome. In some cases, the orthogonal proteasome / tag pair can be synthetically derived, for example, by mutation of the proteasome and / or the tag, making the pair orthogonal. In some cases, interspecies proteasome / tag pairs can be used in orthogonal proteasome systems, such as a proteasome and a degradation tag from a first species (e.g., a first bacterial species, a first eukaryotic species, etc.) used in a second species (e.g., a second bacterial species, a second eukaryotic species, etc.).

[0104] Useful examples of orthogonal proteasome systems that can be adapted for use in the circuits of the present disclosure include, for example, those derived from bacteria (e.g., E. coli, M. florum, etc.), such as, but not limited to, the ClpXP proteasome and ssrA tag system, the mfLon proteasome and pdt tag system, etc. Useful systems and components that can be adapted for use in the circuits of the present disclosure include, for example, but not limited to, those described in Cameron & Collins, Nat Biotechnol. 2014; 32 (12): 1276-1281 and Grilly et al., Molecular Systems Biology. 2007; 3: 127, the disclosures of which are incorporated herein by reference in their entirety. Thus, in some cases, the switch polypeptide can include a proteasome of an orthogonal proteasome / tag pair, and the targeting signaling protein can include a degradation tag of an orthogonal proteasome / tag pair. Circuits using orthogonal proteasome / tag pairs can be introduced into cells where the proteasome and / or tag are heterologous (ie, not endogenous) to the cell.

[0105] As will be readily appreciated, the strategies and components for orthogonal proteasome-based inducible degradation of target signaling proteins in the circuits of the present disclosure are not limited to those specifically described herein, and in some cases, other orthogonal proteasome systems can be adapted for use in the described circuits.

[0106] In some embodiments, the induced degradation strategy used in the disclosed circuits may include a system of induced localization to the proteasome. For example, in some embodiments, the switch polypeptide may include a domain that, when expressed, localizes or can induce the localization of the signaling protein to the proteasome, thereby inducing degradation of the signaling protein. Induced localization of the signaling protein to the proteasome may, in some cases, be ubiquitin-independent. In other words, induced localization of the signaling protein to the proteasome may bypass the ubiquitination step.

[0107] As an example, the switch polypeptide is configured to include a first member of a dimerization pair fused to a proteasome, and the target signaling protein is modified to include a second member of the dimerization pair. The first and second members of the dimerization pair may directly bind to each other (i.e., dimerize directly) or may dimerize via a dimerization mediator (i.e., dimerizer). Any two convenient polypeptide domains that can form a complex with each other can be selected for use as the first and second dimerization domains. For example, when the first and second members of the dimerization pair directly bind to each other, expression of the switch polypeptide that includes the first member of the dimerization pair fused to the proteasome induces the localization of the target signaling protein to the proteasome by direct binding between the first and second members of the dimerization pair, thereby resulting in degradation of the signaling protein. When the switch polypeptide is not expressed, the target signaling protein is not localized to the proteasome.

[0108] When the first and second members of the dimerization pair are dimerized by the dimerization mediator, the targeted signaling protein (including the second member of the dimerization pair) can be localized to the proteasome in the presence of both the switch polypeptide including the first member of the dimerization pair fused to the proteasome and the dimerization mediator. Thus, when a dimerization chemical inducer (CID) is used to induce the dimerization of the proteasome-fused switch polypeptide and the targeted signaling protein, the presence or absence of the CID can control whether the circuit can generate feedback or not, respectively. When dimerization domains that directly bind to each other are used, the generation of feedback can be independent of the presence of any dimerization inducer molecule.

[0109] As a non-limiting example of the induced localization to proteasome strategy of induced degradation, Fpr1 (a peptide shown to bind with high affinity to the lipophilic macrolide rapamycin) is fused (e.g., C-terminal fusion) to a proteasome subunit, and the ligand-binding domain of Tor1 (which binds to Fpr1-bound rapamycin) is fused to a target signaling protein. Upon expression of Fpr1-tagged proteasome subunits in the presence of rapamycin, the Tor1-tagged signaling protein is localized to the proteasome, resulting in localized induced degradation of the signaling protein. In some cases, Fpr1 and Tor1 can be substituted for dimerization domains that directly bind to each other, thereby eliminating the need for a dimerization mediator (e.g., rapamycin). In some cases, the dimerization domains of a direct dimerization pair can be substituted, eliminating the need for a dimerization mediator. Examples of inducible proteasome localization strategies and Fpr1 and Tor1 domains that can be adapted for use in the circuits of the present disclosure include, but are not limited to, Janse et al., J Biol Chem. 2004;279(20):21415-20, the disclosure of which is incorporated herein by reference in its entirety.

[0110] Non-limiting examples of useful dimerization domains include, but are not limited to, protein domains of the iDimerize inducible homodimer (e.g., DmrB) and heterodimer systems (e.g., DmrA and DmrC), and the iDimerize antidimerization system (e.g., DmrD) (Takara Bio Inc.) (Clackson et al., (1998) Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. USA 95(18):10437-10442; Crabtree, GR & Schreiber, SL (1996) Three-part inventions: intracellular signaling and induced proximity. Trends Biochem. Sci. 21(11):418-422; Jin et al., (2000) In vivo selection using a cell-growth switch. Nat. Genet. 26(1):64-66; Castellano et al., (1999) Inducible recruitment of Cdc42 or WASP to a cell-surface receptor triggers actin polymerization and filopodium formation. Curr. Biol. 9(7):351-360; Crabtree et al., (1997) Proximity and orientation underlie signaling by the non-receptor tyrosine kinase ZAP70. Embo. J. 16(18):5618-5628; Muthuswamy et al., (1999) Controlled dimerization of ErbB receptors provides evidence for differential signaling by homo-and heterodimers. Mol. Cell. Biol. 19(10):6845-6857.As one of ordinary skill in the art would readily appreciate, a variety of other dimerization domains may be used.

[0111] As will be readily understood, the strategies and components for induced proteasome localization-based inducible degradation of target signaling proteins in the circuits of the present disclosure are not limited to those specifically described herein, and in some cases, other induced proteasome localization systems can be adapted for use in the described circuits.

[0112] A variety of degrons can be used in the inducible degradation strategies described herein. A degron comprises a portion of a protein that signals and / or targets for degradation (or otherwise increases the rate of degradation) the protein to which it is bound or otherwise associated (e.g., grafted). Non-limiting examples of degrons include short amino acid sequences, structural motifs, exposed amino acids, and the like. Degrons can be of prokaryotic or eukaryotic origin and can be used in naturally occurring or non-naturally occurring forms (i.e., recombinant). Degrons can be post-translationally modified to target proteins for degradation, including, but not limited to, ubiquitination, proteolytic cleavage, phosphorylation, methylation, ADP-ribosylation, ampylation, lipidation, alkylation, nitrosylation, succinylation, sumoylation, neddylation, isylation, and the like.

[0113] Useful degrons include ubiquitin-dependent and ubiquitin-independent degrons. For example, in some cases, a protein can be targeted for ubiquitin-independent proteasomal degradation by attachment of an ornithine decarboxylase (ODC) degron (e.g., but not limited to, a mammalian ODC, such as, but not limited to, a rodent ODC, such as, but not limited to, a C-terminal mouse ODC (cODC)). In some cases, useful degrons include those described in Takeuchi et al., Biochem. J (2008) 410:401-407, and / or Matsuzawa et al., PNAS (2005) 102(42):14982-7, the disclosures of which are incorporated herein by reference in their entireties. In some cases, proteins can be targeted for ubiquitin-independent proteasomal degradation by post-translational modifications of degrons (such as, but not limited to, proteolytic cleavage, phosphorylation, methylation, ADP-ribosylation, ampylation, lipidation, alkylation, nitrosylation, succinylation, sumoylation, neddylation, ishilation, etc.), which directly or indirectly result in partial or complete unfolding of the protein, or other mechanisms that result in protein degradation.

[0114] In some cases, the degrons used in the circuits described herein may include ubiquitin-independent degradation signals, and such signals may vary. For example, in some cases, the ubiquitin-independent degradation signals may include a dipeptide motif, such as a cysteine-alanine (i.e., CA) dipeptide motif. In some cases, the ubiquitin-independent degradation signals may include only a dipeptide motif. In some cases, the ubiquitin-independent degradation signals may include amino acid residues in addition to the dipeptide motif, such as, but not limited to, an LXMSCAQE motif, where X may be any amino acid or an LXMSCAQES motif, where X may be any amino acid. In some cases, the LXMSCAQE motif or LXMSCAQES motif may include where X is any amino acid except proline.

[0115] Thus, in some cases, the degradation signal of the degron may comprise a sequence selected from LPMSCAQES with or without a final S, LAMSCAQES with or without a final S, LVMSCAQES with or without a final S, LSMSCAQES with or without a final S, LEMSCAQES with or without a final S, and LKMSCAQES with or without a final S. In some cases, the degradation signal of the degron may comprise an MSCAQE sequence or an MSCAQES sequence.

[0116] Ubiquitin-dependent degrons include, but are not limited to, for example, PEST (proline (P), glutamic acid (E), serine (S) and threonine (T)) sequence-containing degrons and those described in Melvin et al. (PLoS One. (2013) 29;8(10):e78082), the disclosure of which is incorporated herein by reference in its entirety, identified as Bonger, and degrons described as derived from TAZ, HIF-1α, iNOS, SRC3, cyclin D1, IFNAR1, p53, and β-catenin.

[0117] Useful degrons may also contain an E3 ubiquitin ligase domain, which is often defined as a substrate site recognized by an E3 ubiquitin ligase, and a variety of such degrons have been characterized that contain short peptide motifs and specific structural elements. Non-limiting examples of E3 ligases / degrons and corresponding motif patterns include APC / C(DBOX), primary motif.R..L..[LIVM].; APC / C(KEN), primary motif.KEN.; APC / C(ABBA), primary motif [FIVL].[ILMVP][FHY].[DE].{0,3}[DEST]; APCC_TPR_1, primary motif.[ILM]R$; CBL(PTK), primary motif [DN].Y[ST]..P; CBL(MET), primary motif DYR; COP1, primary motif [DE][DE].{2,3}VP[DE]; CRL4_CDT2_1, primary motif [NQ]{0,1}..[ILMV][ST][DEN][FY][FY].{2,3}[KR]{2,3}[^DE]; CRL4_CDT2_2, primary motif [NQ]{0,1}..[ILMV]T[DEN][HMFY][FMY].{2,3}[KR]{2,3}[^DE];Kelch_KEAP1_1,primary motif [DNS].[DES][TNS]GE;Kelch_KEAP1_2,primary motif QD.DLGV;Kelch_actinfilin,primary motif [AP]P[MV][IM]V;Kelch_KLHL3,primary motif E.EE.E[AV]DQH;MDM2_SWIB,primary motif F[^P]W[^P]{2,3}[VIL];Nend_Nbox_1,primary motif ^M{0,1}[FYLIW][^P];Nend_UBRbox_1,primary motif ^M{0,1}[RK][^P].;Nend_UBRbox_2,primary motif ^M{0,1}([ED]).;Nend_UBRbox_3,primary motif ^M{0,1}([NQ]).;Nend_UBRbox_4,primary motif ^M{0,1}(C).;ODPH_VHL_1,primary motif [IL]A(P).{6,8}[FLIVM].[FLIVM];SCF_COI1_1,primary motif..[RK][RK].SL..F[FLM].[RK]R[HRK].[RK].;SCF_FBW7_1,primary motif [LIVMP].{0,2}(T)P..([ST]);SCF_FBW7_2,primary motif [LIVMP].{0,2}(T)P..E;SCF_SKP2-CKS1_1,primary motif..[DE].(T)PK;SCF_TIR1_1,primary motif.[VLIA][VLI]GWPP[VLI]...R.;SCF-TRCP1,primary motif D(S)G.{2,3}([ST]);SIAH,primary motif.PAVP[^P];SPOP,primary motif [AVP].[ST][ST][ST];" indicates any amino acid type, "[X]" indicates the amino acid type allowed at that position, "^X" at the beginning of the pattern indicates that the sequence starts with amino acid type X, "[^X]" means that the position can contain any amino acid other than type X, followed by numbers designated as "X{x,y}", where x and y indicate the minimum and maximum number of "X" amino acid type required at that position. The symbol "$" refers to the C-terminus of the protein chain. Degrons containing E3 ubiquitin ligase domains are described in Guharoy et al., Nature Communications (2016) 7:10239, the disclosure of which is incorporated herein by reference in its entirety. In some cases, useful degrons can include degrons that contain a signal for ER-associated degradation (ERAD), such as, but not limited to, those described in Maurer et al., Genes Genomes & Genetics (2016) 6:1854-1866, the disclosure of which is incorporated herein by reference in its entirety. In some cases, useful degrons can include drug-inducible degrons, such as, but not limited to, auxin-inducible degrons (AIDs) that utilize a specific E3 ubiquitin ligase (e.g., those described in Nishimura et al., Nature Methods (2009) 6(12):917-922, the disclosure of which is incorporated herein by reference in its entirety).

[0118] As will be readily appreciated, degrons that contain an E3 ubiquitin ligase domain are varied, and the circuits of the present disclosure are not limited to the use of those E3 ubiquitin degrons specifically described herein.

[0119] In some cases, inducible degradation in the disclosed circuits may employ direct caging of ubiquitin, which, upon uncaging, localizes the uncaged ubiquitin-containing polypeptide to the proteasome. For example, degrons can be tailored by altering the relative spacing between the components of the degron (see, e.g., Inobe et al., Nature Chemical Biology, 7(3), 161-167, the disclosure of which is incorporated herein by reference in its entirety). Such modifications can thus render the degron non-functional, or at least minimally functional. The degradative function of the modified degron can then be restored by direct localization or ligation (e.g., via dimerization) of ubiquitin to a latent inactivation domain-containing signaling protein. In such embodiments, any convenient method of ligating / localizing a ubiquitin protein to the modified degron can be used. For example, in some cases, a dimerizer pair can be used with one member of the pair present in the latent inactivation domain and the other member bound to the ubiquitin protein. Optionally, a leucine zipper pair can be used with one member of the pair present in the latent inactivation domain and the other member bound to a ubiquitin protein. Optionally, a SpyCatcher / SpyTag pair can be used with one member of the pair present in the latent inactivation domain and the other member bound to a ubiquitin protein. Such examples are for illustrative purposes only and are not intended to be limiting.

[0120] Other useful examples of degrons that can be used in inducible degradation strategies adapted for use in the circuits of the present disclosure include, for example, N-terminal degrons (e.g., but not limited to, those described in Tasaki & Kwon, Trends in Biochemical Sciences (2007) 32(11):520-528, the disclosure of which is incorporated by reference in its entirety), unstructured regions (e.g., but not limited to, those described in Chung et al., Nat Chem Biol. 2015;11(9):713-720, the disclosure of which is incorporated by reference in its entirety), ligand-induced degradation (LID) domains, and destabilization domain (DD) domains (e.g., but not limited to, those described in Bonger et al., Nat Chem Biol. 2015;11(9):713-720, the disclosure of which is incorporated by reference in its entirety). Biol. 2012;7(8):531-537; Grimley et al., Bioorg.Med.Chem.Lett. (2008) 18:759-761; and Chu et al., Bioorg.Med.Chem.Lett. (2008) 18:5941-5944; Iwamoto et al., Chemistry & Biology (2010) 17:981-988; the disclosures of which are incorporated by reference in their entireties herein), prokaryotic proteasome recognition sequences such as ssrA and mf-Lon (e.g., those described in Cameron et al., (2014) Nature biotechnology 32(12):1276-1281, the disclosures of which are incorporated by reference in their entireties herein), and the like.

[0121] An example of an inducible degradation system adapted for use in a circuit that demonstrates feedback control of the subject circuit is the degronLOCKR system, which comprises a caged degron uncaged by an expressed key polypeptide. The degronLOCKR system and circuits using degronLOCKR are described in co-pending U.S. Provisional Application Nos. 62 / 789,418 (filed January 7, 2019), 62 / 850,336 (filed May 20, 2019), and 62 / 789,351, filed January 7, 2019, and U.S. Provisional Application Nos. 62 / 700,681 (filed July 19, 2018), 62 / 785,537 (filed December 27, 2018), and 62 / 788,398 (filed January 4, 2019), the disclosures of which are incorporated herein by reference in their entirety. In some cases, the circuits and / or methods of the present disclosure exclude the use of a caged degron system and / or components of a caged degron system and / or a degronLOCKR system and / or components of a degronLOCKR system. Thus, in some cases, the latent inactivation domain of the present disclosure is not a caged degron. In some cases, the latent inactivation domain of the present disclosure is not a degronLOCKR. In some cases, the latent inactivation domain of the present disclosure does not include a LOCKR domain. In some cases, the latent inactivation domain of the present disclosure does not include a degronLOCKR polypeptide.

[0122] As summarized above, in some cases, a useful strategy for inactivation can include inducible localization. Thus, the latent inactivation domain of the circuits of the present disclosure can include a domain that inducibly localizes a signaling protein to a location that inactivates the signaling protein. In other words, in the presence of a switch polypeptide, the latent inactivation domain can be activated, such that the inactivation domain localizes the bound signaling protein to a part of the cell where the signaling protein is inactive. A variety of different strategies for inducible localization-based inactivation can be used in the circuits described herein.

[0123] For example, in some embodiments, a switch polypeptide can be configured to include a first member of a binding pair linked to a sequestering domain, and the targeted signaling protein can include a second member of the binding pair. Thus, upon expression of the switch polypeptide and binding of the first and second members of the binding pair, the targeted signaling protein can be sequestered and inactivated. In the absence of the switch polypeptide, the targeted signaling protein is not sequestered and thus remains active (i.e., not inactivated).

[0124] As used herein, the term "sequestering domain" generally refers to any protein domain that, when bound to a polypeptide and available (i.e., uncaged or otherwise inaccessible), results in the localization of the polypeptide to a location in the cell where it cannot perform its primary function. For example, if the polypeptide is a transcription factor whose primary function is to drive expression of one or more target genes, the sequestration domain may function to localize the polypeptide to a location in the cell away from the nucleus to prevent the polypeptide from performing its function of driving expression of one or more target genes. However, a polypeptide having transcription factor function may be configured such that, in the absence of a switch polypeptide, it is able to localize to the nucleus of a cell and drive expression of one or more of its target genes.

[0125] The strategy used for inducible localization may depend on the mechanism of action of the polypeptide, and useful strategies are not limited to sequestering transcription factors from the nucleus. Thus, various sequestration domains can be used in the inducible localization-based inactivation strategies described herein. For example, in some cases, the sequestration domain used can localize the bound polypeptide to various locations in the cell, including, but not limited to, the plasma membrane, mitochondria, peroxisomes, vacuoles, actin cytoskeleton, etc. Thus, the sequestration domain can include a tag that, when present and accessible, induces the localization of the bound polypeptide to a specific location in the cell. Useful examples of such tags can include, but are not limited to, for example, plasma membrane targeting tags, mitochondrial membrane targeting tags, peroxisome targeting tags, vacuole targeting tags, actin cytoskeleton targeting tags, etc.

