Small‐Molecule Regulators for Gene Switches to Program Mammalian Cell Behaviour

Synthetic or natural small molecules have been extensively employed as trigger signals or inducers to regulate engineered gene circuits introduced into living cells in order to obtain desired outputs in a controlled and predictable manner. Here, we provide an overview of small molecules used to drive synthetic‐biology‐based gene circuits in mammalian cells, together with examples of applications at different levels of control, including regulation of DNA manipulation, RNA synthesis and editing, and protein synthesis, maturation, and trafficking. We also discuss the therapeutic potential of these small‐molecule‐responsive gene circuits, focusing on the advantages and disadvantages of using small molecules as triggers, the mechanisms involved, and the requirements for selecting suitable molecules, including efficiency, specificity, orthogonality, and safety. Finally, we explore potential future directions for translation of these devices to clinical medicine.


Introduction
A key aim of synthetic biology is to engineer living cells in order to introduce novel capabilities or to enhance existing functions by repurposing genetic elements found in nature. [1]Typically, an engineered gene circuit comprises three main components: a sensing module for signal detection, a processing module that processes biological information, and a production module that generates a customized output. [2]Often, the user-defined stimulus is a small molecule that is supplied to trigger the desired output, or alternatively is depleted to turn the circuit off when its function is no longer needed. [3,4]These features of switchability and reversibility enable spatiotemporal regulation of engineered functions with high levels of specificity and safety. [5]Furthermore, by varying the intensity and dynamics of the input signal, the output can be fine-tuned in a doseresponse manner. [6]Orthogonality is also an important consideration.Small molecule-triggered gene circuits can utilize hostspecific modulators (e. g., transcription factors) or endogenous signaling pathways, but orthogonal circuits using genetic elements sourced from other species are less likely to interfere with endogenous functions of the host cell. [2]It is also important to employ triggers with minimal off-target effects. [7]esides small molecules, physical inputs can be used to trigger gene circuits. [9]However, although these physical strategies, including light, [8] temperature, [9] mechanical forces, [10] magnetic and electric fields, [11] offer non-invasive, traceless, precise, and efficient means to activate a biological sensor domain, they may require expensive equipment, are often limited to specific applications, and can have other disadvantages.For example, the limited tissue penetration of light restricts the therapeutic applications of optogenetically engineered cell implants. [8,12,13]On the other hand, chemical inducers, including organic and inorganic compounds, peptides, and odorants, provide great diversity, high induction potential, and ease of use. [14]Nevertheless, chemical compounds may encounter difficulties such as poor biodistribution, insufficient bioavailability, or inappropriate pharmacodynamics, which can increase the likelihood of cross-reactivity, off-target effects, and the need for invasive access to the targeted tissue or organ. [15]owever, in contrast to other trigger modalities, small molecules generally possess higher bioavailability and greater membrane permeability, and also are easier to manufacture. [16]herefore, in this review we focus on small-molecule inducers.
The regulation of small molecule-responsive synthetic circuits can occur at various levels, including DNA, transcription (RNA synthesis), post-transcription (RNA modification or editing), translation (protein synthesis), and post-translation (protein modification or trafficking) (Figure 1).The level of control can be chosen according to the intended application of the engineered cell.For example, regulation at the DNA level often results in permanent change, whereas control at the RNA or protein level is preferable when reversibility is crucial.Furthermore, regulation at the transcriptional level is often robust, but involves a significant lag time in producing the desired output, so post-translational control might be preferable when rapid output is essential. [2,17]ere, we present an overview of the small molecules most frequently employed by the synthetic biology community to regulate engineered mammalian cells, including examples of applications at different levels of control (DNA, RNA and protein) and the mechanisms involved.We also discuss the requirements for selecting suitable small molecules.In addition, we explore the advantages and disadvantages of employing small-molecule-programmed genetic circuits in designer mammalian cells for therapeutic applications and we discuss challenges facing their clinical translation and future prospect.