[0126] In some embodiments of the circuits described herein, the targeting signaling protein can include a first member of a binding pair and the switch polypeptide can include a second member of a binding pair linked to a sequestration domain, including, but not limited to, where the sequestration domain includes a plasma membrane targeting tag, a mitochondrial membrane targeting tag, a peroxisome targeting tag, a vacuolar targeting tag, an actin cytoskeleton targeting tag, etc. Other useful targeting tags in some cases can include, but are not limited to, for example, a nuclear localization tag, a nuclear export tag, etc.

[0127] In some embodiments, a useful inducible localization strategy for use in the disclosed circuits may include a switch polypeptide comprising a first leucine zipper domain fused to a localization tag and a second leucine zipper domain fused to a target signaling protein of a signaling pathway. For example, the target signaling protein may be, but is not limited to, a transcription factor member of a signaling pathway, and the localization tag may localize to a cellular location other than the nucleus, such as, but is not limited to, the plasma membrane. In this manner, inactivation of the transcription factor target signaling protein may be controlled by the presence of the switch polypeptide. In the presence of the switch polypeptide, the first leucine zipper binds to the second leucine zipper, and the transcription factor target signaling protein is sequestered from (e.g., outside) the nucleus, thereby inactivating the signaling protein. In the absence of the switch polypeptide, the signaling protein is not sequestered and inactivation is not induced. Thus, the signaling protein may perform its signaling function. This strategy of induced sequestration may in some cases be referred to elsewhere herein as "anchor away."

[0128] Useful leucine zipper binding pairs and sequestration domains that may be used in the anchor-away strategies used in the circuits of the present disclosure include, but are not limited to, those described in Chen et al., ACS Synth.Biol., 2015,4(11):1205-1216, the entire disclosure of which is incorporated herein by reference. Examples of cryptic heterodimerization domains are described in the following publications: These domains can be used to recruit degrons, to localize (anchor-away) proteins, or as dominant negatives. See, for example, Thompson et al., ACS Synthetic Biology 2012 1:118-129 and Chen et al., Nature 2019 565:106-111.

[0129] Useful binding pairs that can be used in the inducible localization strategy are not limited to leucine zipper domains, and the useful domains can be diverse. The first and second members of the binding pair can be directly bound to each other (i.e., can be directly bound) or can be bound via a binding mediator. Any two convenient polypeptide domains that can form a complex with each other can be selected for use as the first and second members of the binding pair. In some cases, polypeptide dimerization domains, including but not limited to those described elsewhere herein, can be used as the first and second members of the binding pair, including when the first and second members of the dimerization pair are directly bound to each other (i.e., can be directly dimerized) or can be dimerized via a dimerization mediator (i.e., a dimerizer).

[0130] Useful examples of binding pairs and first and second members thereof include, but are not limited to, the specific binding pairs and dimerization pairs described, for example, in WO 2014 / 127261 and WO 2017 / 120546, the disclosures of which are incorporated herein by reference in their entireties.

[0131] In some cases, the strategy for inducible localization used in the circuits of the present disclosure may include a caged sequestration domain. As used herein, a "caged sequestration domain" generally refers to a multi-domain polypeptide that includes a localization tag and a cage domain that prevents the localization tag from triggering the localization of the polypeptide and any associated proteins according to the normal function of the localization tag. For example, a polypeptide that includes a caged sequestration domain that includes a plasma membrane targeting tag cannot localize to the plasma membrane when the tag remains caged, but can localize the polypeptide and any associated proteins to the plasma membrane when the tag is uncaged.

[0132] In another example, a polypeptide that includes a caged sequestration domain that includes a nuclear localization tag (such as, but not limited to, a nuclear localization sequence (NLS)) may not localize to the nucleus if the tag remains caged, but may localize the polypeptide and any binding protein to the nucleus if the tag is uncaged. In some cases, this strategy for directed localization may find use in targeting signaling proteins to signaling pathways outside the nucleus of a cell, for example, for function in the cytoplasm and / or plasma membrane. In such embodiments, any convenient nuclear localization sequence may be used.

[0133] In another example, a polypeptide comprising a caged sequestration domain that includes a nuclear export tag (such as, but not limited to, a nuclear export sequence (NES)) may localize to the nucleus when the tag remains caged, but may localize the polypeptide and any binding protein outside the nucleus when the tag is uncaged. In some cases, this strategy for directed localization may find use when a signaling protein targets function in the nucleus of a cell. In such embodiments, any convenient nuclear export sequence may be used. In some cases, a caged sequestration domain that includes a nuclear export tag may exclude a LOCKR domain, including, but not limited to, when the caged sequestration domain does not include a nesLOCKR polypeptide.

[0134] In related embodiments, any useful strategy for caging / uncaging the localization tag can be used, including but not limited to, protection of the localization tag by a domain containing a proteolytic cleavage site cleavable by a protease contained in the switch polypeptide. For example, the localization tag can be attached to the signaling protein, and the localization tag can be caged by a protection domain containing a proteolytic cleavage site cleavable by a protease contained in the switch polypeptide. In the absence of the switch polypeptide, the protease is not present, and the protection domain prevents the localization tag from triggering the localization of the signaling protein. In the presence of the switch polypeptide, the protease cleaves the proteolytic cleavage site, uncaging or otherwise deprotecting the localization tag, triggering the localization of the signaling protein according to the localization tag.

[0135] In some embodiments, LOCKR-based cages can be used in the inducible localization strategy of the disclosed circuits. For example, in some cases, a signaling protein can be configured to include a localization tag (such as, but not limited to, NLS or NES) that can be caged by LOCKR and uncaged by a switch polypeptide that includes a key polypeptide. In some embodiments, LOCKR-based cages can be not used in the inducible localization strategy of the disclosed circuits. For example, in some cases, a signaling protein can be configured to include a localization tag (such as, but not limited to, NLS or NES) that is not caged by a LOCKR domain, and a latent inactivation domain-containing protein can not include a LOCKER domain, such as nesLOCKR or nlsLOCKER. Circuits utilizing the LOCKR domain and the LOCKR system are described in co-pending provisional applications identified by Attorney Docket Nos. UCSF-578 PRV and MBHB 18-1783-PRO, filed January 7, 2019, as well as U.S. Provisional Patent Applications Nos. 62 / 700,681 (filed July 19, 2018), 62 / 785,537 (filed December 27, 2018), and 62 / 788,398 (filed January 4, 2019), the disclosures of which are incorporated herein by reference in their entireties.

[0136] As summarized above, a useful strategy for induced inactivation of signaling proteins of the disclosed circuits may include protein splitting. As used herein, "protein splitting" generally refers to splitting a polypeptide to render it non-functional or to abolish at least one function of the polypeptide. In some cases, a polypeptide may be split reversibly so that the split parts of the polypeptide can be reunited to render the polypeptide functional or to restore at least one function of the polypeptide that was abolished by the splitting. In some cases, a polypeptide may be split irreversibly so that the split parts of the polypeptide cannot be reunited to render the polypeptide functional or to restore a function of the polypeptide that was abolished by the splitting.

[0137] In some embodiments, the polypeptide of the signaling protein is split (disabling at least one function of the signaling protein) and each half of the split polypeptide is fused to a member of a binding pair such that the split signaling protein can be reconstituted upon binding of the members of the binding pair. For example, the signaling protein can be split into a first half and a second half, the first half can be fused to a first member of the binding pair, and the second half of the split signaling protein can be used for the second member of the binding pair. When the first and second members of the binding pair bind to each other, the signaling protein is reconstituted, restoring at least one function of the abolished signaling protein.

[0138] In the circuits of the disclosure, a switch polypeptide can be configured to include a binding pair member that competitively outcompetes one or both of the binding pair members used to reconstitute the split-polypeptide. Thus, in the presence of the switch polypeptide, binding of the first and second members of the binding pair can be disrupted by the switch polypeptide, thereby preventing reconstitution of the signaling protein and resulting in inactivation of the signaling protein.

[0139] In such cases, the binding domain of the switch polypeptide may be referred to herein as a "dominant negative" domain. The dominant negative domain may competitively outcompete binding between domains of a latent inactivation domain bound to or otherwise integrated into a signaling protein of the disclosed circuit. Thus, in some cases, the latent inactivation domain may be said to contain a competitive binding domain that non-covalently binds to a domain of the signaling protein. Such a "competitive binding domain" may be displaced in the presence of the binding domain of the switch polypeptide, resulting in inactivation of the signaling protein. In the absence of the switch polypeptide, the competitive binding domain binds to its binding partner of the split signaling protein, reconstituting the signaling protein and allowing it to perform its function in the signaling pathway.

[0140] In some embodiments, a signaling protein, such as a transcription factor or kinase, can be split such that the signaling protein does not perform its function (i.e., does not induce transcription of a target, does not catalyze phosphorylation of a target, etc.) when the split portions of the protein are not associated with each other. A first half of the split signaling protein can be fused to a first leucine zipper and a second half of the split signaling protein can be fused to a second leucine zipper such that the signaling protein can be reconstituted when the first leucine zipper binds to the second leucine zipper. A dominant negative leucine zipper, i.e., a third leucine zipper that binds to either the first or second leucine zipper with higher affinity than the interaction between the first and second, is incorporated into the switch polypeptide. Thus, the switch polypeptide can competitively outcompete one half of the split signaling protein rendering it non-functional (e.g., renders transcription factor function ineffective, renders kinase function ineffective, etc.). Useful examples of the use of a leucine zipper and a dominant negative leucine zipper to competitively outcompete the binding of two low affinity leucine zippers include, for example and without limitation, the heterologous mammalian bZIP (CEBPα) transactivator and high affinity 3HF dominant negative inhibitor described in Buchler & Cross. Molecular Systems Biology (2009) 5:272, the disclosure of which is incorporated herein by reference in its entirety.

[0141] In some embodiments, a useful protein splitting strategy involves cleaving a protease cleavage site located between dimerization domains that dimerize portions of the split signaling protein to reconstitute the signaling protein. For example, a signaling protein can be split into a first portion having a first member of a dimerization pair and a second portion comprising a second member of the dimerization pair. One or both members of the dimerization pair can be configured to include a protease cleavage site that is cleaved by a protease. Thus, upon expression of a switch polypeptide that includes a protease, the protease cleavage site is cleaved and the reconstituted signaling protein is split, thereby rendering it inactive.

[0142] In some cases, the dimerization pair includes a leucine zipper, such as an antiparallel leucine zipper, that dimerizes the split signaling protein. Thus, the signaling protein is split into a first portion having a first leucine zipper of the antiparallel leucine zipper pair and a second portion including a second leucine zipper of the pair. One or both of the leucine zippers of the pair can be configured to include a protease cleavage site that is cleaved by a protease. Thus, upon expression of the switch polypeptide that includes the protease, the protease cleavage site is cleaved and the reconstituted signaling protein is split, thereby rendering it inactive.

[0143] Useful examples of protease strategies that can be used include, but are not limited to, for example, where the switch polypeptide can include a TEV protease that cleaves a protease cleavage site present in the dimerization domain of the split signaling protein. Cleavage by the TEV protease destroys the ability of the dimerization domain to reconstitute the split signaling protein, thereby inactivating and re-splitting the reconstituted split signaling protein. Thus, in the absence of the TEV protease-containing switch polypeptide, the protease cleavage site remains uncleaved and the split signaling protein remains reconstituted and active. In some cases, components and configurations that can proteolytically cleave the signaling protein to result in an inactivated split signaling protein can include those used in the CHOMP (Circuit of Hacked Orthogonal Modular Proteases) system described in Gao et al., Science. 2018;361(6408):1252-1258, the disclosure of which is incorporated herein by reference in its entirety. As will be readily understood, in some cases, other encoded proteases, corresponding protease cleavage sites, and / or dimerization domains may be substituted for those explicitly described.

[0144] Also, as will be readily appreciated, various configurations of induced inactivation of signaling proteins using protein cleavage may be used in the circuits of the present disclosure.

[0145] As summarized above, the circuits of the present disclosure can include a switch polypeptide, the expression of which can be controlled by a regulatory sequence to which a sequence encoding the switch polypeptide is operably linked. As used herein, the term "switch polypeptide" generally refers to a polypeptide that, when expressed in the presence of a corresponding latent inactivation domain, activates the latent activation domain. Activation of the latent inactivation domain thereby triggers inactivation of the polypeptide to which the inactivation domain is linked or otherwise incorporated, and any other associated proteins, e.g., associated signaling proteins. The composition of the switch polypeptide will vary, e.g., depending on the latent element that the switch is designed to activate.

[0146] A switch polypeptide configured to function with a particular latent inactivation domain can, in some cases, be configured as an orthogonal system. By "orthogonal system" it is generally meant that a particular switch polypeptide functions with a particular latent inactivation domain, but a switch polypeptide does not necessarily function with other latent inactivation domains and / or a latent inactivation domain does not necessarily function with other switch polypeptides. Thus, two or more different orthogonal systems of switch polypeptides and latent inactivation domains can function together (e.g., simultaneously) in the same organism or cell without interference. In other words, a first switch polypeptide of a first orthogonal system can function to activate a first latent inactivation domain of the first system, while the switch polypeptide does not substantially interfere with the function of any element of the second orthogonal system (e.g., a second switch polypeptide, a second latent inactivation domain, etc.). In some cases, an orthogonal system can be used to enable parallel operation of two or more molecular feedback circuits (e.g., including two or more molecular feedback circuits that each regulate a different signaling pathway, two or more molecular feedback circuits that each regulate a different element of the same signaling pathway, etc.).

[0147] Each switch polypeptide and latent inactivation domain need not necessarily be configured as an orthogonal pair. For example, in some cases, two or more different switch polypeptides can function to activate the same latent inactivation domain. Correspondingly, in some cases, two or more different latent inactivation domains can be configured to be activated by the same switch polypeptide.

[0148] Linker The polypeptide used in the circuit of the present disclosure may or may not include a peptide linker.For example, in some cases, the two domains of the subject polypeptide can be linked by a peptide linker.Correspondingly, the nucleic acid sequence that codes for the components of the circuit of the present disclosure can be linked by a sequence that codes for a peptide linker.

[0149] The peptide linker can vary in length from about 3 amino acids (aa) or less to about 200 aa or more, including, but not limited to, 3 aa to 10 aa, 5 aa to 15 aa, 10 aa to 25 aa, 25 aa to 50 aa, 50 aa to 75 aa, 75 aa to 100 aa, 100 aa to 125 aa, 125 aa to 150 aa, 150 aa to 175 aa, or 175 aa to 200 aa. The peptide linker can have a length of 3 aa to 30 aa, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aa. The peptide linker can have a length of 5aa to 50aa, e.g., 5aa to 40aa, 5aa to 35aa, 5aa to 30aa, 5aa to 25aa, 5aa to 20aa, 5aa to 15aa, or 5aa to 10aa.

[0150] Suitable linkers can be readily selected and can be any of a number of suitable lengths, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, can contain 4 to 10 amino acids, from 5 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, can be 1, 2, 3, 4, 5, 6, or 7 amino acids.

[0151] Exemplary linkers include glycine polymers (G), glycine-serine polymers (e.g., including (GS), (GSGGS) (SEQ ID NO: 1) and (GGGS) (SEQ ID NO: 2), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art; furthermore, glycine and glycine-serine polymers can be used, with Gly and Ser both being relatively unstructured and therefore capable of acting as neutral tethers between components. Glycine polymers can be used, with glycine having access to much more phi-psi space than alanine and being much less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Exemplary linkers may include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO:3), GGSGG (SEQ ID NO:4), GSGSG (SEQ ID NO:5), GSGGG (SEQ ID NO:6), GGGSG (SEQ ID NO:7), GSSSG (SEQ ID NO:8).

[0152] Signaling pathways As summarized above, the molecular circuits described herein can be used to regulate a variety of signaling pathways, including natural and synthetic signaling pathways. Suitable signaling pathways include those that are regulated (e.g., activated, inhibited, etc.) by one or more inputs to produce one or more outputs. The inputs and outputs of a signaling pathway can be diverse and can include endogenous (e.g., natural) inputs or outputs of a signaling pathway, as well as inputs and outputs of a heterologous (e.g., engineered or synthetic) signaling pathway.

[0153] In some cases, the input of a signaling pathway associated with a circuit of the present disclosure may include an intracellular signal, including, for example, where the output of the pathway may be intracellular or intercellular. In some cases, the output of a signaling pathway associated with a circuit of the present disclosure may include an intracellular signal, including, for example, where the input of the pathway may be intracellular or intercellular. In some cases, the input of a signaling pathway associated with a circuit of the present disclosure may include an intercellular signal, including, for example, where the output of the pathway may be intracellular or intercellular. In some cases, the output of a signaling pathway associated with a circuit of the present disclosure may include an intercellular signal, including, for example, where the input of the pathway may be intracellular or intercellular.

[0154] In some cases, both the inputs and outputs of a signaling pathway associated with a circuit of the present disclosure may include intracellular signals. In some cases, both the inputs and outputs of a signaling pathway associated with a circuit of the present disclosure may include intercellular signals.

[0155] Suitable non-limiting examples of native signaling pathways that may be modulated using the circuits of the present disclosure include, but are not limited to, e.g., the AKT signaling pathway, the Akt / PKB signaling pathway, the AMPK signaling pathway, the apoptosis signaling pathway, the BMP signaling pathway, the cAMP-dependent pathway, the estrogen signaling pathway, the Hedgehog signaling pathway, the hippo signaling pathway, the immune activation pathway, the immune suppression pathway, the immune cell differentiation pathway, the insulin signaling pathway, the JAK-STAT signaling pathway, the MAPK / ERK signaling pathway, the mTOR signaling pathway, the NF-κB signaling pathway, the nodal signaling pathway, the Notch signaling pathway, the p53 signaling pathway, the PI3K signaling pathway, the TGF beta signaling pathway, the TLR signaling pathway, the TNF signaling pathway, the VEGF signaling pathway, the Wnt signaling pathway, and the like.

[0156] Suitable, non-limiting examples of pathways whose elements may be engineered to contain a potential inactivation domain as described herein include the PANTHER (Protein Analysis Through Evolutionary Analysis) pathways described as part of the Gene Ontology Phylogenetic Annotation Project. Also included are the 5HT1 type receptor-mediated signaling pathway, 5HT2 type receptor-mediated signaling pathway, 5HT3 type receptor-mediated signaling pathway, 5HT4 type receptor-mediated signaling pathway, 5-hydroxytryptamine biosynthesis, 5-hydroxytryptamine degradation, acetate utilization, activin beta signaling pathway, adenine and hypoxanthine salvage pathway, adrenaline and noradrenaline biosynthesis, alanine biosynthesis, allantoin degradation, ALP23B signaling pathway, alpha adrenergic receptor signaling pathway, Alzheimer's disease-amyloid secretase pathway, Alzheimer's disease-presenilin pathway, aminobutyric acid degradation, anandamide biosynthesis, anandamide degradation, androgen / estrogenic / progesterone pathway. steroid biosynthesis, angiogenesis pathway, angiotensin II-stimulated signaling via G proteins and beta-arrestins, apoptosis signaling pathway, arginine biosynthesis, ascorbate degradation, asparagine and aspartate biosynthesis, ATP synthesis, axon guidance via netrin, axon guidance via semaphorins, axon guidance via slit / robo, B cell activation pathway, beta1 adrenergic receptor signaling pathway, beta2 adrenergic receptor signaling pathway, beta3 adrenergic receptor signaling pathway, biotin biosynthesis, blood coagulation, BMP / activin signaling pathway, bupropion degradation, cadherin signaling pathway, coenzyme A-bound carnitine metabolism, carnitine metabolism, CCKR signaling, cell cycle, cholesterol biosynthesis, chorismate biosynthesis, circadian clock system, cobalamin biosynthesis, coenzyme A biosynthesis, corticotropin-releasing factor receptor signaling pathway,Cysteine ​​biosynthesis, Rho Cytoskeleton regulation by GTPases, de novo purine biosynthesis, de novo pyrimidine deoxyribonucleotide biosynthesis, de novo pyrimidine ribonucleotide biosynthesis, DNA replication, dopamine receptor-mediated signaling pathway, DPP-SCW signaling pathway, DPP signaling pathway, EGF receptor signaling pathway, endocannabinoid signaling, endothelin signaling pathway, enkephalin release, FAS signaling pathway, FGF signaling pathway, flavin biosynthesis, tetrahydrofolate biosynthesis, formyltetrahydroformate biosynthesis, fructose galactose metabolism, GABA-B receptor II signaling, gamma aminobutyric acid synthesis, GBB signaling pathway, general transcription by RNA polymerase I, general transcription regulation, glutamine glutamate conversion, glycolysis, gonadotropin-releasing hormone receptor pathway, hedgehog signaling pathway, heme biosynthesis, heterotrimeric G protein signaling pathway-Gi alpha and Gs alpha mediated pathway, heterotrimeric G protein signaling pathway-Gq alpha and Go alpha mediated pathway, heterotrimeric G protein signaling pathway delivery pathway-rod outer segment phototransduction, histamine H1 receptor-mediated signaling pathway, histamine H2 receptor-mediated signaling pathway, histamine synthesis, histidine biosynthesis, Huntington's disease pathway, hypoxic response via HIF activation, inflammation via chemokine and cytokine signaling pathways, insulin / IGF pathway-mitogen-activated protein kinase kinase / MAP kinase cascade, insulin / IGF pathway-protein kinase B signaling cascade, integrin signaling pathway, interferon gamma signaling pathway, interleukin signaling pathway, ionotropic glutamate receptor pathway, isoleucine biosynthesis, JAK / STAT signaling pathway, leucine biosynthesis, lipoate biosynthesis, lysine biosynthesis, mannose metabolism, metabotropic glutamate receptor group III pathway, metabotropic glutamate receptor group II pathway, metabotropic glutamate receptor group I pathway, methionine biosynthesis, methyl citrate cycle, methylmalonyl pathway, mRNA splicing, muscarinic acetylcholine receptor 1 and 3 signaling pathway.Muscarinic ethylcholine receptor 2 and 4 signaling pathway, MYO signaling pathway, N-acetylglucosamine metabolism, nicotine degradation, nicotine pharmacokinetic pathway, nicotinic acetylcholine receptor signaling pathway, Notch signaling pathway, O-antigen biosynthesis, opioid-prodynorphin pathway, opioid-proenkephalin pathway, opioid-proopiomelanocortin pathway, ornithine degradation, oxidative stress response, oxytocin receptor-mediated signaling pathway, p38 MAPK pathway, p53 pathway, p53 pathway induced by glucose deprivation, P53 pathway feedback loop 1, p53 pathway feedback loop 2, pantothenate biosynthesis, Parkinson's disease, PDGF signaling pathway, pentose phosphate pathway, peptidoglycan biosynthesis, phenylacetate degradation, phenylalanine biosynthesis, phenylethylamine degradation, phenylpropionate degradation, PI3 kinase pathway, plasminogen activation cascade, pyridoxal-5-phosphate biosynthesis, proline biosynthesis, PRPP biosynthesis, purine metabolism, pyridoxal phosphate salvage pathway, pyrimidine metabolism, pyruvate metabolism, Ras pathway, S-adenosylmethionine biosynthesis, salvage pyrimidine deoxyribonucleotides, salvage pyrimidine ribonuclease leotide, SCW signaling pathway, serine glycine biosynthesis, conversion of succinate to propionate, sulfate fixation, transport of synaptic vesicles, TCA cycle, T cell activation pathway, TGF-beta signaling pathway, thiamine biosynthesis, thiamine metabolism, threonine biosynthesis, thyrotropin releasing hormone receptor signaling pathway, Toll pathway, Toll receptor signaling pathway, transcriptional regulatory bZIP transcription factors, triacylglycerol metabolism, tryptophan biosynthesis, tyrosine biosynthesis, ubiquitin proteasome pathway, valine biosynthesis, vasopressin synthesis, VEGF signaling pathway, vitamin B6 biosynthesis, vitamin B6 metabolism, vitamin D metabolism and pathway, Wnt signaling pathway, xanthine and guanine salvage pathway, etc.

[0157] Further non-limiting examples of signaling pathways and their descriptions include the following: the AKT signaling pathway (AKT is a serine / threonine kinase involved in mediating various biological responses such as the inhibition of apoptosis); angiopoietin-TIE2 signaling (angiopoietins are a new family of growth factor ligands that bind to the TIE2 / TEK RTKs (receptor tyrosine kinases)); antigen processing and presentation by MHC (antigen processing and presentation is the process that results in the binding of proteins to major histocompatibility complex (MHC) molecules for recognition by T cells); death receptor-mediated apoptosis (certain cells have their own sensors called death receptors (DRs) that detect the presence of extracellular death signals and rapidly ignite the cell's intrinsic apoptotic machinery); the APRIL pathway (in the immune response, APRIL functions as a costimulator of B and T cell proliferation and supports class switching); the B cell development pathway (the B cell receptor mediated apoptosis pathway, which is a pathway that mediates the proliferation and proliferation of B cells and T cells), and the B cell development pathway (the B cell receptor mediated apoptosis pathway, which is a pathway that mediates the proliferation and proliferation of B cells and T cells). The BCR complex is typically composed of an antigen-binding subunit, which is made up of two Ig heavy chains, two Ig light chains, and a signaling subunit; the BMP pathway (bone morphogenetic proteins (BMPs) are a large subclass of the transforming growth factor-beta (TGF-beta) superfamily); cancer immunoediting (the immune system attempts to suppress tumor growth, but tumor cells can escape or attenuate this immune pressure); the CCR5 pathway in macrophages (the CC motif chemokine receptor type 5 (CCR5) is a member of the chemokine receptor subclass of the G protein-coupled receptor (GPCR) superfamily); CD4 and CD8 T cell lineage (each mature T cell typically retains expression of a co-receptor molecule (CD4 or CD8) with binding characteristics that match the major histocompatibility complex (MHC) binding characteristics of its T cell receptor (TCR)), cell apoptosis pathway (apoptosis is a naturally occurring process in which cells are induced into programmed cell death), CTL-mediated apoptosis (cytotoxic T lymphocytes (CTLs), also known as killer T cells, are produced during cell-mediated immunity designed to eliminate somatic cells that display foreign epitopes), CTLA4 signaling pathway (co-stimulatory CTLA4 pathway attenuates or downregulates T cell activation. CTLA4 is adesigned to remove somatic cells that display foreign epitopes), cytokine network (cytokines are classified based on the biological response to pro- or anti-inflammatory cytokines depending on their effect on immune cells), ErbB family pathway (the ErbB family of transmembrane receptor tyrosine kinases (RTKs) plays an important role during organ growth and development), Fas signaling (FAS (also called APO1 or CD95) is a death domain-containing member of the tumor necrosis factor (TNF) receptor superfamily), FGF pathway (one of the best-characterized modulators of angiogenesis is heparin-binding fibroblast growth factor (FGF)), granulocyte adhesion and dialysis (granulocyte adhesion and extravasation have been analyzed mainly in the context of non-lymphatic endothelial cells), Granzyme pathway (Granzyme A (GzmA) activates a caspase-independent cell death pathway with morphological characteristics of apoptosis), GSK3 signaling (GSK3 is a ubiquitously expressed, highly conserved, serine / threonine protein kinase found in all eukaryotes), hematopoiesis from pluripotent stem cells (hematopoietic stem cells are classified into long-term, short-term, and multipotent progenitor cells based on the degree of self-renewal capacity), hematopoiesis from pluripotent stem cells (pluripotent stem cells can form nearly every conceivable tissue type found in humans), IL-2 gene expression in activated and resting T cells (IL-2 is a cytokine that stimulates the growth, proliferation, and differentiation of T cells, B cells, NK cells, and other immune cells), I The I-6 pathway (IL-6 is a pleiotropic cytokine that affects many physiological events in the immune system and various organs), the IL-10 pathway (IL-10 is a pleiotropic cytokine with important immunoregulatory functions whose activity affects many immune cell types), the IL-22 pathway (IL-22 is a member of the IL-10 family of cytokines that exerts multiple effects in the immune system), the interferon pathway (interferons are pleiotropic cytokines best known for their ability to induce cellular resistance to viral infections), the JAK / STAT pathway (the JAK / STAT pathway is a signal transduction cascade whose evolutionarily conserved roles include cell proliferation and hematopoiesis),The MAPK family pathway (mitogen-activated protein kinases (MAPKs) belong to a large family of serine / threonine protein kinases that are conserved in diverse organisms such as yeast and humans), Nanog in mammalian ESC pluripotency (NANOG is a transcription factor that is transcribed in pluripotent stem cells and downregulated during cell differentiation), and the p53-mediated apoptosis pathway (tumor protein p53 is a nuclear transcription factor that regulates the expression of diverse genes involved in apoptosis, growth arrest, or senescence in response to genotoxic or cellular stress). ), the pathogenesis of rheumatoid arthritis (Rheumatoid arthritis (RA) is a chronic symmetric polyarticular disease that primarily affects the small joints of the hands and feet), PI3K signaling in B lymphocytes (Phosphoinositide 3-kinase (PI3K) regulates many biological processes including cell growth, differentiation, survival, proliferation, migration, and metabolism), the RANK pathway (RANKL and its receptor RANK are key regulators of bone remodeling and are essential for osteoclast development and activation), and RANK signaling in osteoclasts (RANKL induces differentiation of osteoclast precursor cells). The TGF-beta pathway (members of the transforming growth factor (TGF)-beta family play important roles in the development, homeostasis, and repair of most tissues), the THC differentiation pathway (type 1 (TH1) and type 2 (TH2) T helper cells are derived from T helper cells and provide help to cells of both the innate and adaptive immune systems), the TNF signaling pathway (tumor necrosis factor (TNF) is a multifunctional proinflammatory cytokine that affects lipid metabolism, coagulation, insulin resistance, and endothelial function), and the TNF-stimulation pathway (TNF-stimulation pathway). the tumor necrosis factor (TNF) superfamily pathway (the tumor necrosis factor (TNF) superfamily is composed of 19 members that signal through 29 receptors that are members of the TNF receptor (TNFR) superfamily); transendothelial migration of leukocytes (transport of plasma proteins and solutes across the endothelium involves two distinct pathways (transcellular and paracellular junctions)); the tumoricidal effect of hepatic NK cells (the liver is a major site of tumor formation and metastasis); the TWEAK pathway (TWEAK is a cell surface-associated protein that belongs to the tumor necrosis factor (TNF) superfamily and is involved in the regulation of tumor cell proliferation and metastasis);Vascular endothelial growth factor (VEGF) is a highly conserved genetic pathway that has evolved from a simple to a complex system.

[0158] As summarized above, elements of a signaling pathway (including, but not limited to, those described herein) can be modified to contain a latent inactivation domain, such that inactivation of the signaling pathway member can be controlled by expression of a switch polypeptide. Suitable pathway components that can be used include, for example, input-receiving members, intermediate members, and output-producing members, including, but not limited to, the corresponding members of the pathways listed above.

[0159] Similarly, essentially any synthetic pathway can be regulated using the molecular circuits described herein. Suitable, non-limiting examples of synthetic signaling pathways that can be regulated using the circuits of the present disclosure include, but are not limited to, pathways controlled by synthetic or engineered receptors, such as, but not limited to, CAR, engineered TCR, synNotch, etc.

[0160] In some cases, the pathways modulated using the circuits of the present disclosure can include immunomodulatory pathways, such as immunostimulatory or immunosuppressive pathways. Such immunomodulatory pathways can be natural or synthetic pathways and can be endogenous to the cells in which the circuits are used or heterologous to the cells in which the circuits are used.

[0161] Suitable, non-limiting examples of synthetic signaling pathways that may be regulated using the circuits of the present disclosure also include biosynthetic and / or bioproduction pathways, which may be natural or synthetic pathways and may be used in cells and / or organisms to which the pathway is endogenous or heterologous.

[0162] Non-limiting examples of biosynthetic pathways that can be regulated using the circuits of the present disclosure include, but are not limited to, hormone production pathways (e.g., insulin production pathway, estrogen / progesterone production pathway, androgen production pathway, growth hormone production pathway, etc.), opioid production pathways, isobutanol production pathways, non-ribosomal polyketide synthetase (NRPS) production pathways, antibiotic production pathways, chemotherapeutic drug production pathways, artemisinic acid production pathways, terpenoid production pathways, polyketide production pathways, and the like.

[0163] Non-limiting examples of synthetic biosynthetic pathways include, but are not limited to, synthetic hormone production pathways, synthetic opioid production pathways, synthetic antibiotic production pathways, synthetic chemotherapeutic drug production pathways, synthetic artemisinic acid production pathways, synthetic terpenoid production pathways, synthetic polyketide production pathways, and the like.

[0164] nucleic acid As summarized above, the present disclosure also provides nucleic acids encoding molecular feedback circuits. The subject nucleic acids include, for example, sequences encoding switch polypeptides, sequences encoding signaling proteins comprising latent inactivation domains, and the like. Such nucleic acids can be configured such that one or more sequences are operably linked to regulatory sequences. For example, the nucleic acids can be configured such that a sequence encoding a switch polypeptide is operably linked to a regulatory sequence responsive to the output of a signaling pathway. Nucleic acids are provided that encode essentially any circuit that uses a latent inactivation domain, including, but not limited to, those specifically described herein. Included are isolated nucleic acids encoding the subject circuits, as well as various constructs, such as vectors (e.g., expression cassettes, recombinant expression vectors, viral vectors, and the like), that include such nucleic acids.

[0165] Recombinant expression vectors of the present disclosure include those that contain one or more of the described nucleic acids. Nucleic acids that contain nucleotide sequences encoding all or a portion of the components of the circuits of the present disclosure are, in some embodiments, DNA, including, for example, recombinant expression vectors. Nucleic acids that contain nucleotide sequences encoding all or a portion of the components of the circuits of the present disclosure are, in some embodiments, RNA, including, for example, in vitro synthesized RNA.

[0166] As summarized above, in some cases, the subject circuits may utilize a coding nucleic acid (e.g., a nucleic acid encoding a switch polypeptide or a latent inactivation domain-binding signaling protein) operably linked to a regulatory sequence, such as a transcriptional control element (e.g., a promoter, enhancer, etc.). In some cases, the transcriptional control element is inducible. In some cases, the transcriptional control element is constitutive. In some cases, the promoter is functional in eukaryotic cells. In some cases, the promoter is functional in prokaryotic cells. In some cases, the promoter is a cell type specific promoter. In some cases, the promoter is a tissue specific promoter.

[0167] Depending on the host / vector system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc., can be used in the expression vector (see, e.g., Bitter et al. (1987), Methods in Enzymology, 153:516-544).

[0168] The promoter may be a constitutively active promoter (i.e., a promoter that is constitutively active / "ON" state), it may be an inducible promoter (i.e., a promoter whose state, active / "ON" or inactive / "OFF", is controlled by an external stimulus, e.g., temperature, the presence or absence of a particular compound or protein), it may be a spatially restricted promoter (i.e., a transcriptional control element, enhancer, etc., e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the "ON" or "OFF" state during a particular stage of embryonic development or during a particular stage of a biological process, e.g., the cell cycle in mammals, the hair follicle cycle, the circadian cycle in mammals, etc.).

[0169] Suitable promoters and enhancer elements are known in the art. For expression in bacterial cells, suitable promoters include, but are not limited to, lacI, lacZ, T3, T7, gpt, lambda P and trc. For expression in eukaryotic cells, suitable promoters include, but are not limited to, yeast promoters (e.g., promoters of yeast mating pathway genes, yeast galactose-inducible promoter, etc.), light and / or heavy chain immunoglobulin gene promoters and enhancer elements, cytomegalovirus immediate early promoter, herpes simplex virus thymidine kinase promoter, early and late SV40 promoters, promoters present in long terminal repeat sequences from retroviruses, mouse metallothionein-I promoter, and various tissue-specific promoters known in the art.

[0170] In some cases, transcriptional control elements of various strengths can be used. For example, promoters of various strengths (e.g., weak promoters, intermediate promoters, and strong promoters), such as, for example, constitutive promoters or inducible promoters, can be used, including, but not limited to, constitutive promoters pREV1, pRNR2, pRET2, etc. In some cases, the strength of a promoter can be adjusted, e.g., made weaker or stronger, by decreasing or increasing, respectively, the number of binding sites (e.g., DBD binding sites) in the promoter. Thus, the number of binding sites present in a promoter of interest can vary and can range from 1 to 6 or more, including, but not limited to, 1, 2, 3, 4, 5, 6, etc.

[0171] In some cases, the transcriptional control element of the nucleic acid described herein may include a cis-acting regulatory sequence. Any suitable cis-acting regulatory sequence may be used in the nucleic acid described herein. For example, in some cases, the cis-acting regulatory sequence may be or include an upstream activating sequence or an upstream activating sequence (UAS). In some cases, the UAS of the nucleic acid described herein may be a Gal4-responsive UAS. In some cases, useful transcriptional control elements may include immune-related transcriptional control elements, such as, but not limited to, the nuclear factor of activated T cells (NFAT) promoter.

[0172] In some cases, transcriptional control of the disclosed circuits may include the use of one or more regulatory elements responsive to synthetic transcription factors. Synthetic transcription factors and the regulatory elements responsive thereto may vary, including, but not limited to, estradiol ligand binding domain (LBD)-based synthetic transcription factors, progesterone LBD-based synthetic transcription factors, zinc finger-based synthetic transcription factors, and the like. Synthetic transcription factors may be chimeric and may include various domains, such as DNA binding domains (DBDs), activation domains, zinc finger domains, and the like. Useful domains, such as LBDs, DBDs, activation domains, and the like, may vary, including, but not limited to, Gal4p DBD, Zif268 transcription factor DBD, viral activation domains (e.g., VP16, VP64, and the like), Msn2p activation domain, and the like. Non-limiting examples of useful synthetic transcription factors include, but are not limited to, GEM (Gal4 DNA binding domain-estradiol hormone binding domain-Msn2 activation domain), Z3PM (Z3 zinc finger-progesterone hormone binding domain-Msn2 activation domain), and the like. Correspondingly, useful regulatory elements can vary and include promoters responsive to synthetic transcription factors, including, but not limited to, pZ promoter, pZ3 promoter, pGAL1 promoter, and the like. Examples of suitable promoters and synthetic transcription factors include, for example, those described herein and those described in Aranda-Diaz et al., ACS Synth Biol. (2017) 6(3):545-554, the disclosure of which is incorporated herein by reference in its entirety.