Small molecules commonly used in synthetic biology
The drug discovery process for small molecules typically begins with extensive screening campaigns, utilizing institutional or commercial compound collections to identify suitable chemicals for lead optimization efforts. [18,19]22] In this section, we highlight drugs that have been repurposed for synthetic biology applications, categorized according to their primary functions in medicine, agriculture, or the research fields for which they were originally developed.

Antibiotics
Tetracycline and its derivatives, including oxytetracycline and doxycycline, [18] are broad-spectrum antibiotics that inhibit protein synthesis in bacteria through binding to the bacterial ribosome, [24] and are widely used in synthetic biology to regulate gene expression in mammalian cells, both in vitro and in vivo. [23]Tetracycline itself shows reduced bioavailability when taken with food or dairy products and has a risk of gastro-Maysam Mansouri is a synthetic biologist focused on engineering synthetic genetic systems in mammalian cells.He has designed a range of therapeutic genetic circuits, including DNA, RNA, and protein switches, to control and program mammalian cell behaviour within the context of cell and gene therapy.Small molecules can regulate gene circuits implanted in engineered mammalian cells at various biological levels: DNA (1), RNA synthesis (2), RNA maturation (3), protein synthesis (4), and protein maturation and trafficking (5).Examples of small molecules enabling regulation at different biological levels are provided: Tam (tamoxifen), Dox (doxycycline), Rapa (rapamycin), ABA (abscisic acid), Tet (tetracycline), Brana (branaplam), Graz (grazoprevir), and VA (vanillic acid).
intestinal side effects, [23,24] whereas doxycycline has a longer half-life and improved bioavailability, enabling less frequent dosing. [25]In synthetic biology, tetracycline and doxycycline serve as inducers by binding to a regulatory protein known as Tet repressor (TetR).This interaction mediates the connection between TetR and its target DNA sequence (Tet operator), resulting in modulation, either activation or suppression, of the target gene. [26]The system's appeal lies in the specificity of the TetR-operator sequence interaction, substantial induction levels, extensive safety studies on tetracycline, and its high affinity for TetR, making it an attractive choice for gene regulation. [15]ther antibiotics utilized as signals to program gene circuits include streptogramin and various macrolides.Streptogramin is frequently employed to target Gram-positive bacteria by inhibiting bacterial growth.This antibiotic can modulate the binding of a repressor protein known as Pip (pristinamycininduced protein) to a specific synthetic sequence upstream of a desired transgene. [27]Erythromycin, a macrolide antibiotic, is also commonly used to treat bacterial infections, particularly those caused by Gram-positive bacteria, and functions by inhibiting protein synthesis in bacteria.In synthetic biology, this antibiotic has been repurposed to regulate the DNA-binding activity of a bacterial repressor with respect to its own DNAbinding sequence, ultimately leading to either gene expression or suppression. [28]Trimethoprim (TMP) is a well-characterized antibiotic drug that inhibits bacterial growth by blocking folate synthesis.It effectively penetrates the blood-brain barrier and has been safely used in humans for both therapeutic purposes and long-term prophylactic regimens for over 50 years.In synthetic biology, TMP has been employed as a trigger signal to program the half-life of targeted proteins by conditionally stabilizing chimeric proteins fused to a dihydrofolate reductase (DHFR) degron from E. coli. [29,30]oumermycin and novobiocin are antibiotics that bind to the amino-terminal subdomain (24 K) of the bacterial DNA gyrase B subunit (GyrB) and inhibit bacterial growth. [31]A coumarin-based transcriptional gene switch was developed in mammalian cells to control gene expression in a reversible and robust way.In the presence of the inducer coumermycin, the constitutively produced chimeric transactivator (λOP-GyrB-p65) is dimerized and bound to the λOP-responsive element on a synthetic expression unit to turn on the transgene.Addition of novobiocin causes dissociation of the dimerized transactivator (λOP-GyrB-p65) and switches off transgene expression. [32]