[0173] Suitable promoters may optionally include suitable reversible promoters. Reversible promoters can be isolated and derived from many organisms, such as eukaryotes and prokaryotes. It is well known in the art to modify the reversible promoter from a first organism for use in a second organism (e.g., a first prokaryote and a second eukaryote, a first eukaryote and a second prokaryote, etc.). Such reversible promoters and systems based on such reversible promoters but also including additional regulatory proteins include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator protein (AlcR), etc.), tetracycline-regulated promoters (e.g., promoter systems including TetActivators, TetON, TetOFF, etc.), steroid-regulated promoters (e.g., rat glucocorticoid receptor promoter system, human estrogen receptor promoter system, retinoid promoter system, thyroid promoter system, ecdysone promoter system, mifepristone promoter system, etc.), metal-regulated promoters (e.g., metallothionein promoter system, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid-regulated promoters, ethylene-regulated promoters, benzothiadiazole-regulated promoters, etc.), temperature-regulated promoters (e.g., heat shock-inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light-regulated promoters, synthetic inducible promoters, etc.

[0174] Inducible promoters suitable for use include any inducible promoter described herein or known to one of skill in the art. Examples of inducible promoters include chemically / biochemically regulated promoters and physically regulated promoters, such as alcohol-regulated promoters, tetracycline-regulated promoters (e.g., anhydrotetracycline (aTc)-responsive promoters and other tetracycline-responsive promoter systems including the tetracycline repressor protein (tetR), tetracycline operator sequence (tetO) and tetracycline transactivator fusion protein (tTA)), steroid-regulated promoters (e.g., rat glucocorticoid receptor, human estrogen receptor activator (tTA)), and the like). These include, but are not limited to, promoters based on the receptor, moth ecdysone receptor, and promoters from the steroid / retinoid / thyroid receptor superfamily), metal-regulated promoters (e.g., promoters derived from metallothionein (a protein that binds and sequesters metal ions) genes from yeast, mouse and human), pathogenesis-regulated promoters (e.g., induced by salicylic acid, ethylene or benzothiadiazole (BTH)), temperature / heat-inducible promoters (e.g., heat shock promoters), and light-regulated promoters (e.g., light-responsive promoters from plant cells).

[0175] In some cases, a useful promoter may be an immune cell promoter. For example, in embodiments in which components of the circuit are expressed in immune cells, immune cell promoters may be used. Suitable immune cell promoters include, but are not limited to, for example, CD8 cell-specific promoters, CD4 cell-specific promoters, neutrophil-specific promoters, and NK-specific promoters. For example, the CD4 gene promoter may be used, see, for example, Salmon et al. (1993) Proc. Natl. Acad. Sci. USA 90:7739, and Marodon et al. (2003) Blood 101:3416. As another example, the CD8 gene promoter may be used. NK cell-specific expression may be achieved by using the Ncr1 (p46) promoter, see, for example, Eckelhart et al. (2011) Blood 117:1565.

[0176] In some cases, the immune cell-specific promoter of the nucleic acids of the disclosure can be a promoter such as a B29 gene promoter, a CD14 gene promoter, a CD43 gene promoter, a CD45 gene promoter, a CD68 gene promoter, an IFN-β gene promoter, a WASP gene promoter, a T cell receptor β chain gene promoter, a V9γ (TRGV9) gene promoter, a V2δ (TRDV2) gene promoter, or the like.

[0177] In some cases, the nucleic acid comprising a nucleotide sequence encoding the circuit of the present disclosure or one or more elements thereof is a recombinant expression vector or is included in a recombinant expression vector. In some embodiments, the recombinant expression vector is a viral construct, such as a recombinant adeno-associated virus (AAV) construct, a recombinant adenovirus construct, a recombinant lentivirus construct, a recombinant retrovirus construct, etc. In some cases, the nucleic acid comprising a nucleotide sequence encoding the circuit of the present disclosure, or one or more elements thereof, is a recombinant lentivirus vector. In some cases, the nucleic acid comprising a nucleotide sequence encoding the circuit of the present disclosure, or one or more elements thereof, is a recombinant AAV vector.

[0178] Suitable expression vectors include viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., Hum Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655); adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998;Flannery et al., PNAS 94:6916 6921, 1997;Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997;Jomary et al., Gene Ther 4:683 690, 1997;Rolling et al., Hum Gene Ther 10:641 648, 1999;Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93 / 09239; Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617), SV40, herpes simplex virus, human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999); and retroviral vectors (e.g., vectors derived from murine leukemia virus, spleen necrosis virus, and retroviruses (e.g., Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus). In some cases, the vector is a lentiviral vector.Also suitable are transposon-mediated vectors, such as the piggyBac vector and the sleeping beauty vector.

[0179] In some cases, the nucleic acid of the present disclosure may have a single sequence encoding two or more polypeptides, where the expression of two or more polypeptides is enabled by the presence of a sequence element between the individual coding regions that facilitates the individual expression of the individual polypeptides. Such a sequence element may be referred to herein as a bicistronic promoting sequence, and when a bicistronic promoting sequence is present between two coding regions, it allows the expression of a separate polypeptide from each coding region present in a single nucleic acid sequence. In some cases, the nucleic acid may include two coding regions that encode two polypeptides present in a single nucleic acid with a bicistronic promoting sequence between the coding regions. Any suitable method may be used to individually express multiple individual polypeptides from a single nucleic acid sequence, as well as any suitable method of bicistronic expression.

[0180] In some cases, the bicistronic-promoting sequence may allow for expression of two polypeptides from a single nucleic acid sequence that are temporarily linked by a cleavable linking polypeptide. In such cases, the bicistronic-promoting sequence may contain one or more encoded peptide cleavage sites. Suitable peptide cleavage sites include those of self-cleaving peptides as well as those that are cleaved by separate enzymes. In some cases, the peptide cleavage site of the bicistronic-promoting sequence may include a furin cleavage site (i.e., the bicistronic-promoting sequence may encode a furin cleavage site).

[0181] In some cases, the bicistronic enhancing sequence may encode a self-cleaving peptide sequence. Useful self-cleaving peptide sequences include, but are not limited to, peptide 2A sequences, such as, but not limited to, the T2A sequence.

[0182] Optionally, the bicistronic-promoting sequence may include one or more spacer-encoding sequences. Spacer-encoding sequences generally encode amino acid spacers, sometimes also referred to as peptide tags. Useful spacer-encoding sequences include, but are not limited to, V5 peptide-encoding sequences, including, for example, sequences encoding the V5 peptide tag.

[0183] Multiple or bicistronic expression of multiple coding sequences from a single nucleic acid sequence may utilize methods that include, but are not limited to, furin cleavage, T2A, and V5 peptide tag sequences. For example, an internal ribosome entry site (IRES)-based system may be used in some cases. Any suitable method of bicistronic expression may be used, including, but not limited to, those described in Yang et al. (2008) Gene Therapy. 15(21):1411-1423, and Martin et al. (2006) BMC Biotechnology. 6:4, the disclosures of which are incorporated herein by reference in their entireties.

[0184] cell As summarized above, the present disclosure also provides cells containing nucleic acids encoding molecular feedback circuits. Cells modified to contain one or more nucleic acids encoding one or more molecular feedback circuits and / or one or more components thereof are considered genetically modified herein, and such modifications can be stable or transient, as desired. Useful cells can include prokaryotic and eukaryotic cells, including, but not limited to, bacterial cells, plant cells, animal cells, yeast cells, mammalian cells, rodent cells, non-human primate cells, human cells, and the like.

[0185] Suitable cells include stem cells, progenitor cells, and partially and fully differentiated cells. Suitable cells include neurons, liver cells, kidney cells, immune cells, cardiac cells, skeletal muscle cells, smooth muscle cells, lung cells, etc.

[0186] Suitable cells include stem cells (e.g., embryonic stem (ES) cells, induced pluripotent stem (iPS) cells), germ cells (e.g., oocytes, sperm, oogonia, spermatogonia, etc.), somatic cells such as fibroblasts, oligodendrocytes, glial cells, hematopoietic cells, neurons, muscle cells, bone cells, liver cells, pancreatic cells, etc.

[0187] Suitable cells include human embryonic stem cells, fetal cardiomyocytes, myofibroblasts, mesenchymal stem cells, autologous transformed expanded cardiomyocytes, adipocytes, totipotent cells, pluripotent cells, blood stem cells, myoblasts, adult stem cells, bone marrow cells, mesenchymal cells, embryonic stem cells, parenchymal cells, epithelial cells, endothelial cells, mesothelial cells, fibroblasts, osteoblasts, chondrocytes, exogenous cells, endogenous cells, stem cells, hematopoietic stem cells, bone marrow derived progenitor cells, cardiomyocytes, skeletal cells, fetal cells, undifferentiated cells, multipotent progenitor cells, unipotent progenitor cells, monocytes, cardiac myoblasts, skeletal myoblasts, macrophages, capillary endothelial cells, xenogeneic cells, allogeneic cells, and postnatal stem cells.

[0188] Optionally, the cell is a stem cell. Optionally, the cell is an induced pluripotent stem cell. Optionally, the cell is a mesenchymal stem cell. Optionally, the cell is a hematopoietic stem cell. Optionally, the cell is an adult stem cell.

[0189] Suitable cells include bronchoalveolar stem cells (BASCs), bulge epithelial stem cells (bESCs), corneal epithelial stem cells (CESCs), cardiac stem cells (CSCs), epidermal neural crest stem cells (eNCSCs), embryonic stem cells (ESCs), endothelial progenitor cells (EPCs), hepatic oval cells (HOCs), hematopoietic stem cells (HSCs), keratinocyte stem cells (KSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), pancreatic stem cells (PSCs), retinal stem cells (RSCs), and skin-derived progenitors (SKPs).

[0190] In some cases, the cell is an immune cell. Suitable mammalian immune cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. In some cases, the cell is not an immortalized cell line, but instead is a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell, immune cell precursor, or immune stem cell obtained from an individual. In one example, the cell is a lymphoid cell, e.g., a lymphocyte or precursor thereof, obtained from an individual. In another example, the cell is a cytotoxic cell or precursor thereof, obtained from an individual. In another example, the cell is a stem cell or progenitor cell obtained from an individual.

[0191] As used herein, the term "immune cells" generally includes white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow. "Immune cells" include, for example, lymphoid cells, i.e., lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), T regulatory cells (Tregs), and gamma delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which can mediate cytotoxic responses. "B cells" include mature and immature cells of the B cell lineage, including pre-B cells, immature B cells, mature B cells, memory B cells, and cells expressing CD19, such as plasmablasts. Immune cells include B cell precursors, such as pro-B cells, and B cell lineage derivatives, such as plasma cells.

[0192] Cells encoding the circuits of the present disclosure can be generated by any convenient method. Nucleic acids encoding one or more elements of the subject circuits can be stably or transiently introduced into the subject immune cells, including cases where the subject nucleic acids are only present transiently, maintained extrachromosomally, or integrated into the host genome. Introduction of the subject nucleic acids and / or genetic modification of the subject immune cells can occur in vivo, in vitro, or ex vivo.

[0193] In some cases, the subject nucleic acid introduction and / or genetic modification is performed ex vivo. For example, immune cells, stem cells, etc. are obtained from an individual, and the cells obtained from the individual are modified to express components of the circuit of the present disclosure. Thus, the modified cells can be modified by regulatory feedback to one or more optimal signaling pathways as defined by one or more molecular feedback circuits present on the introduced nucleic acid. In some cases, the modified cells are regulated ex vivo. In other cases, the cells are introduced into (e.g., the individual from which the cells were obtained) and / or are already present in the individual, and the cells are regulated in vivo, for example, by administering a nucleic acid or vector to the individual in vivo.

[0194] In some cases, cells using the feedback circuits of the present disclosure can be therapeutic cells useful for targeted cell therapy. For example, in applications such as cell therapy using immune cells, the immune cells are engineered to deliver a therapeutic payload of interest to the human body. If the output of these engineered cells is too high, toxic effects can occur (e.g., cytokine release syndrome (CRS) observed in CAR T cell therapy), while on the other hand, if the output is too low, the treatment may be ineffective. Therapeutic cells can be fine-tuned to achieve a desired level of output (i.e., set point) under well-controlled laboratory conditions. However, the dynamic environment in which engineered therapeutic cells function makes it difficult to ensure that the output remains constant over time. Using the molecular circuits described herein to implement feedback control, engineered cells have the ability to automatically correct for disturbances they encounter in the environment (e.g., including disturbances that cause the output to drift). In one aspect, the self-regulating engineered cells are more robust in in vivo scenarios, thereby improving existing cell therapy applications of synthetic biology.

[0195] In some cases, cell therapies such as CAR T cells or synthetic receptor (e.g., SynNotch)-enabled T cells greatly benefit from feedback control as a safety mechanism. The feedback controller of CAR T cells can regulate the level of T cell activation and inhibit toxic effects such as CRS caused by overstimulation of immune cells. Similarly, in SynNotch T cells, for example, feedback control can allow the delivery of the desired payload at a precise concentration regardless of what disturbances exist or are introduced to the engineered cells. As can be easily understood, the use of feedback control in therapeutic cells is not limited to these approaches and includes other approaches.

[0196] Useful cells that can use the circuits of the present disclosure are not limited to therapeutic cells. For example, in some cases, cells used for bioproduction can be used. As used herein, "biological production" generally refers to the process of using cells to produce desired elements for various applications, such as industrial, commercial, biomedical, research, etc. The biological products produced in a biological production process can be diverse, and such products can be endogenous or heterologous to the cells and / or organisms used in their production. In some cases, biological products of interest include, but are not limited to, recombinant therapeutic proteins, viruses (e.g., recombinant viruses for gene therapy), vaccines, antibodies, proteins and peptides (e.g., enzymes, growth factors, etc.), polysaccharides, nucleic acids (including DNA and RNA), cells, and nutritional products. The circuits and / or methods of the present disclosure can be used in conjunction with a number of different production techniques known in the art, such as production of biological products using cells (e.g., mammalian, yeast, bacterial and / or insect cells) in bioreactors, methods involving the use of transgenic animals (e.g., goats or chickens), methods involving the use of transgenic plants (e.g., tobacco, seeds or moss), and other methods known to those of skill in the art.

[0197] Where used, cells suitable for biological production include, but are not limited to, for example, COS cells, NS0 cells, SP2 / 0 cells, YB2 / 0 cells, etc. Useful cells can be of prokaryotic origin (e.g., bacterial) or eukaryotic origin (including, for example, mammalian, yeast, plant, etc.), and in some cases can be established cell culture lines. Suitable cells may optionally include HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.

[0198] In some cases, useful biological production cells can include yeast cells. Suitable yeast cells include, but are not limited to, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia spp. sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium graminoum gramineum, Fusarium venenatum, Neurospora crassa, Chlamydomonas reinhardtii, and the like.

[0199] In some cases, useful biological production cells may include prokaryotic cells. Suitable prokaryotic cells include, but are not limited to, any of a variety of laboratory strains of Escherichia coli, Lactobacillus species, Salmonella species, Shigella species, and the like. See, for example, Carrier et al. (1992) J. Immunol. 148:1176-1181, U.S. Patent No. 6,447,784, and Sizemore et al. (1995) Science 270:299-302. Examples of Salmonella strains that can be used include, but are not limited to, Salmonella typhi and Salmonella typhimurium. Suitable Shigella strains include, but are not limited to, Shigella flexneri, Shigella sonnei, and Shigella disenteriae. Typically, the laboratory strain is a non-pathogenic strain. Non-limiting examples of other suitable bacteria include, but are not limited to, Bacillus subtilis, Pseudomonas pudita, Pseudomonas aeruginosa, Pseudomonas mevalonii, Rhodobacter sphaeroides, Rhodobacter capsulatus, Rhodospirillum rubrum, Rhodococcus sp., and the like. In some embodiments, the cell is Escherichia coli.

[0200] In some cases, feedback control is useful for cells used in metabolic engineering where the balance of enzymes in metabolic pathways is essential to obtain optimal titers of products. It is well known that intermediates of metabolic pathways, or even their end products, have at least some level of toxicity to host cells. Therefore, optimizing the ratio of enzymes is beneficial to maximize the amount of product produced while maintaining effective cell growth. Furthermore, due to the large size of reactors used in industrial fermentation, during fermentation, cells may experience a highly variable environment and may be exposed to a variety of different stress factors at different levels. These disturbances may shift the activity of enzymes and force a "rebalance" of pathway activity. The feedback controller using the molecular circuit of the present disclosure mitigates the effects of disturbances and maximizes titers by dynamically rebalancing enzyme ratios.

[0201] method As summarized above, the present disclosure also provides methods of using the latency inactivation-based molecular feedback circuit. Such methods include, but are not limited to, for example, methods of regulating a signaling pathway in a cell in which the cell is or has been genetically modified with the latency inactivation-based molecular feedback circuit. Any of the above circuits and their components can be used in the methods described herein.

[0202] A variety of inactivation strategies can be used to inactivate the signaling proteins of the circuits used in the methods of the present disclosure. For example, in some cases, the inactivation of the signaling protein can use a degradation-based strategy, including, for example, where the inactivation domain used results in the degradation of the signaling protein. In some cases, the inactivation domain can be or can include a degradation domain.

[0203] In some cases, inactivation of a signaling protein may use a protease-based strategy, including, for example, where a latent inactivation domain is activated by a proteolytic cleavage event mediated by a switch polypeptide that is or includes a protease. In some cases, a signaling protein may be inactivated by an expressed protease, including, but not limited to, where the signaling protein is cleaved by a protease.

[0204] In some cases, the inactivation of the signaling protein can use a localization-based strategy, for example, inactivation of the signaling protein by the inactivation domain includes relocalization of the signaling protein mediated by a switch polypeptide. In some cases, inactivation of the signaling protein by the inactivation domain includes sequestering the signaling protein. In some cases, relocalization of the signaling protein can involve a binding event that associates a localization signal with the signaling protein. For example, in some cases, the inactivation domain can include a first member of a binding pair, and the switch domain can include a second member of the binding pair linked to a sequestering domain.

[0205] In some cases, inactivation of a signaling protein can use dominant-negative inhibition of the signaling protein, including, for example, when a switch polypeptide comprises a dominant-negative domain. For example, the subject method can include a split signaling protein that is reconstituted by binding of two members of a binding pair, where the switch polypeptide comprises a member of a binding pair that includes or is linked to a dominant-negative domain. In such methods, binding of the switch polypeptide can disrupt the reassociation of the halves of the split signaling protein, thereby inactivating the split signaling protein. Thus, in some cases, dominant-negative inhibition of a signaling protein can include, but is not necessarily limited to, competitive binding of a non-covalent binding domain to a member of a binding pair that is linked to or otherwise incorporated into the signaling protein.

[0206] As explained above, in some cases, useful members of a binding pair that can be used in the various methods of the disclosure can include members of a leucine zipper binding pair, i.e., the first and second members of the binding pair can include first and second portions of a leucine zipper.

[0207] Methods used to regulate signaling of a cellular signaling pathway can serve a variety of purposes. For example, in some cases, the circuits of the present disclosure can be used in methods to provide feedback control of a signaling pathway of interest. In some cases, the feedback control can include, among other aspects, negative feedback control, which can prevent a pathway from remaining active, for example, when a particular pathway output is generated and / or produced above a threshold level. In some cases, the feedback control can include, among other aspects, positive feedback control, which can amplify a particular pathway output. In some cases, feedback control can result in a more stable output of a signaling pathway, including, but not limited to, for example, when the signaling output of the pathway is insulated from uncertainties such as, for example, environmental factors and inputs.

[0208] As explained above, the cells of the disclosed methods can vary and can include in vitro and / or ex vivo cells genetically modified with one or more nucleic acids encoding one or more components of one or more circuits described herein. In some cases, the cells are primary cells obtained from a subject. In some cases, the cells are obtained from a cell culture.

[0209] Thus, the disclosed method may include obtaining cells for use in the method, including when such cells are unmodified or when such cells have already been genetically modified to contain the disclosed circuitry. In some cases, the disclosed method may include performing a genetic modification. In some cases, the disclosed method may include harvesting the cells, including when the cells are harvested before and / or after genetic modification. Methods for harvesting cells may vary and may include, for example, harvesting cells from a cell culture, harvesting a cell sample from a subject that contains the cells of interest, etc.

[0210] In some cases, the disclosed methods may include modulating (e.g., increasing and / or decreasing) signaling of a signaling pathway, including activating a latent inactivation domain to cause inactivation of a signaling protein of the pathway. As described herein, the disclosed circuits may include feedback, including positive and negative feedback. The feedback of the methods may depend, at least in part, on the output of the signaling pathway. Thus, once the circuit is introduced and / or cells containing the circuit are delivered, the regulation of the signaling pathway by the circuit may not require further manipulation; in other words, the feedback regulation of the signaling pathway by the circuit may be essentially automatic.

[0211] Thus, in methods using cells that include a molecular feedback circuit of the present disclosure, the cells can optionally be administered to a subject without the need for further manipulation of the circuit. For example, when a subject is treated with cells that include a molecular feedback circuit of the present disclosure, the treatment can include administering the cells to the subject, which can include cases where such administration is the only therapeutic intervention for treating the subject.

[0212] In such methods, the cells that may be administered include, but are not limited to, immune cells. In such methods, the circuitry may be configured to optionally regulate signaling of a natural or synthetic signaling pathway of an immune cell, such as, but not limited to, an immune activating pathway or an immune suppressing pathway. Non-limiting examples of suitable immune activating pathways include cytokine signaling pathways, B cell receptor signaling pathways, T cell receptor signaling pathways, and the like, whether regulated by natural or synthetic means. Non-limiting examples of suitable immune suppressing pathways include inhibitory immune checkpoint pathways, and the like, whether regulated by natural or synthetic means.

[0213] The disclosed methods may include administering to a subject cells expressing a therapeutic agent. Such cells may include the disclosed molecular feedback circuitry and may or may not be immune cells. For example, in some cases, the methods may include administering to a subject non-immune cells that endogenously or heterologously produce a therapeutic agent, where the production of the therapeutic agent is controlled in whole or in part by a molecular feedback circuitry. In some cases, the methods may include administering to a subject immune cells that endogenously or heterologously produce a therapeutic agent, where the production of the therapeutic agent is controlled in whole or in part by a molecular feedback circuitry. Non-limiting examples of suitable encoded therapeutic agents include, but are not limited to, hormones or elements of a hormone production pathway, such as insulin or elements of the insulin production pathway, estrogen / progesterone or elements of the estrogen / progesterone production pathway, testosterone or elements of the androgen production pathway, growth hormone or elements of the growth hormone production pathway, and the like.

[0214] Such methods may optionally be used to treat a subject for a condition, including for example, a metabolic or hormonal deficiency, in which case the molecular feedback circuit may be configured such that the output of the molecular feedback circuit controls, in whole or in part, metabolic or hormonal production and / or secretion.

[0215] In some cases, the method may include contacting a cell with one or more nucleic acids encoding the circuit, such contact being sufficient to introduce the nucleic acid into the cell. Any convenient method of introducing a nucleic acid into a cell may find use herein, including, but not limited to, viral transfection, electroporation, lipofection, irradiation, chemical transformation, the use of a transducing carrier (e.g., a transducible carrier protein), and the like. The nucleic acid may be introduced into a cell maintained or cultured in vitro or ex vivo. The nucleic acid may also be introduced into a cell of a living subject in vivo, for example, by using one or more vectors (e.g., viral vectors) that deliver the nucleic acid to the cell, without the need to isolate, culture, or maintain the cell outside the subject.

[0216] Any convenient method of delivering a circuit encoding a component may be used in the subject methods. Optionally, a subject circuit may be delivered by administering to a subject cells expressing the circuit. Optionally, a subject circuit may be delivered by administering to a subject a nucleic acid comprising one or more nucleotide sequences encoding the circuit. Administering a nucleic acid encoding a circuit to a subject may include administering to a subject cells containing the nucleic acid, where the nucleic acid may or may not be expressed. Optionally, administering a nucleic acid encoding a circuit to a subject may include administering to a subject a vector designed to deliver the nucleic acid to a cell.

[0217] The subject methods can include introducing into a subject in need thereof a cell containing a nucleic acid sequence encoding a therapeutic agent, the expression of which is controlled at least in part by a molecular feedback loop. The therapeutic agent can be a therapeutic agent for the treatment of cancer. The introduced cell can be an immune cell, including, for example, a myeloid cell or a lymphoid cell.

[0218] Non-limiting examples of cancers that may be treated include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, AIDS-related cancers (e.g., Kaposi's sarcoma, lymphoma, etc.), anal cancer, appendix cancer, astrocytoma, atypical teratoma / rhabdoid tumor, basal cell carcinoma, bile duct cancer (extrahepatic), bladder cancer, bone cancer (e.g., Ewing's sarcoma, osteosarcoma, and malignant fibrous histiocytoma, etc.), brain stem glioma, brain tumors (e.g., astrocytoma, central nervous system embryonal tumor, central nervous system germ cell tumor, craniopharyngioma, ependymoma, etc.), breast cancer (e.g., female breast cancer, male breast cancer, pediatric breast cancer, etc.), bronchial tumors, Burkitt's lymphoma, carcinoma-like tumors (e.g., childhood, gastrointestinal, etc.), cancer of unknown primary, cardiac (heart) tumors, central nervous system (e.g., atypical teratomas / rhabdoid tumors, embryonal tumors, germ cell tumors, lymphomas, etc.), cervical cancer, childhood cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), chronic myeloproliferative neoplasms, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, breast ducts (e.g., bile duct, extrahepatic, etc.), ductal carcinoma in situ (DCIS), embryonal tumors, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, unilateral -ing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer (e.g., intraocular melanoma, retinoblastoma, etc.), fibrous histiocytoma of bone (e.g., malignant, osteosarcoma, etc.), gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (e.g., extracranial, extragonadal, ovarian, testicular, etc.), gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, cardiac cancer, hepatocellular (liver) cancer, histiocytosis (e.g., Langerhans cell, etc.), Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor (e.g., pancreatic neuroendocrine tumors, etc.), Kaposi's sarcoma, kidney cancer (e.g., renal cell, Wilms' tumor, childhood kidney tumors, etc.), Langerhans cell histiocytosis, laryngeal cancer, leukemia (e.g., acute lymphoblastic (ALL), acute myeloid (AML), chronic lymphocytic (CLL), chronic myeloid (CML), hairy cell, etc.), lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer (e.g., non-small cell, small cell, etc.), lymphoma (e.g., AIDS-related, Burkitt, cutaneous T cell, Hodgkin, non-Hodgkin, primary central nervous system (CNS), etc.), macroglobulinemia (e.g.,Waldenstrom, etc.), male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of occult primary, NUT midline carcinoma, oral cancer, midline carcinoma with multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell neoplasm, mycosis mycosis, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia (e.g. chronic (CML), etc.), myeloid leukemia (e.g. acute Myeloproliferative neoplasms (e.g., chronic), nasal and paranasal cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin's lymphoma, non-small cell lung cancer, oral cavity cancer, oral cavity cancer (e.g., lip), oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer (e.g., epithelial, germ cell tumors, tumors of low malignant potential), pancreatic cancer, pancreatic neuroendocrine tumors (islet cell tumors), papillomatosis, paraganglioma, paranasal sinus and nasal cancer, and parathyroid gland. Cancer of the penis, cancer of the pharynx, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcomas (e.g., Ewing, Kaposi, osteosarcoma, rhabdomyosarcoma, soft tissue, uterine, etc.), Sezary syndrome, skin cancer (e.g., childhood, melanoma, Merkel cell carcinoma, non-melanoma etc.), small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer (e.g., with latent primary, metastatic, etc.), gastric cancer, T-cell lymphoma, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, ureter and renal pelvis cancer, urethral cancer, uterine cancer (e.g., endometrium), uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor, etc.,

[0219] In some cases, the method of the present disclosure can be used to treat a subject for immune dysfunction (e.g., including but not limited to, when the condition is an autoimmune disease). For example, in some cases, the molecular feedback circuit of the present disclosure can be configured to regulate the immune activation level of a subject with an autoimmune disease, thereby controlling the subject's autoimmune response, in order to treat the subject for the autoimmune disease. In some cases, a cell configured to include the molecular feedback circuit of the present disclosure, the output of which is immune suppression, can be administered to a subject with an autoimmune disease.

[0220] The present disclosure further includes methods of making the nucleic acids, circuits, and cells used in the methods described herein. In making the subject nucleic acids and circuits and their components, any convenient method of nucleic acid manipulation, modification, and amplification (e.g., collectively referred to as "cloning") can be used. In making the subject cells containing nucleic acids encoding the described circuits, any convenient method of transfection, transduction, culture, and the like can be used.

[0221] The nucleotide sequences encoding all or part of the components of the disclosed circuits may be present in an expression vector and / or a cloning vector. When the subject circuit or its components are divided between two or more separate polypeptides, the nucleotide sequences encoding the two or more polypeptides may be cloned into the same vector or into separate vectors. Expression vectors may include selectable markers, origins of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like.

[0222] Numerous suitable vectors and promoters are known to those of skill in the art, and many are commercially available for making the subject recombinant constructs. By way of example, the following vectors are provided: Bacterial: pBs, phagescript, PsiX 174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia).

[0223] Expression vectors generally have a restriction site located near the promoter sequence for convenient insertion of a nucleic acid sequence encoding a heterologous protein. A selectable marker operable in the expression host may also be present.Suitable expression vectors include viral vectors (e.g., vaccinia virus, poliovirus, adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5:1088 1097, 1999; WO 94 / 12649; WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984; and WO 95 / 00655), adeno-associated virus (e.g., Ali et al., Hum Gene Ther 9:81 86, 1998;Flannery et al., PNAS 94:6916 6921, 1997;Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997;Jomary et al., Gene Ther 4:683 690, 1997;Rolling et al., Hum Gene Ther 10:641 648, 1999;Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166:154-165; and Flotte et al., PNAS (1993) 90:10613-10617 in WO 93 / 09239), SV40, herpes simplex virus, human immunodeficiency virus (e.g., Miyoshi et al., PNAS 94:10319 23, 1997; Takahashi et al., J Virol 73:7812 7816, 1999) and retroviral vectors (e.g., vectors derived from retroviruses such as murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0224] As described above, in some embodiments, the nucleic acid comprising a nucleotide sequence encoding the disclosed circuit or a component thereof is, in some embodiments, DNA or RNA, e.g., in vitro synthesized DNA, recombinant DNA, in vitro synthesized RNA, recombinant RNA, etc. Methods for in vitro synthesis of DNA / RNA are known in the art, and any known method can be used to synthesize DNA / RNA comprising a desired sequence. Methods for introducing DNA / RNA into a host cell are known in the art. Introduction of DNA / RNA into a host cell can be performed in vitro, ex vivo, or in vivo. For example, a host cell (e.g., NK cell, cytotoxic T lymphocyte, etc.) can be transduced, transfected, or electroporated in vitro or ex vivo with DNA / RNA comprising a nucleotide sequence encoding all or a portion of the disclosed circuit.

[0225] The disclosed method may further include culturing the cells genetically modified to encode the disclosed circuit, including, but not limited to, culturing the cells prior to administration, culturing the cells in vitro or ex vivo (e.g., in the presence or absence of one or more antigens), etc. Any convenient method for cell culture may be used, and such methods will vary based on a variety of factors, including, but not limited to, the type of cells to be cultured, the intended use of the cells (e.g., whether the cells are cultured for research or therapeutic purposes), etc. In some cases, the disclosed method may further include the general process of cell culture, including, but not limited to, seeding the cell culture, feeding the cell culture, passaging the cell culture, splitting the cell culture, analyzing the cell culture, treating the cell culture with drugs, harvesting the cell culture, etc.

[0226] The disclosed methods may optionally further include obtaining and / or collecting cells to be used in the subject methods. Optionally, the cells are collected from a subject. Collecting cells from a subject may include obtaining a tissue sample from the subject and enriching, isolating and / or growing the cells from the tissue sample. Cell isolation and / or enrichment may be performed using any convenient method, including, for example, isolation / enrichment by culture (e.g., adherent culture, suspension culture, etc.), cell sorting (e.g., FACS, microfluidics, etc.), and the like. Cells may be collected from any convenient cell-tissue sample, including, for example, but not limited to, blood (including, for example, peripheral blood, umbilical cord blood, etc.), bone marrow, biopsy, skin sample, buccal swab, and the like. Optionally, the cells are obtained from a source, including, for example, a blood bank, a tissue bank, and the like. The obtained cells may have been previously isolated or may have been obtained as part of a tissue sample, e.g., isolation / enrichment may be performed after obtaining the cells and prior to use. In certain instances, the cells obtained may be non-primary cells, including, for example, cells of a cultured cell line. Cells suitable for use in the methods described herein are further detailed herein.

[0227] Examples of Non-Limiting Aspects of the Disclosure The aspects (including embodiments) of the present subject matter described above may be beneficial alone or in combination with one or more other aspects or embodiments. Without limiting the foregoing, certain non-limiting aspects of the present disclosure, numbered 1-[xxx], are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each individually numbered aspect can be used or combined with any of the preceding or succeeding individually numbered aspects. This is intended to support all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. 1. A signaling protein that drives an output of a signaling pathway when activated by an input of the signaling pathway, the signaling protein comprising a latent inactivation domain; a regulatory sequence responsive to said output, operably linked to a nucleic acid sequence encoding a switch polypeptide whose expression triggers said inactivation domain to inactivate said signaling molecule; A molecular feedback circuit comprising: 2. The circuit of aspect 1, wherein the input or the output or both comprise an intracellular signal. 3. The circuit of aspect 1, wherein the input or the output or both comprise an intercellular signal. 4. The circuit of any one of aspects 1 to 3, wherein the inactivation domain is a degradation domain. 5. The circuit of embodiment 4, wherein the degradation domain comprises a degron. 6. The circuit of aspect 4 or 5, wherein the latent inactivation domain comprises a protection domain that prevents degradation of the signaling protein and is deprotected by the switch polypeptide. 7. The circuit of embodiment 6, wherein the switch polypeptide comprises a protease. 8. The circuit of any of aspects 1 to 3, wherein the inactivation domain comprises a first member of a binding pair. 9. The circuit of embodiment 8, wherein the switch polypeptide comprises a second member of the binding pair linked to a sequestration domain. 10. The circuit of aspect 9, wherein the sequestration domain comprises a plasma membrane targeting tag, a mitochondrial membrane targeting tag, a peroxisome targeting tag, a vacuolar targeting tag, or an actin cytoskeleton targeting tag. 11. The circuit of embodiment 8, wherein said switch domain comprises a second member of said binding pair that comprises a dominant negative domain. 12. The circuit of aspect 11, wherein the latent inactivation domain comprises a competitive binding domain non-covalently bound to the first member of the binding pair. 13. The circuit of any of aspects 8 to 12, wherein the first and second members of the binding pair comprise first and second portions of a leucine zipper. 14. The circuit of any of aspects 1 to 13, wherein the signaling protein is a positive regulator of the signaling pathway. 15. The circuit of any of aspects 1 to 14, wherein the signaling protein is a negative regulator of the signaling pathway. 16. The circuit of any of aspects 1 to 15, wherein the signaling protein is an intermediate member of the signaling pathway or a transcription factor. 17. The circuit of aspect 16, wherein the transcription factor is a synthetic transcription factor. 18. The circuit of any of aspects 1 to 17, wherein the regulatory sequence comprises a binding site for a transcription factor of the output. 19. The circuit of aspect 18, wherein the regulatory sequence comprises multiple binding sites for the transcription factor. 20. The circuit of aspect 19, wherein the plurality of binding sites is between 2 and 10 binding sites. 21. The circuit of any of aspects 16 to 20, wherein the output is expression of the transcription factor. 22. The circuit of any of aspects 1-15, wherein the signaling protein is a receptor and the input is a ligand for the receptor. 23. The circuit of any of aspects 1 to 22, wherein the signaling pathway is selected from the group consisting of AKT signaling pathway, Akt / PKB signaling pathway, AMPK signaling pathway, apoptosis signaling pathway, BMP signaling pathway, cAMP-dependent pathway, estrogen signaling pathway, hedgehog signaling pathway, hippo signaling pathway, immune activation pathway, immune suppression pathway, immune cell differentiation pathway, insulin signaling pathway, JAK-STAT signaling pathway, MAPK / ERK signaling pathway, mTOR signaling pathway, NF-kappaB signaling pathway, nodal signaling pathway, notch signaling pathway, p53 signaling pathway, PI3K signaling pathway, TGF beta signaling pathway, TLR signaling pathway, TNF signaling pathway, VEGF signaling pathway, and Wnt signaling pathway. 24. The circuit of any of aspects 1 to 23, further comprising a regulatory sequence operably linked to the nucleic acid sequence encoding said signaling protein. 25. The circuit of aspect 24, wherein the regulatory sequence operably linked to the nucleic acid sequence encoding the signaling protein is the native promoter of the signaling protein. 26. The circuit of any of aspects 1 to 22, wherein the signaling pathway is a synthetic signaling pathway. 27. The circuit of aspect 26, wherein the receptor is a synthetic receptor. 28. The circuit of aspect 27, wherein the synthetic receptor is a synNotch receptor. 29. The circuit of aspect 27, wherein the synthetic receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). 30. The circuit of aspect 29, wherein the output is immune activation or immune suppression. 31. One or more nucleic acid molecules encoding the molecular feedback circuit of any of embodiments 1 to 30. 32. A cell genetically modified to contain one or more nucleic acid molecules according to embodiment 31. 33. The cell according to aspect 32, which is a eukaryotic cell. 34. A method of treating a subject for a condition, comprising administering to the subject an effective amount of a eukaryotic cell according to aspect 33. 35. The method of aspect 34, wherein the condition is cancer and the output of the molecular feedback circuit is immune activation. 36. The method of aspect 34, wherein the condition is an autoimmune disease and the output of the molecular feedback circuit is immunosuppression. 37. The method of aspect 34, wherein the condition is a metabolic or hormonal deficiency and the output of the molecular feedback circuit is the production and / or secretion of the metabolic or hormonal deficiency. 38. A method for modulating signal transduction in a cell signaling pathway, comprising: genetically modifying the cells with a molecular feedback loop; The molecular feedback circuit comprises: a nucleic acid sequence encoding a signaling protein of the signaling pathway, the signaling protein comprising a latent inactivation domain; a regulatory sequence responsive to an output of the signaling pathway, the regulatory sequence operably linked to a nucleic acid sequence encoding a switch polypeptide that, when expressed, activates a latent inactivation domain; wherein said activated inactivation domain inactivates said signaling protein, thereby regulating signaling of said signaling pathway. 39. The method of embodiment 38, wherein the regulating comprises negative feedback. 40. The method of embodiment 38, wherein the regulating comprises positive feedback. 41. The method of any of aspects 38 to 40, wherein inactivation of the signaling protein by the inactivation domain comprises degradation of the signaling protein. 42. The method of embodiment 41, wherein the inactivation domain is a degradation domain. 43. The method of embodiment 41 or 42, wherein the latent inactivation domain is activated by a proteolytic cleavage event mediated by the switch polypeptide. 44. The method of any of aspects 38 to 40, wherein inactivation of the signaling protein by the inactivation domain comprises sequestration of the signaling protein. 45. The method of embodiment 44, wherein the inactivation domain comprises a first member of a binding pair and the switch domain comprises a second member of the binding pair linked to a sequestration domain. 46. ​​The method of any of aspects 38 to 40, wherein inactivation of the signaling protein by the inactivation domain comprises dominant-negative inhibition of the signaling protein. 47. The method of embodiment 46, wherein the switch domain comprises a second member of the binding pair linked to a dominant negative domain. 48. The method of aspect 46 or 47, wherein the latent inactivation domain comprises a competitive binding domain non-covalently bound to the first member of the binding pair. 49. The method of any of aspects 45 to 48, wherein the first and second members of the binding pair comprise first and second portions of a leucine zipper. 50. The method of any of aspects 38 to 49, wherein the cell is an in vitro cell or an ex vivo cell. 51. The method of any of aspects 38 to 50, wherein the signaling pathway is a native signaling pathway of the cell. 52. The method of embodiment 51, wherein the native signaling pathway is a native biosynthetic pathway. 53. The method of embodiment 52, wherein the native biosynthetic pathway is a hormone production pathway. 54. The method of embodiment 53, wherein the hormone production pathway is selected from the group consisting of the insulin production pathway, the estrogen / progesterone production pathway, the androgen production pathway, and the growth hormone production pathway. 55. The method of aspect 44, wherein the cell is an immune cell and the native signaling pathway is an immune stimulatory or inhibitory pathway. 56. The method of aspect 48, wherein the immune activation pathway is selected from the group consisting of a cytokine signaling pathway, a B cell receptor signaling pathway, and a T cell receptor signaling pathway. 57. The method of aspect 48, wherein the immunosuppressive pathway is an inhibitory immune checkpoint pathway. 58. The method of any of aspects 38 to 50, wherein the signaling pathway is a synthetic signaling pathway. 59. The method of aspect 58, wherein the signaling protein is a synNotch receptor and the output is release of the intracellular domain of the synNotch receptor. 60. The method of aspect 58, wherein the cell is an immune cell and the signaling pathway is a synthetic immune stimulatory pathway or a synthetic immune inhibitory pathway. 61. The method of aspect 60, wherein the immune cell is a myeloid cell or a lymphoid cell. 62. The method of embodiment 61, wherein the immune cell is a lymphoid cell selected from the group consisting of a T lymphocyte, a B lymphocyte, and a natural killer cell. 63. The method of any of aspects 60 to 62, wherein the signaling protein is a synthetic immune receptor. 64. The method of aspect 63, wherein the synthetic immune receptor is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). 65. The method of any of aspects 58 to 64, wherein the output is immune activation or immune suppression. 66. The method of embodiment 58, wherein the synthetic signaling pathway is a synthetic biosynthetic pathway. 67. The method of embodiment 66, wherein the synthetic biosynthetic pathway is selected from the group consisting of a hormone production pathway, an opioid production pathway, an antibiotic production pathway, a chemotherapeutic drug production pathway, an artemisinic acid production pathway, a terpenoid production pathway, and a polyketide production pathway. EXAMPLES