Antivirals
Protease inhibitors, such as GRL-0617 and GC-376, have recently attracted interest as antiviral agents against SARS-CoV-2. [33]GRL-0617 is a potent, selective, and competitive noncovalent inhibitor of the PLpro protease, which plays a critical role in viral replication and assembly. [34]GC-376 targets the Mpro protease by covalently binding to Cys145 within the active site, effectively blocking SARS-CoV-2 replication in cultured cells in vitro. [33]We have harnessed these proteases to function as gene expression tools in synthetic biology circuits.Several inhibitors targeting the hepatitis C virus (HCV) NS3/4A protease, which is involved in the production of various proteins crucial for viral replication, [30] have also been used in engineering mammalian cells, including asunaprevir, grazoprevir, ledipasvir, and telaprevir. [31,32]They are used to block cleavage at NS3 target sites linked to a synthetic module (e. g., transactivator or repressor), so that the module remains functional as long as cleavage is inhibited. [35]

Plant-derived compounds
Plant-derived compounds generally represent a safe option as inducers in humans.Purified or synthetic plant-derived small molecules that have found applications in synthetic biology include salicylic acid (SA), abscisic acid (ABA), vanillic acid (VA), caffeine, gibberellic acid (GA), and auxin.
Salicylic acid (SA), which plays a role in plant defense mechanisms against environmental stresses, has been employed to control gene expression in human cells by regulating a bacterial repressor derived from E. coli. [36]Abscisic acid (ABA), also involved in environmental stress management as well as the regulation of growth and development, [35] is widely used in the synthetic biology community to mediate interactions between two ABA-responsive heterodomains (ABI/PYL). [17,37]anillic acid (VA) a food additive found in vanilla beans and certain plants, is employed in synthetic biology applications for the regulation of gene circuits at the transcription and posttranslation levels within an orthogonal setup. [38,39]This compound has also been utilized to regulate VA receptor-based lineage control networks that activate transcription factors to program cell fate by triggering endogenous signaling pathways, differentiating induced pluripotent stem cells (iPSCs) into insulin-producing beta-cells. [40]affeine, a natural stimulant present in coffee beans, tea leaves, and various other plant products, has been used as an input signal to trigger the production of GLP1, a clinically licensed peptide used to treat type II diabetes, in engineered cells. [41]Gibberellic acid (GA), a phytohormone regulating plant growth and development, can be used to induce interactions between two different moieties fused to GA-responsive domains (GID/GAI).GA-based gene circuit systems have been designed to regulate mammalian cells at the transcription and post-translational levels. [17,37]Auxin, a plant hormone, acts as a molecular glue to induce the degradation of targeted proteins in synthetic biology.If a protein of interest is conjugated with an auxin-inducible degron (AID) domain, auxin binds the AIDtagged protein to E3 ubiquitin ligase, ultimately leading to the degradation of the fusion protein. [42]

Anticancer agents
Well-known anticancer drugs that have been repurposed in synthetic biology to activate designer cells include rapamycin, tamoxifen, and cisplatin.Rapamycin, a macrolide antibiotic isolated from Streptomyces hygroscopicus, is used for immuno-suppression and cancer treatment. [43]It inhibits the mammalian target of rapamycin (mTOR), a protein kinase that regulates cell growth, proliferation, and metabolism, and consequently interferes with various cellular processes, including protein synthesis and cell cycle progression. [44]In synthetic biology, rapamycin was a pioneering inducer of gene circuits, based on its ability to interact with the FK506-binding protein (FKBP) and the FKBPrapamycin-binding (FRB) domain. [45,46]amoxifen is a selective estrogen receptor modulator (SERM) commonly employed in the treatment of hormone receptor-positive breast cancer.In synthetic biology, it is used as an inducer of the Cre/loxP system, especially for in vivo applications. [47]Cisplatin, a platinum-containing compound, inhibits the growth of cancer cells by inducing DNA damage, but in synthetic biology, it is employed to activate the HSP70 promoter [47] in a synthetic expression unit.