[0228] The following examples are provided to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise specified, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; kb, kilobase; bp, base pairs; nt, nucleotides; im, intramuscular (intramuscularly); ip, intraperitoneal (intraperitoneally); sc, subcutaneous (subcutaneously); and the like.

[0229] Example 1: A feedback loop strategy using latent inactivation of signaling pathway components Various strategies were used to construct feedback circuits that use latent inactivation of components of signaling pathways. Schematic examples of signaling pathways, referred to in these examples as "biological networks," are shown in the left panel of FIG. 7. As shown, signaling proteins "A," "B," and "Z," which are components of the signaling pathway, transform inputs to result in transcriptional "outputs." Examples of strategies used include induced degradation using degronLOCKR feedback (FIG. 7, second panel), sequestration feedback (FIG. 7, third panel), and competitive feedback (FIG. 7, fourth panel). In some cases, as described below, examples using LOCKR-based systems (e.g., degronLOCKR, nesLOCKR, and nlsLOCKR) provide proof-of-concept for the general strategies of latent inactivation used in the circuits described herein. However, in such examples, the use of a LOCKR-based system is merely exemplary and / or comparative, and in light of the present disclosure, one could readily substitute a LOCKR-independent system (i.e., a system that does not use LOCKR generally, or, for example, that does not use degronLOCKR, nesLOCKR, or nlsLOCKR in particular).

[0230] In the degronLOCKR feedback example, the circuit is configured such that the caged degron binds to signaling protein "B". The circuit is further configured to include a nucleic acid having a regulatory sequence operably linked to a sequence encoding a key polypeptide. As shown, the regulatory sequence used is responsive to an intermediate element of a signaling pathway. Thus, when the signaling pathway transmits a signal and the intermediate element of the signaling pathway is expressed or otherwise activated, the key polypeptide is expressed. Expression of the key polypeptide uncages the caged degron, resulting in degradation of the signaling pathway element "B" and negative feedback on pathway signaling. Further examples of degronLOCKR feedback are provided in Example 4.

[0231] In an example of sequestration feedback, the circuit is configured such that a first member of the binding pair, also referred to as "prey," binds to signaling protein "B." The circuit is further configured to include a nucleic acid having a regulatory sequence operably linked to a sequence encoding a second member of the binding pair, referred to as "bait" in FIG. 7, bound to a protein motif localized to the plasma membrane (PM). As shown, the regulatory sequence used is responsive to an intermediate element of the signaling pathway. Thus, when the signaling pathway transmits a signal and the intermediate element of the signaling pathway is expressed or otherwise activated, a "bait PM" polypeptide is expressed. The expressed bait PM polypeptide binds to a prey domain bound to signaling pathway element "B." As a result, signaling pathway element "B" is sequestered to the plasma membrane away from the intracellular location where element "B" functions in the signaling pathway. Thus, sequestration of element "B" results in a negative feedback on pathway signaling. One example of sequestration feedback includes an "anchor-away" circuit, which is described in more detail below.

[0232] In a competitive feedback example, a circuit is constructed such that a signaling pathway element "B" is split and the split parts are reassembled by a leucine zipper domain incorporated into each split part. As a result, the reassembled split protein can transmit a signal in the signaling pathway in the same way as the corresponding element of the unsplit element "B". The circuit is further constructed to include a nucleic acid having a regulatory sequence operably linked to a sequence encoding a dominant negative leucine zipper domain. The dominant negative leucine zipper domain binds to one or both of the element "B" leucine zipper domains with a higher affinity than the affinity with which the element "B" leucine zipper domain binds. Thus, when present, the dominant negative leucine zipper domain competitively outcompetes the reassembly of the split parts of element "B". Thus, the signaling pathway element "B" is inactivated when the dominant negative leucine zipper domain is expressed, resulting in a negative feedback on pathway signaling. An example of competitive feedback includes the circuit described in Example 3 below.

[0233] Example 2: Anchor Away: Isolation-Based Feedback Circuit A sequestration-based feedback circuit, named "AnchorAway", was constructed as shown generally in Figure 8. Specifically, a synthetic transcription factor ("SynTF") GEM was fused to one half of a leucine zipper ("prey") and induced by E2 to activate transcription from the pGAL1 promoter. GEM activates the production of Z3PM and RFP as a sensor. Z3PM is induced by Pg to activate transcription from the pZ3 promoter. Z3PM activates the production of the other half of the leucine zipper fused to YFP and a plasma membrane targeting domain ("Prey PM"). Feedback localizes GEM to the plasma membrane, preventing it from activating transcription. An alternative diagram of the AnchorAway circuit showing the "Anchor", "Controller", "Process", and "Control Output" portions of the circuit is provided in Figure 9.

[0234] To measure the circuit output, the anchor-away feedback circuit was constructed and tested in combination with a corresponding "no feedback" control circuit using YFP. In FIG. 10, steady-state YFP output as a function of progesterone is shown for the anchor-away feedback, with the no feedback behavior shown at the top. Increasing promoter strength (from top to bottom) corresponds to an increasing number of Z3 binding sites that activate the inhibitor (i.e., bait). These data indicate that a stronger promoter results in a greater feedback effect, resulting in a lower maximum output and a change in the slope of the feedback curve shown.

[0235] Example 3: Competitive-Based Feedback CCAAT / enhancer-binding protein alpha (CEBPα) is a leucine zipper transcription factor that dimerizes and activates the GCAAT promoter. "DN" is a dominant negative that binds with high affinity to CEBPα monomers, preventing the transcription factor from binding to DNA and activating transcription (FIG. 11; see also Buchler & Cross. Molecular Systems Biology (2009) 5:272, the disclosure of which is incorporated herein by reference in its entirety).

[0236] The anchor-away feedback circuit was redesigned to replace the sequestration-based feedback with components for competition-based feedback, as shown diagrammatically in Figure 12. GEM (and E2) determine the set point of the circuit via the production of CEBPα. CEBPα activates transcription of Z3PM. Z3PM is induced by Pg and activates transcription from the pZ3 promoter. Z3PM activates the production of YFP and the other half of the leucine zipper (DN) that splits the CEBPα TF dimer. Thus, the feedback inactivates CEBPα, adjusting the production of Z3PM as output increases.

[0237] The closed loop feedback circuit shown in FIG. 12 was constructed and compared to the corresponding open loop feedback circuit using YFP to measure the circuit output. In FIG. 13, the steady state YFP output as a function of progesterone is shown with feedback (left) and without feedback (right). As shown, increasing the amount of E2 increased the output of the circuit. Feedback reduced the maximum output of the circuit and also reduced the slope of the dependence on Pg. Taken together, the data show that the closed loop feedback circuit using DN changes the slope of the Pg dose response compared to the corresponding circuit without feedback (open loop). This example demonstrates that dominant negative production can be used to implement negative feedback on leucine zipper transcription factors in a competition-based feedback circuit.

[0238] Example 4: Modular and tunable biological feedback control using de novo protein switches In this example, degronLOCKR, a novel protein switch engineered via a host-unrecognized moiety with modular connectivity and predictable regulatability, is used to implement feedback control over endogenous pathways and synthetic circuits in the yeast S. cerevisiae.

[0239] The degronLOCKR device is based on LOCKR (Latching Orthogonal Cage Key pRoteins) technology and consists of a designer degSwitch and a Key protein. The degSwitch is a six-helix bundle with a cODC degron embedded in a destabilized sixth helix (the Latch), which is occluded via interactions with a five-helix scaffold (the Cage). The Key, a genetically encoded peptide, can competitively exclude the Latch for binding to the Cage. This exposes the cODC degron, which in turn targets the degSwitch and any fused cargo to the proteasome for degradation. As protein degradation is a universal method for post-translational regulation, degronLOCKR is a powerful device for synthetic biology. It has been shown that degronLOCKR can control gene expression by modulating the stability of transcription factors. Here, this functionality is exploited to implement modular feedback control on biological networks using degronLOCKR by expressing the Key as a function of the network's output (Figure 14, panel a). The degronLOCKR feedback strategy offers several advantages over other approaches for implementing feedback control. First, the modularity of degronLOCKR allows degSwitches to be fused directly to any protein of interest to produce on-target effects. Modifying endogenous genes with degSwitches also maintains the native transcriptional and translational regulation of the signaling protein. Finally, degronLOCKR is a completely de novo designed protein, thus allowing predictable modification to tune its properties. Negative feedback of degronLOCKR synthesis in an endogenous yeast pathway

[0240] As a qualitative proof of concept, degronLOCKR was used to implement synthetic negative feedback in the yeast MAPK mating pathway (Figure 14, panel b), a complex signaling pathway with many intrinsic feedback loops. The ability of degronLOCKR to modulate pathway output was tested by adding degSwitches to the endogenous loci of several positive pathway molecules in a ΔFAR1 ΔBAR1 background strain and expressing the KEYs using an inducible system (Aranda-Diaz et al., ACS Synth. Biol. 6, 545-554 (2017)) (Figure 14, panel c). The KEYs were targeted to either the cytosol or nucleus using nuclear localization sequences, causing degradation of each molecule in a specific compartment of the cell (Figure 15). This localized inducible degradation is a unique feature of degronLOCKR that allows for location-specific action in cells. The mating pathway was stimulated with a saturating dose of α-factor (100 nM) and pathway activity was monitored using a pAGA1-YFP-cODC (McCullagh et al., Nat. Cell Biol. 12, 954-962 (2010)) transcriptional reporter (cODC degron (Hoyt et al., J. Biol. Chem. 278, 12135-12143 (2003)) which destabilizes a long-lived fluorescent reporter and allows dynamics to be observed). Degradation of STE20 (MAPKKKK), STE11 (MAPKKK) and FUS3 (MAPK) had a moderate effect, whereas degradation of STE12 (TF) completely eliminated mating pathway output (Figure 14, panel c, bottom). These data indicate that the degron LOCKR is an effective tool for regulating intrinsic pathways.

[0241] Next, synthetic negative feedback control of the mating pathway was implemented by expressing key-CFP-NLS from a mating pathway responsive promoter (pFIG1) in a strain with endogenous STE12 fused to degSwitch (Figure 16, panel a). The effect of this feedback was compared to a feedback-free strain in which STE12 is still fused to degSwitch, but key is driven by a constitutive promoter. Automated flow cytometry was used to follow pAGA1-YFP-cODC kinetics after stimulation with high (25 nM), medium (6.25 nM), and low (3.13 nM) doses of alpha-factor (Figure 16, panel b). For comparison, a feedback-free strain (pREV1-key-CFP-NLS) was measured simultaneously. After stimulation with each dose of alpha-factor, the output of the synthetic feedback strain and the output of the feedback-free strain initially tracked closely together. After approximately 2 hours, the output of the feedback-free strain increased until it reached different steady states corresponding to different doses of alpha-factor. Strains with degronLOCKR synthetic feedback showed a larger transient overshoot with larger doses of α-factor but ultimately converged to the same steady-state output regardless of the size of the input. These data suggest that synthetic feedback desensitizes the steady-state output to α-factors in the mating pathway in this input regime. This result is likely not due to saturation of signaling because the observed transients are different.

[0242] To obtain a more global comparison of the steady-state behavior of the synthetic feedback and no-feedback strains, the output dose response of each was measured as a function of α-factor. The feedback strains showed a decay in maximum output amplitude and a decrease in slope in the linear region of the dose response (Figure 16, panel c). Comparing the synthetic feedback strains to no-feedback strains with various constitutive promoter strengths (Lee et al., ACS Synth. Biol. 4, 975-986 (2015)) (pREV1, pRNR2, pRET2) shows that the behavior generated by feedback cannot be achieved by expressing different constitutive amounts of the key. Taken together, the dynamic adaptive behavior and dose responses clearly demonstrate the effect of synthetic negative feedback and the utility of degronLOCKR as a tool for rapid rewiring of complex endogenous signaling pathways. DegronLOCKR feedback in synthetic transcriptional cascades

[0243] Next, the quantitative capacity and operational scope of the degronLOCKR feedback module was mapped using a simple synthetic transcriptional cascade (Aranda-Diaz et al.) consisting of two inducible synthetic transcription factors (Figure 17, panel a). First, GEM (Gal4 DNA binding domain-estradiol hormone binding domain-Msn2 activation domain) is induced by estradiol (E2) and activates pGAL1 to produce Z3PM (Z3 zinc finger-progesterone hormone binding domain-Msn2 activation domain). Z3PM is then induced by progesterone (Pg) and activates the transcription of pZ3-YFP-cODC. To implement the feedback, we used the same modular strategy that was successful in controlling the mating pathway. Namely, we fused GEM to the degSwitch and used pZ3 to express key-CFP-NLS (synthetic feedback). Because the amount of KEY produced, and therefore the rate of GEM degradation, is a function of Z3PM activity, feedback makes the concentration of GEM dependent on the output of Z3PM. The circuit can be perturbed by the addition of Pg or by the induction of a blue-light-inducible degron (psd) fused to Z3PM (Renicke et al., Chem. Biol. 20, 619-626 (2013)), increasing or decreasing the output, respectively. Feedback buffers these disturbances by regulating the concentration of Z3PM. This kind of disturbance-rejection experiment is an essential test of feedback in technological systems.

[0244] A simple computational model of the circuit predicts that, whereas increasing Pg leads to a monotonic increase in output without feedback (a constitutively expressed key), feedback leads to a transient increase in output, followed by adaptation to a steady state whose value is closer to the pre-disturbance value than the circuit without feedback when Pg is increased in the same way (Figure S17, panel b, left). Feedback attenuates the dependence of output on the Pg disturbance by decreasing the rate of Z3PM production, thereby compensating for the increase in Z3PM activity after an increase in Pg with a decrease in its concentration (Figure S17, panel b, right; Figure S18).

[0245] These predictions were experimentally verified by first inducing cells with 7.5 nM E2 and 0.78 nM Pg and growing them until their output reached a steady state (Figure 17, panel c). At that point, cells were perturbed with high (6.25 nM), medium (3.13 nM) or low (1.56 nM) step inputs of Pg, and the kinetics of pZ3-YFP-cODC were measured using an automated flow cytometry- and optogenetics-enabled continuous culture platform. As a control, the same series of inductions were performed on a no-feedback strain (pRNR2-expressing KEY), which has a similar YFP steady-state output as the feedback strain at pre-perturbation concentrations of E2 and Pg. In the absence of feedback, a step input of Pg increased the activity of Z3PM, which in turn increased the expression of YFP until the output reached a new steady state commensurate with the disturbance. In contrast, the synthetic feedback circuit increased the expression of KEY as Z3PM activity increased, leading to degradation of GEMs and, consequently, reduced Z3PM production. This buffering effect is evident starting 2 hours after the disturbance, when the output of the composite feedback circuit starts to decrease while the output of the feedback-free circuit continues to rise. This adaptation behavior is qualitatively similar to the composite negative feedback loop constructed for the joint pathway. Because the inputs and disturbances are well defined, we can quantify the adaptation using an accuracy metric calculated by taking the inverse of the absolute difference between the post-disturbance output and the pre-disturbance output normalized by the pre-disturbance output (Ma et al., Cell 138, 760-773 (2009)) (Figure 17, panel e). The feedback circuit yields much higher accuracy than the feedback-free circuit for a positive disturbance of Pg, demonstrating the benefits of feedback control.

[0246] A similar experiment was performed, exposing cells to a negative perturbation. Cells were grown to steady state with 30 nM E2 and 1.57 nM Pg, then induced with blue light to activate the degradation of Z3PM (Figure 17, panel d). As a control, a feedback-free circuit was constructed with the key constitutively expressed from pRPL18B to match the steady-state expression of the synthetic feedback circuit before the perturbation. As a result of the degradation of Z3PM, YFP expression immediately decreased in both the synthetic feedback and feedback-free circuits, after which the feedback-free circuit settled to a new, lower steady state. However, the feedback circuit, after a slight overshoot, returned to a steady state closer to the pre-perturbation value than the feedback-free circuit. The amount of adaptation in the synthetic feedback circuit to a negative perturbation is not as dramatic as a positive perturbation (Figure 17, panel f). Model simulations show that the negative perturbation pushes the output of the circuit to a lower expression level, resulting in a smaller relative difference between the feedback and feedback-free circuits. Thus, even if the feedback still actively buffers against negative disturbances, its effects are more difficult to observe. This highlights the fact that any feedback circuit, whether it is built with biological molecules or electronic components, has properties that need to be explored through thorough prototyping to enable productive modular use.

[0247] To further characterize the degronLOCK feedback module, feedback and no-feedback circuits were induced with a full range of E2 and Pg concentrations and flow cytometry was used to measure pZ3-YFP-cODC output at steady state (Figure 19 and Figure 20). In these experiments, pGAL1-RFP was measured to obtain more proximal information about the activity of GEM, i.e., its feedback action. At a fixed concentration of E2 (7.5 nM E2), increasing Pg increases the YFP output of the no-feedback circuit until saturation is reached (Figure 21, Figure 17, panel e). RFP fluorescence is insensitive to Pg, since KEY is expressed from a constitutive promoter in this strain. In contrast, in the synthetic feedback circuit, RFP fluorescence decreases as a function of Pg, a result of degronLOCKR induced degradation of GEM. This effect eventually saturates above 6.25 nM Pg, as indicated by constant RFP expression above this concentration. The difference between these two regimes of operation can be clearly seen in the YFP output, which shows that sensitivity to Pg decreases in the regime of active feedback and increases dramatically as feedback saturates. These results are qualitatively reproduced by a model showing feedback that saturates when complex formation between the Key and the degSwitch saturates (Figures S16 and S17).

[0248] We next investigated the tunability of the degronLOCKR feedback control. One useful feature of the designed feedback controller is the ability to tune the gain to suit the application. We evaluated two ways of tuning the feedback gain: changing the strength of the feedback promoter and changing the binding affinity of the key to the cage. (Figure 22, panel a). The computational model predicts that either method for tuning the feedback gain is qualitatively similar and therefore interchangeable (Figure 22, panel b). To test this, we created moderate and weak mutants of the pZ3 promoter with four and three Z3 binding sites (BS), respectively. To test the effect of weakening the strength of the feedback promoter, we performed Pg dose responses at a fixed concentration of E2 for the different circuit mutants (Figure 22, panel b). We observed that weakening the promoter indeed changed the dependence of the steady-state output on Pg. As the number of binding sites was reduced, the output dose response of the feedback circuit converged to that of a circuit without feedback (Figure 23). We then reduced the affinity of the key to the cage by shortening the length of the key. The full-length key was truncated by 4 (medium) or 12 (short) residues, and each key variant was tested in a feedback circuit using the full strength pZ3 promoter (6xZ3 BS) (Figure 22, panel c; Figure 23). Similar to decreasing the strength of the feedback promoter, shortening the key length altered the dependence of steady-state output on Pg (Figure 22, panel d). Reducing the strength of the feedback gain by either method resulted in more transient events and reduced adaptation (Figure 24). Tuning feedback gain by key length is an attractive alternative to promoter engineering and illustrates the unique strengths of de novo proteins.

[0249] To demonstrate the generalizability of these tuning methods, we modified the splicing pathway and performed combinatorial tuning of a synthetic feedback loop by varying both the strength of the feedback promoter and the length of the key (Figure 22, panel d). Because pAGA1 is a much stronger promoter than pFIG1, expressing the key using pAGA1 produced an expression pulse after induction with alpha factor (Figure 22, panel e). The size of the pulse, as well as the subsequent steady-state output, were both increased by shortening the length of the key, decreasing the amount of feedback in the system. Similarly, decreasing the length of the key while using the weaker promoter pFIG1 to drive the feedback resulted in a larger transient output and a higher steady-state output. Measurements of steady-state output as a function of alpha factor for different promoters and key lengths (Figure 22, panel f, Figure 25) clearly demonstrate that decreasing promoter strength or key length increases the steady-state output and the slope of the dose response of the pathway, indicating a decrease in feedback gain. Taken together, these data demonstrate the facile tunability that is a hallmark of the degronLOCKR feedback strategy.

[0250] method Construction of DNA circuits

[0251] Hierarchical Golden Gate Assembly was used to construct plasmids for yeast strain construction according to Lee et al. (2015). Individual parts had BsaI, BsmBI and NotI sites removed to facilitate downstream assembly and linearization. Parts were generated by PCR or purchased as gBlocks from IDT. These parts were then assembled into transcription units (promoter gene terminator) on cassette plasmids. These cassettes were then assembled together to form multigene plasmids for insertion into the yeast genome.

[0252] Yeast strains and growth media

[0253] The S. cerevisiae strain used in all experiments was BY4741 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0). All yeast cultures were grown in YPD medium (10 g / L Bacto Yeast Extract, 20 g / L Bacto peptone, 20 g / L dextrose). Selection for auxotrophic markers (URA3, LEU2, and / or HIS3) was performed on synthetic complete medium (6.7 g / L Bacto-yeast nitrogen base without amino acids, 2 g / L complete supplemental amino acid mix, 20 g / L dextrose).

[0254] Knockout of FAR1 and BAR1

[0255] A modified version of BY4741 (yAHN797) was generated for mating pathway experiments with FAR1 and BAR1 knocked out using the CRISPR / Cas9 method outlined in Lee et al. First, FAR1 was targeted using two sgRNAs designed using Benchling biology's design tools to target the ORF of each gene. These sgRNAs were expressed on a CEN6 / ARS4 plasmid containing Cas9 with two nuclear localization sequences and a URA3 auxotrophic marker. Repair DNA with homology to 50 bp upstream and downstream of the ORF was generated by annealing oligos. Yeast was transformed with the plasmid containing sgRNA / Cas9 and repair DNA using a standard lithium acetate procedure. The efficacy of the sgRNA was assessed by comparing the colony number of transformants that received repair DNA to those that did not. Colonies were screened by colony PCR to verify the knockout, and successful clones were grown in overnight cultures of YPD. 5 μl of the overnight culture was then plated onto synthetic complete medium containing 5-fluoroorotic acid (5-FOA) to counterselect for the URA3 auxotrophic marker on the CEN6 / ARS4 plasmid, and the knockout process was then repeated to knock out BAR1.

[0256] Integration of degSwitch into the yeast genome

[0257] Overlap extension PCR was used to generate linear DNA consisting of the degSwitch with a 5xGS linker and the URA3 auxotrophic marker. This linear DNA was then used as a PCR template to add 80 bp of homology targeting the 3' end of the MAT pathway regulators GPA1, MSG5, SST2, STE5, STE7, STE11, and STE50. Individual lithium acetate yeast transformations were then performed with each of the linear DNA fragments to insert the degSwitch downstream of each of the seven genes into the parent strain yAHN797 and selectively plated on synthetic complete medium lacking uracil. Insertions were confirmed using colony PCR.

[0258] Yeast cell culture and induction

[0259] Yeast strains were streaked from glycerol stocks onto SDC plates containing the appropriate auxotrophic marker, or onto YPD plates if no auxotrophic marker was present. Individual colonies from these plates were used to inoculate cultures in YPD and grown to saturation for 12–24 h.

[0260] Alpha factor induction

[0261] Saturated cultures were diluted 1:500 in fresh YPD and 450 μl was dispensed into individual wells of a 2 mL 96-well storage block (Corning) and grown for 3 hours at 30°C and 900 RPM in a Multitron shaker (Infors HT). Alpha-factor mating pheromone was prepared at 10x concentration by making appropriate dilutions into YPD from a 50 uM stock solution (Zymo Research). After 3 hours of growth, 50 μl of alpha-factor solution was added to the 96-well block and the block was returned to the shaker for 4 hours of growth.

[0262] Estradiol and progesterone induction

[0263] Saturated cultures were diluted 1:500 in fresh YPD and 400 μl was dispensed into individual wells of a 2 mL 96-well storage block (Corning) and grown for 3 hours at 30°C and 900 RPM in a Multitron shaker (Infors HT). Estradiol (Sigma-Aldrich) and progesterone (Fisher Scientific) were prepared at 10x concentrations by appropriate dilution into YPD from 3.6 mM (estradiol) and 3.2 mM (progesterone) stock solutions. After 3 hours of growth, 50 μl of estradiol and progesterone inducers were added to the 96-well block in the appropriate combination and the block was returned to the shaker for 10 hours of growth.

[0264] yeast culture

[0265] Saturated cultures were diluted 1:200 or 1:100 for conjugative pathway cultures into 10 mL or 15 mL YPD. Cultures were grown in glass tubes at 30°C in a shaker for 2 hours. Cultures were then diluted to 0.01 OD600 and dispensed into individual Falcon tubes in a total volume of 30 mL YPD. Growth was continued for an additional hour in a custom bioreactor at 30°C, agitated with a magnetically controlled stir bar. All cultures were grown in YPD with 0.5x penicillin-streptomycin.

[0266] Hardware

[0267] To collect measurements over time, a platform for automated flow cytometry and continuous culture was constructed. An existing automated experimental platform was adapted to perform small molecule induction at various concentrations and long-term culture. Yeast cultures were grown in 50 mL optically clear conical tubes (Falcon) held in eight temperature-controlled magnetic stirring chambers. Liquid handling was performed using two syringe pumps (Cavro XCalibur pumps, TECAN) of a BD high-throughput sampler. This setup allowed sampling from individual cultures into a BD LSRII flow cytometer for measurements. To achieve continuous culture, a certain volume of culture was first discarded and replaced with different ratios of hormone medium and fresh medium. Commands for HTS were controlled using LABVIEW 2013.

[0268] The sampling period consisted of three main steps: extraction of the sample, the dilution volume, and replenishing the dilution volume with the respective hormone concentration. The sampling period was chosen to keep the event rate approximately constant during long time-course experiments. A doubling time of 90 min was assumed, therefore 4 mL of culture was extracted and then replaced with fresh medium and hormones every 25 min (dilution rate 0.16 mL min -1 ). The shorter experiments performed on the conjugation pathway were not performed in continuous culture, allowing for a higher sampling frequency of every 10 min.

[0269] Estradiol and progesterone induction (single induction)

[0270] To test conditions where [E2] and [Pg] concentrations remained the same throughout the experiment, only one induction was required. Three stocks were made: (1) inducer, (2) supplemented stocks at 1X [E2] and 1X [Pg] concentrations, and (3) supplemented stocks without hormone. During the induction time point, cultures were induced to each concentration with different ratios of (1) and (3). Cultures were maintained at each concentration with adjusted ratios of (2) and (3).

[0271] Estradiol and progesterone induction (two inductions)

[0272] To test disturbance removal with the same [E2] but different [Pg], cultures were induced twice. The first induction allowed all cultures to grow to steady state at the same pre-disturbance concentration. After the cultures reached steady state (t=0 hr), the cultures were either induced with more Pg or kept at the same concentration and grown to steady state again. Four stocks were made: (1) inducer to achieve pre-disturbance concentration, (2) inducer to achieve different disturbance [Pg], (3) a supplement stock of 1X [E2] / [Pg] to maintain the desired concentration, and (4) a supplement stock of 1X [E2] but no Pg. Cultures were induced with (1) at t=-10 hr. All cultures were kept at the same pre-disturbance concentration for 10 hr by supplementing with a 1:8 dilution of (3) and (4). At t=0, cultures were induced with different ratios of (2) and (4). By adjusting the ratio of (3) and (4) to maintain the concentration, the highest disturbance [Pg] was achieved without dilution and the lowest [Pg] was maintained at 1:8 dilution.

[0273] Alpha factor induction

[0274] To test the dynamic response of degronLOCKR-mediated feedback on the mating pathway, cultures were induced with input (alpha-factor) at t0. Different volumes of YPD 1x25 nM alpha-factor stock and YPD without alpha-factor were combined to achieve different concentrations.

[0275] light induction

[0276] Each bioreactor is equipped with an individual blue LED connected to a USB-controllable LED driver (Mightex). Starting from the light induction time point, cultures were exposed to a saturating light dose (45 s on / 15 s off at an intensity amplitude of 25 mA). This light regime was maintained until expression reached a steady state.

[0277] Flow cytometry

[0278] Analysis of fluorescent protein reporter expression was performed on a BD LSRII flow cytometer (BD Biosciences) equipped with a high-throughput sampler. Cultures were diluted in TE and then passed through the instrument to obtain acceptable cell densities. The FITC channel was used to measure YFP (Venus) fluorescence, the PE-Texas Red channel (for steady-state measurements) or the mCherry channel (for dynamic measurements) to measure RFP (mKate2), and the DAPI channel to measure CFP. For steady-state measurements, 5,000–10,000 results were collected per sample. For dynamic measurements, 2,000–10,000 results were collected per sample. Fluorescence values ​​were calculated as a measure of height (H) in the appropriate channel and normalized to cell size by dividing by side scatter (SSC-H).

[0279] Figure 14. degronLOCKR is a modular tool for controlling biological pathways. a) Schematic of degronLOCKR as a modular tool for implementing synthetic feedback control on endogenous or synthetic biological networks by fusing degSwitches to effector molecules and driving expression of Keys from the output of the network. b) Simplified schematic of the yeast mating pathway that does not exhibit complex endogenous feedback. The pathway is activated by addition of α-factor and signaling activity is measured using a pAGA1-YFP-cODC reporter. c) degronLOCKR induced degradation of positive signaling molecules to control mating pathway activity. Endogenous copies of the indicated signaling molecules were fused to degSwitches and Keys were expressed using a progesterone-inducible system. Cells were induced with a saturating dose of α-factor and pathway activity with and without Keys was compared. After 4 h of growth, pAGA1-YFP-cODC was measured in a flow cytometer. Data represent the mean ± sd of three biological replicates.

[0280] Figure 16. The degronLOCKR module successfully implements synthetic feedback control of the mating pathway. a) Schematic of synthetic negative feedback in which an endogenous copy of STE12 is fused to degSwitch and KEY-CFP-NLS is expressed using either the pathway reporter pFIG1 (synthetic feedback) or a constitutive promoter (no feedback). All output measurements are with pAGA1-YFP-cODC. b) Measurement of pAGA1-YFP-cODC kinetics. Synthetic feedback and no feedback (pREV1) strains were induced at time t = 0 hours with high (25 nM), medium (6.25 nM) or low (3.13 nM) doses of α-factor and flow cytometry measurements (points) were performed every 10 minutes. The line represents a running average taken over three data points. c) α-factor dose response of synthetic feedback (pFIG1) and four no feedback (no key, pREV1, pRNR2, pRET2) strains. pAGA1-YFP-cODC fluorescence was measured 4 hours after α-factor induction using flow cytometry. Points represent the mean ± sd of three biological replicates. The solid line is a Hill function fit to the data. High, medium and low doses of α-factor from the experiment in (b) are shown on the graph.

[0281] Figure 17. Operational characteristics of the degronLOCKR feedback module quantified by control of the synthetic circuit. a) Schematic of the synthetic feedback circuit. GEM-degSwitch is constitutively expressed and activated by estradiol (E2) to drive expression of pGAL1-Z3PM-psd. Z3PM is activated by progesterone (Pg) to drive expression from pZ3. Degradation of Z3PM-psd can be induced using blue light. pZ3-YFP-cODC is the measured output of the circuit, and pZ3-Key-CFP-NLS drives feedback in the circuit (synthetic feedback) by activating degradation of GEM-degSwitch. In the feedback-free circuit, a constitutive promoter is used to express Key-CFP-NLS. b) Model simulations of the feedback and feedback-free circuits (see Supplementary Information). Simulated kinetics (left) and steady-state changes (right) of output following a Pg perturbation show that feedback buffers the increase in Pg concentration by lowering GEM and decreasing Z3PM concentration. c) Dynamic measurements of pZ3-Venus-cODC using automated flow cytometry for a synthetic feedback and no-feedback strain (pRNR2-key-CFP-NLS) after a positive perturbation. Cells were grown to steady-state expression with 0.78 nM Pg and 7.5 nM E2. At time 0 h, cells were kept at the same Pg concentration or induced to a new final concentration of Pg of 1.56 nM (low), 3.13 nM (medium), or 6.25 nM (high). Kinetics were measured for an additional 8 h. The solid line represents a moving average taken over three data points. d) Dynamic measurements of pZ3-Venus-cODC using automated flow cytometry in synthetic feedback and no-feedback strains (pRPL18B-Key-CFP-NLS-Drive Key) after negative perturbations. Cells were grown to steady-state expression with 1.57 nM Pg and 30 nM E2 and then exposed to blue light at 0 hours to activate psd. Kinetics were measured 8 hours after perturbation. Growth and sampling conditions are as in c). e) Precision of the synthetic feedback circuit versus the no-feedback circuit for each perturbation.f) Comparison of steady-state circuit behavior (10 h post-stimulation) with and without feedback (pRNR2-KEY-CFP-NLS) as a function of Pg at a fixed concentration of 7.5 nM E2. RFP fluorescence is a proxy for Z3PM concentration and YFP fluorescence is the output of the circuit. The Pg doses used for the positive perturbations in c) are indicated. Points represent the mean ± s.d. of three biological replicates.

[0282] Figure 22. The DegronLOCKR synthetic feedback method is predictably tunable. a) (Top) Examination of various ways to tune the feedback gain of the synthetic feedback circuit. (Bottom) Model simulations of circuit output and Z3PM as a function of Pg perturbations for decreasing key generation rate or key / cage affinity (see Supplementary Information). b and c) Experimental validation of the tuning. b) (Top) Tuning the feedback gain by varying the number of Z3 binding sites on pZ3 with a fixed length key. (Bottom) RFP and YFP fluorescence as a function of Pg for strong (pZ3-6x), medium (pZ3-4x), and weak (pZ3-3x) feedback strains versus a no feedback (pREV1-key-CFP-NLS) strain. Points represent the mean ± s.d. of three biological replicates. c) (Top) Tuning the feedback gain by varying key length with feedback promoter strength fixed in pZ3-6x. (Bottom) RFP and YFP fluorescence as a function of Pg for feedback strains with long (55aa), medium (51aa) and short (43aa) keys versus a no feedback (pREV1-NLS-key-CFP) strain. Points represent the mean ± sd of three biological replicates. d) Alteration of promoter strength and key length to tune feedback gain in a synthetic negative feedback loop in the mating pathway. pAGA1 is a stronger reporter of the mating pathway than pFIG1. ​​e) (Top) Kinetic measurements of pAGA1-YFP-cODC for various feedback and no feedback strains after stimulation with 25 nM α-factor. Points represent flow cytometry measurements and lines represent running averages obtained across three data points. (Bottom) α-factor dose response of feedback strains versus no feedback (pREV1-NLS-key-CFP) strains. Four hours after α-factor induction, YFP fluorescence was measured using flow cytometry. Points represent the mean of three biological replicates and error bars represent standard error. The solid line is a Hill function fit to the data. The doses of α-factor used in the kinetic experiments (top) are indicated on the graph.