Synthetic compounds
In this section, we highlight synthetic ligands that are frequently employed to program gene circuits.
Cumate was designed as a small molecule to regulate gene expression in mammalian cells [48] by facilitating interaction between a bacterial CymR repressor protein from Pseudomonas putida and its synthetic operator DNA sequence (p-cym and pcmt operons).This interaction can either activate (Cumate-On) or suppress (Cumate-Off) gene expression in mammalian cells.
Two noteworthy synthetic small-molecule-based degron technologies are Shield-1 and dTAG13. [48]Shield-1 is a synthetic compound that controls the stability and activity of specific proteins.In this system, the target protein is fused to a destabilizing domain (DD) from FKBP12 (FK506-binding protein 12) mutant, ensuring unfolding and rapid degradation of the fused protein upon translation.Shield-1 specifically binds to the DD domain, preventing proteasomal degradation by stabilizing the DD protein fold and rescuing the target protein from degradation. [49,50]Conversely, dTAG13 is a small molecule that induces the degradation of the target protein.dTAG13 consists of AP1867, a ligand for FKBP F36V, and another ligand that binds to the CRBN E3 Ub ligase.dTAG13 binds to FKBP12 F36V, which is fused to the desired protein, and the CRBN E3 Ub ligase, leading to ubiquitination followed by degradation of the target protein fused to FKBP12 F36V.In this system, the fused protein remains stable and functional upon translation unless dTAG13 is supplemented, when proteasome-mediated degradation of the target protein occurs. [51]ifepristone, also known as RU-486, is a synthetic steroid that acts as a progesterone receptor antagonist and a glucocorticoid receptor antagonist.This FDA-approved compound is commonly used for medically induced abortion. [52]RU-486 has been employed to trigger a synthetic transactivator comprising a truncated human progesterone ligand-binding domain and VP16, to regulate the expression of human growth hormone (hGH) from a synthetic construct. [53]uristrone A is a synthetic analog of ecdysone, an insect moulting hormone that stimulates metamorphosis in Drosophila melanogaster.An ecdysone-induced gene expression system was developed through co-expression of a heterodimer transactivator consisting of human RXR and a chimeric ecdysone receptor fused to VP16.The transactivator drives the expression of the desired gene from a synthetic hybrid promoter, which consist of ecdysone and glucocorticoid response element halfsites (E/GRE), fused to a Drosophila heat-shock protein minimal promoter (ΔHSPmin) to activate transgene expression. [54]mall molecules that regulate alternative splicing can be repurposed to control gene circuits at the post-transcriptional level.For example, LMI070, RG7800, and RG7619 were developed for the treatment of spinal muscular atrophy (SMA), a hereditary neuromuscular disorder.LMI070, also known as branaplam, developed by Novartis, was used in a synthetic biology-inspired gene therapy study to induce alternative splicing of a synthetic SMN2 gene locus harboring desired gene, thereby triggering on-demand expression of functional protein. [55]

Small-molecule-programmed gene circuits
In the previous section, we provided an overview of the small molecules most commonly employed to regulate gene circuits in synthetic biology.Here we provide illustrative examples of the use of small molecules to generate desired outputs from engineered biological units at different levels of control, i. e., DNA, RNA, or protein.