[0283] Figure 15: Panel of mating pathway regulators tested in degronLOCKR. degSwitch was fused to the C-terminus of the endogenous copy of each regulator. Keys with or without the SV40 NLS were expressed using the Pg-inducible system. STE20, STE11 and PTP3 were degraded using a cytoplasmic key (Key-CFP), and STE12, DIG1 and DIG2 were degraded using a nuclear key (Key-CFP-NLS). MSG5 and FUS3 were degraded using either a cytoplasmic (cyto) or nuclear (nuc) key. Cells were induced with 1 nM (low) or 100 nM (high) α-factor and 50 nM or 0 nM Pg and grown for 4 h before measuring YFP fluorescence using a flow cytometer. Data represent the mean ± sd of three biological replicates.

[0284] Figure 18: Steady-state solutions in response to positive or negative disturbances. Degradation rates of a) progesterone (Pg) or b) ZPM (γ Z ) varies according to our Hill-type model. The solid line corresponds to a feedback system (FB), while the dashed line shows the example where the feedback is removed (i.e., f instead of Eq. 12). K =μ K* , without FB). The grey box indicates the range of the region where the feedback is considered to be “active”, which is determined by the relative change in the disturbance (a) ΔP / P or b) Δγ Z / γ Z It is defined as the relative change of all GEMs to the key or GEMs (Δ(G+C) / (G+C)) greater than 0.15. Notably, in the absence of feedback, Δ(G+C) is equal to zero for any disturbance except for the direct synthesis or degradation rate of the key or GEMs.

[0285] Figure 19: Diagram showing circuit behavior as a function of Pg for a fixed dose of E2. Comparison of steady-state circuit behavior (10 hours post-stimulation) as a function of Pg at all E2 concentrations with and without feedback (pRNR2-Key-CFP-NLS). YFP fluorescence is the output of the circuit, RFP fluorescence is a proxy for Z3PM concentration, and BFP fluorescence is the amount of Key produced. Points represent the mean ± s.d. of three biological replicates.

[0286] Figure 20: Circuit behavior as a function of E2 for a fixed dose of Pg. Comparison of steady-state circuit behavior (10 hours post-stimulation) as a function of E2 at all Pg concentrations with and without feedback (pRNR2-key-CFP-NLS). YFP fluorescence is the output of the circuit, RFP fluorescence is a proxy for Z3PM concentration, and BFP fluorescence is the amount of key produced. Points represent the mean ± s.d. of three biological replicates.

[0287] Figure 21: Circuit behavior when constitutively expressing different amounts of KEY. Comparison of steady-state circuit behavior with feedback (10 hours post-stimulation) and various levels of expression of KEY (pREV1, pRNR2, pRET2, pRPL18B) without feedback as a function of Pg at a fixed concentration of 7.5 nM E2. YFP fluorescence is the output of the circuit, RFP fluorescence is a proxy for Z3PM concentration, and BFP fluorescence is the amount of KEY produced. Points represent the mean ± s.d. of three biological replicates.

[0288] Figure 23: Changing promoter strength or key length modulates feedback gain. Comparison of steady-state circuit behavior (10 hours post-stimulation) as a function of Pg for various levels of feedback gain (left, modulation by changing feedback promoter strength; right, modulation by changing key length) at a fixed concentration of 7.5 nM E2. Left, modulation by changing feedback promoter strength (x refers to the number of Z3 operator sites). Right, modulation by changing key length (m refers to the number of residues removed from the C-terminus of the key). YFP fluorescence is the output of the circuit, RFP fluorescence is a proxy for Z3PM concentration, and BFP fluorescence is the amount of key produced. Points represent the mean ± s.d. of three biological replicates.

[0289] Figure 24: Adjusting feedback strength changes the dynamic behavior of the circuit output. Dynamic measurements of pZ3-Venus-cODC using automated flow cytometry for synthetic feedback and no feedback strains (pREV1-key-CFP-NLS) with various gains after induction with 3.13 nM Pg and 7.5 nM E2 at time = 0 hours. Solid lines represent running averages taken over three data points.

[0290] Figure 25: Combinatorial regulation of synthetic feedback in the mating pathway. (Top) Kinetic measurements of pAGA1-YFP-cODC for various feedback and no-feedback (pREV1, pRNR2, pRET2, pRPL18B) strains following stimulation with 25 nM α-factor. Points represent flow cytometry measurements and lines represent running averages obtained across three data points. (Bottom) Dose response of α-factor for feedback versus no-feedback (pREV1, pRNR2, pRET2, pRPL18B) strains. Four hours after α-factor induction, YFP fluorescence was measured using flow cytometry. Points represent the mean ± sd of three biological replicates. Solid line is a Hill function fit to the data.

[0291] Example 5: Inducible nuclear export In this example, inducible localization using a nuclear export signal is demonstrated as a functional strategy for feedback circuit design.

[0292] Dynamic shuttling of transcription factors and kinases in and out of the nucleus plays an essential role in cellular function. To obtain inducible control over nuclear localization, we further extended the functionality of LOCKR in vivo by incorporating a nuclear export sequence (NES) into the Switch a The NES sequence (Guttler, T. et al., Nat. Struct. Mol. Biol. (2010) 17:1367-1376) was caged using the same strategy as used for cODC, and the resulting nesSwitch a was fused to YFP, which has a strong nuclear localization sequence (Kosugi, S. et al., J. Biol. Chem. (2009) 284:478-485). An RFP-histone fusion (HTA2) was constitutively expressed in the same cells to act as a nuclear marker (Figure 26, panel a). a To test the switching ability of YFP, we compared YFP localization in the presence and absence of Key expression. a In the absence of -BFP, it was found to colocalize with RFP in the nucleus (Figure 26, panel b, left). a key a When co-expressed with -BFP, YFP fluorescence appeared more cytosolic, indicating uncaging of the nuclear export signal (Figure 26, panel b, right). a and key a Co-expression of -BFP results in a similar pattern (Figure 27, panels a and b), so the YFP specks observed in the cytosol are likely due to aggregation. a The results are independent of the presence of an NLS on the -BFP (Figure 27, panels c and d). Without being bound by theory, the mechanism for maintaining nesLOCKR outside the nucleus may involve nuclear export and the cytosolic binding of the newly translated NLS-YFP-nesSwitch with the KEY in the cytosol. aor residual NLS-YFP-nesSwitch a It can be combined with any of the captures. a -BFP expression is always the same as YFP-nesSwitch a This results in colocalization with (Figure 27).

[0293] Next, we used nesLOCKR to control the localization of SynTF. It was hypothesized that activation of nesLOCKR would result in a decrease in the output of the pSynTF promoter, since SynTF needs to be localized to the nucleus to activate transcription. Using a dual induction system (Figure 26, panel c), we expressed different concentrations of SynTF-RFP-nesSwitch a and key a -BFP was expressed in the same cells as the pSynTF-YFP reporter and steady-state fluorescence was measured using flow cytometry (Figure 26, panel d). Induction of KEY with 31.25 nM E2 caused a 33% decrease in YFP signal, indicating successful activation of nesLOCKR and exclusion of SynTF from the nucleus (Figure 26, panel e). These results further demonstrate the ability to use a localization-based strategy for feedback control in the circuits described herein.

[0294] Figure 26. Control of protein localization using nesLOCKR. a) NLS-YFP-nesSwitch a a) Schematic of Key-induced nuclear export of Key. The nucleus is marked by histone HTA2-RFP. a Colocalization of NLS-YFP-nesSwitcha with nuclear HTA2-RFP fluorescence is observed in the absence of -BFP (left), compared to Key a When -BFP was expressed, the NLS-YFP-nesSwitch was more diffuse outside the nucleus. a Fluorescence microscopy showing that a fluorescent signal is observed (right). c) nesLOCKR against synthetic transcription factor (SynTF). a Schematic of the double induction assay to examine the effect of Pg on the expression of Key a-BFP expression and E2 induces the expression of SynTF-RFP-nesSwitch a The pSynTF promoter is activated by SynTF to express YFP. d) Heatmap of YFP and RFP fluorescence as a function of E2 (0-125 nM) and Pg (0-500 nM) measured by flow cytometry. e) YFP at 31.25 nM E2 as a function of Pg induction, SynTF-RFP-nesSwitch. a and Key a -Line plot comparing BFP fluorescence (black rectangle in Figure 26, panel b). Fluorescence values ​​were normalized to maximum YFP, RFP or BFP fluorescence. Error bars represent the s.d. of three biological replicates.

[0295] Figure 27: Cytosolic aggregation of nesLOCKR when Key is expressed. a) Schematic of cytosolic YFP-nesSwitcha and Key-BFP with nuclear marker HTA2-RFP. b) YFP fluorescence was observed in the YFP-nesSwitcha and Key-BFP. a When expressed without an NLS (left), it shows the expected cytosolic distribution, whereas YFP-nesSwitch a When both nesSwitch and Key-BFP were expressed in the cytosol, puncta of YFP fluorescence were observed, which is consistent with the a The Key-BFP fluorescence was predicted to be due to the aggregation of YFP-nesSwitch a c) NLS-YFP-nesSwitch with Key-BFP-NLS colocalizes with the nuclear marker HTA2-RFP (right). a Schematic diagram of YFP-nesSwitch. a When expressed with a strong (SV40) NLS, it localizes to the nucleus (left). When Key-BFP is expressed with a moderately strong NLS, the same pattern of YFP localization is observed as when Key-BFP is expressed without an NLS (Figure 26, panel b), indicating that NES uncaging is independent of Key-BFP localization. Key-BFP-NLS fluorescence is consistent with that of the NLS-YFP-nesSwitch a Fluorescence colocalizes (right).

[0296] Example 4: Function of degronLOCKR in human primary T cells The ability of degronLOCKR to function in human primary T cells was demonstrated by inducibly degrading the mCherry fluorescent protein. A lentiviral transfer construct was constructed containing mCherry fused to an asymmetric short scaffold degronSwitch with a t8 toehold and cODC degron embedded in the latch. The mCherry-degronSwitch fusion was expressed using the pPGK constitutive promoter. In a second lentiviral construct, fusions of Key and tagBFP were expressed using four different constitutive promoters (pPGK, pSFFV, pCMV(G), pCMV(D)).

[0297] The experiment was performed on human primary CD4+ T cells. The cells were transduced with different combinations of the lentiviruses described above. In some cases, the cells were transduced with only mCherry-degronSwitch. In other cases, the cells received both the mCherry-degronSwitch virus in addition to the virus expressing Key-tagBFP. After transduction with the lentivirus, fluorescence was measured using flow cytometry. The distribution is shown in Figure 28. We observed that when cells were co-transduced with a virus containing any amount of Key production, mCherry fluorescence was almost completely abolished (Key production was quantified using tagBFP fluorescence). This data indicates that Key can trigger the degronSwitch and activate the degradation of mCherry.

[0298] Example 5: degronLOCKR-mediated feedback function in Jurkat T cells Using the inducible humanized synthetic transcription factor ZF3-p65-Ert2 (ZPE) as a model process, we tested whether feedback mediated by the degronLOCKR is functional in human T cells (inducible TF gift of Mo Khalil, Boston U). The output of the circuit is an mCherry fluorescent reporter produced by the pZF3 promoter, with ZPE fused to the degronSwitch driven by a pSFFV constitutive promoter. Two versions of the circuit were constructed, one without feedback and one with feedback via the key. The circuit with feedback has the key driven by a separate pZF3 promoter and fused to mEGFP. Several variants of this feedback circuit were tested by mixing and matching different pZF3 promoter variants and key lengths. These experiments were performed by stably integrating the construct into Jurkat T cells using lentivirus. Cells that received the circuit were gated out as mCherry positive (the pZF3(4x)mCMV promoter is leaky) and, in the feedback version, gated out as BFP positive.

[0299] This experiment was performed by inducing cells with various tamoxifen (4OHT), which activates the ZPE transcription factor by translocating it into the nucleus. Cells were measured 5 days after induction using flow cytometry. Sample distribution of circuit output and key production of the circuit with feedback is shown in Figure 29.

[0300] We compared the dose response of the feedback-free and feedback circuits for the full range of 4OHT concentrations. It appears that the buffering from feedback can be tuned by varying the length of the promoter or key, similar to effects previously observed in yeast. When observing feedback from the mCMV promoter driving the full-length key, we see that the presence of feedback reduces the maximum steady-state output and also reduces the slope of the dose response (Figure 30). These properties are typical characteristics of feedback and suggest that our feedback circuit is influencing the circuit. In the future, mCherry can be replaced with the payload of interest. Our feedback circuit can provide disturbance rejection and tune the kinetics of T cell activation (i.e., production of CAR) or delivery of therapeutic payloads.

[0301] While the present invention has been described with reference to specific embodiments thereof, it will be understood by those skilled in the art that various modifications can be made and equivalents can be substituted without departing from the true spirit and scope of the invention. In addition, many modifications can be made to adapt a particular situation, material, composition of matter, process, process step or steps to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the appended claims.

Claims

1. A cell comprising a molecular feedback circuit, The molecular feedback circuit comprises: a nucleic acid sequence encoding a signaling protein that drives an output of a signaling pathway when activated by an input of the signaling pathway, the signaling protein comprising a cryptic inactivation domain but not a caged degron; a polynucleotide comprising a regulatory sequence responsive to said output operably linked to a nucleic acid sequence encoding a switch polypeptide whose expression triggers said inactivation domain to inactivate said signaling protein; A molecular feedback circuit comprising: (a) the inactivation domain comprises a degradation domain that includes a degron, and a protection domain, and the switch polypeptide is a protease, and cleavage of the protection domain by the protease exposes the degron to trigger degradation of the signaling protein; or (b) the inactivation domain comprises a first member of a binding pair, and the switch polypeptide i. a second member of the binding pair linked to a spacing domain, or ii. A cell comprising a molecular feedback circuit comprising a second member of said binding pair that comprises a dominant negative domain.

2. The cell of claim 1 , wherein the input or the output, or both, comprises an intracellular signal.

3. The cell of claim 1 , wherein the input or the output, or both, comprises an intercellular signal.

4. 4. The cell of any one of claims 1-3, wherein the switch polypeptide comprises a second member of the binding pair linked to a sequestration domain, the sequestration domain comprising a plasma membrane targeting tag, a mitochondrial membrane targeting tag, a peroxisome targeting tag, a vacuolar targeting tag, or an actin cytoskeleton targeting tag.

5. the switch polypeptide comprises a second member of the binding pair that comprises a dominant negative domain; The cell of claim 1 , wherein the latent inactivation domain comprises a competitive binding domain non-covalently bound to the first member of the binding pair.

6. 6. The cell of claim 1 , wherein the first and second members of the binding pair comprise first and second portions of a leucine zipper.

7. the signaling protein is a positive regulator of the signaling pathway, the signaling protein is a negative regulator of the signaling pathway; or The cell of claim 1 , wherein the signaling protein is an intermediate member of the signaling pathway or a transcription factor.

8. The cell of claim 1 , wherein the polynucleotide comprising the regulatory sequence comprises a binding site for the output transcription factor.

9. The cell of claim 8 , wherein the output is expression of the transcription factor.

10. 10. The cell of claim 1, wherein the signaling protein is a receptor and the input is a ligand for the receptor.

11. 11. The cell of claim 10, wherein the receptor is a synthetic receptor, the synthetic receptor being a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

12. The cell of claim 11 , wherein the output is immune activation or immune suppression.

13. One or more nucleic acid molecules encoding a molecular feedback circuit, comprising: The molecular feedback circuit comprises: a nucleic acid sequence encoding a signaling protein that drives an output of a signaling pathway when activated by an input of the signaling pathway, the signaling protein comprising a cryptic inactivation domain but not a caged degron; a polynucleotide comprising a regulatory sequence responsive to said output operably linked to a nucleic acid sequence encoding a switch polypeptide whose expression triggers said inactivation domain to inactivate said signaling protein; A molecular feedback circuit comprising: (a) the inactivation domain comprises a degradation domain that includes a degron, and a protection domain, and the switch polypeptide is a protease, and cleavage of the protection domain by the protease exposes the degron to trigger degradation of the signaling protein; or (b) the inactivation domain comprises a first member of a binding pair, and the switch polypeptide i. a second member of the binding pair linked to a spacing domain, or ii. a second member of the binding pair comprising a dominant negative domain; One or more nucleic acid molecules.

14. One or more nucleic acid molecules described in claim 13, wherein the input or the output or both include an intracellular signal.

15. One or more nucleic acid molecules described in claim 13, wherein the input or the output or both include an intercellular signal.

16. One or more nucleic acid molecules described in any one of claims 13 to 15, wherein the switch polypeptide comprises a second member of the binding pair linked to an isolation domain, the isolation domain comprising a plasma membrane targeting tag, a mitochondrial membrane targeting tag, a peroxisome targeting tag, a vacuolar targeting tag, or an actin cytoskeleton targeting tag.

17. The switch polypeptide comprising a second member of the binding pair comprising a dominant negative domain; 16. One or more nucleic acid molecules according to any one of claims 13 to 15, wherein the latent inactivation domain comprises a competitive binding domain non-covalently bound to the first member of the binding pair.

18. One or more nucleic acid molecules described in any one of claims 13 to 17, wherein the first and second members of the binding pair comprise first and second portions of a leucine zipper.

19. The signaling protein is a positive regulator of the signaling pathway, the signaling protein is a negative regulator of the signaling pathway; or 19. One or more nucleic acid molecules according to any one of claims 13 to 18, wherein the signalling protein is an intermediate member of the signalling pathway or a transcription factor.

20. One or more nucleic acid molecules described in any one of claims 13 to 19, wherein a polynucleotide comprising the regulatory sequence comprises a binding site for the output transcription factor.

21. One or more nucleic acid molecules described in claim 20, wherein the output is expression of the transcription factor.

22. One or more nucleic acid molecules described in any one of claims 13 to 21, wherein the signaling protein is a receptor and the input is a ligand for the receptor.

23. One or more nucleic acid molecules described in claim 22, wherein the receptor is a synthetic receptor, the synthetic receptor being a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

24. One or more nucleic acid molecules described in claim 23, wherein the output is immune activation or immune suppression.

25. 25. A cell genetically modified to contain one or more nucleic acid molecules according to any one of claims 13 to 24.

26. 1. A method for modulating signal transduction of a signal transduction pathway in a cell, comprising genetically modifying said cell with a molecular feedback loop, The molecular feedback circuit comprises: a nucleic acid sequence encoding a signaling protein of the signaling pathway, the signaling protein comprising a cryptic inactivation domain but not a caged degron; a polynucleotide comprising a regulatory sequence responsive to an output of the signaling pathway, the polynucleotide comprising the regulatory sequence operably linked to a nucleic acid sequence encoding a switch polypeptide that, when expressed, activates a latent inactivation domain; Including, the activated inactivation domain inactivates the signaling protein, thereby modulating signaling of the signaling pathway; inactivation of the signaling protein by the inactivation domain comprises sequestration of the signaling protein; or The method, wherein inactivation of the signaling protein by the inactivation domain comprises dominant-negative inhibition of the signaling protein.

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