Small-molecule regulation of DNA modification
DNA modifiers encompass proteins that recognize specific DNA sequences and modify them by adding, deleting, or inverting the targeted sequences.Such modifications are permanent, making them a good choice for applications where long-term circuit activation is desired. [56]Examples include nucleases, as the CRISPR-Cas system, TALEN, and ZFN, and recombinases such as Cre and Flp.
Nucleases are a class of enzymes that cut DNA sequences at specific locations.TALEN and ZFN rely on a DNA-binding domain and a FokI domain that are customized to bind to a specific DNA sequence and to cut the DNA, respectively. [56]In contrast, CRISPR/Cas9 simplifies the recognition process by utilizing a guide RNA (gRNA) sequence to direct Cas9 nuclease to the targeted sequence. [57]However, long-term expression of nucleases increases the likelihood of random and off-target cleavages, which are associated with genotoxicity. [58]Furthermore, prolonged expression of nucleases derived from bacteria, like Cas9, can be immunogenic, potentially enhancing the clearance of genome-edited cells by the immune system. [59,60]xamples in which small molecules specifically trigger the functionality of nucleases include ObLiGaRe, which allows the expression of spCas9 only in the presence of doxycycline (Figure 2a). [61]The expressed spCas9 binds to constitutively expressed gRNA and edits the genome of human and mouse cell lines.Small molecules have also been used to control engineered nucleases that are split into N-terminal and Cterminal moieties.For example, ABA, gibberellin, and rapamycin have been used to reconstitute functional TALEN and Cas9 proteins for genome editing (Figure 2b). [62]Compared to ObLiGaRe, the reconstitution of functional nucleases in the presence of an inducer is rapid, but ObLiGaRe provides tighter control with minimal background in the off state.
Several recombinase systems are regulated by small molecules.As discussed earlier, tamoxifen can be used as a trigger to induce gene expression by regulating translocation of Cre to the nucleus, where it can activate or block expression of the target gene (Figure 2c). [47,63]For example, this strategy has been employed in biotechnology to facilitate the on-demand production of viruses. [64]Similarly, a synthetic biology-based cell consortium (Figure 2d) has been engineered in which Ltryptophan (L-Trp)-induced B3 recombinase removes a stop codon flanked by recognition sites for B3, permitting the expression of a reporter gene. [65]

Small-molecule regulation of transcription
The number of gene circuits regulated at the transcriptional level is vast (Table 1). [15]These circuits often consist of a transactivator/repressor with a DNA-binding domain that controls the expression or repression of a desired gene, either by rewiring an endogenous module or by programing an orthogonal component to regulate gene expression. [2]Tran-scriptional control-based gene circuits typically offer high efficiency, ease of reversibility, and minimal leakiness.However, they involve a delay between induction and the production of output (typically 4-6 hours).Here, we categorize these synthetic circuits into two classes based on the location of their sensory modules: membrane-anchored or cytoplasmic-based systems (Figure 3).Membrane-bound synthetic receptors sense signals from the external environment and transmit them to engineered cells.A notable example in this class is the generalized extracellular molecule sensor (GEM) platform. [66]GEM is a synthetic receptor with an extracellular ligand-binding domain atop the native erythropoietin receptor (EpoR), allowing it to target a ligand of choice, including small molecules and peptides.By employing different intracellular domains that activate endogenous signaling pathways (e. g., JAK/STAT, PI3 K/ Akt, and MAPK), GEM rewires endogenous transcription factors (eTFs) to bind to a synthetic expression unit containing a landing site for the eTFs followed by the gene of interest.In a similar approach, a caffeine-responsive synthetic receptor, triggering the STAT3 pathway, was used to induce expression of hGLP1, a clinically licensed therapeutic peptide for the treatment of type II diabetes.Notably, cell implants with caffeine-stimulated advanced regulatory circuits, known as C-STAR, attenuated hyperglycemia in response to caffeine consumption in an experimental diabetic mouse model. [41]Another class of synthetic receptors regulated by small molecules for the timely expression of therapeutics is the chimeric antigen receptors (CARs). [67,68]CARs are synthetic receptors that enable engineered T cells to recognize specific tumor antigens expressed on cancer cells.Wu and colleagues developed a smallmolecule-gated CAR, allowing control over T cell function and thereby mitigating toxicity by regulating the timing, location, ) to the nucleus, initiating the expression of a desired gene located on a synthetic expression unit with a landing site for the activated transcription factor.b.Inducible CAR: An AND-gated CAR system is activated in the presence of the targeted antigen as well as rapamycin, which triggers CAR signaling.c.SNIP CAR: A designed protease cuts the internal domain in CAR unless the system is activated by a protease inhibitor.d.Tango system: A Chimeric GPCR is Cterminally fused to a transactivator (TA), and in the presence of the inducer drug, chimeric beta-arrestin, fused to a protease, binds to GPCR.The protease cuts the recognition site introduced between GPCR and TA, releasing TA.TA is then translocated to the nucleus and initiates transcription of a transgene from a provided synthetic expression unit.e. POST system: Phosphorylated POST sensor domains in the presence of a small molecule (e. g., caffeine) transfer the phosphate to a bacterial TA, DcuR, which is subsequently translocated to the nucleus and expresses the desired gene from an expression unit.f-g.Transcriptional control mediated by HCV (f) and coronavirus (g) proteases that control the function of an engineered TA (e. g., VP16).h.A small molecule controls the interaction of a split transactivator that initiates the transcription of a transgene in the presence of the inducer.i. Tet-On system: Dox binds to rtTA and mediates the interaction of rtTA with its DNA-binding site downstream of the transgene.j.Small-molecule-responsive aptamer mediating alternative splicing between a suicide exon (in the absence of a small molecule) or the regular exon (in the presence of a drug).k.Xon system: Branaplam mediates splicing to include a synthetic exon containing ATG, which eventually enables proper translation of the desired gene.l.Tetracycline-responsive ribozyme designed in the 3'UTR of a transgene, regulating the stability of synthesized mRNA.
and dosage of T cell activity. [69]This dual-input circuit functions only when both inputs, i. e., the tumor antigen and the smallmolecule inducer (e. g., rapamycin or gibberellin), are present.Another small-molecule-regulated CAR is SNIP-CAR (signal neutralization by an inhibitable protease (SNIP)). [70]In this circuit, NS3 protease located in the CAR receptor (cis) or codelivered to the membrane (trans) is responsible for cutting a designed cleavage site between the extracellular and intracellular functional domains of the CAR, thereby turning off the signal and rendering the system inactive (off).In the presence of an FDA-approved NS3 protease inhibitor, grazoprevir, the protease activity of NS3 is prevented, and CAR remains in the active (on) state.Another protease-mediated receptor-based system is the Tango system. [35,71]This system comprises a chimeric GPCR, an engineered beta-arrestin, and a synthetic expression unit containing the gene of interest.The GPCR is Cterminally fused to a cleavage site and a transactivator, while beta-arrestin is fused to an orthogonal protease (TEVp).Ligandinduced GPCR-beta-arrestin interaction stimulates TEVp cleavage at the cleavage site, releasing the TF, which is translocated to the nucleus and initiates transcription of the desired gene.This circuit can be reformatted with any type of GPCR for ligand-GPCR interaction screening.
Small molecules can also be employed to program gene circuits located in the cytoplasm or nucleus to either activate or repress the transcription of desired elements.One such innovation is our development of a phospho-regulated orthogonal transduction system called POST. [72]POST facilitates phosphorylation and subsequent dimerization of a bacterial histidine kinase.The phosphorylated and thus activated orthogonal module is then translocated to the nucleus and binds to the designed operator DNA sequence, initiating the expression of the desired gene.Another two-component gene expression system involves protease-based transcriptional control. [73]In this system, the NS3 protease is placed between a DNA-binding domain and a transactivator, with a cleavage site introduced at both sites.Only in the presence of the inhibitor can the synthetic module bind to its operator and initiate the expression of the desired gene; otherwise, the transactivator module will be removed by protease cleavage in the absence of the inhibitor.We have also harnessed proteases from coronaviruses and integrated them into synthetic biology circuits to function as gene expression tools. [74]In this setup, the DNAbinding domain (DBD) is equipped with coronavirus proteases at the 3'-site, while the transactivator (TA) is positioned at the 5'-site.The cleavage site is placed at the 5'-site between DBD and TA.Under resting conditions, the protease cleaves the cleavage site, so that the system is in the OFF state.However, in the presence of small-molecule inhibitors, TA can mediate the transcription of the desired gene from a synthetic expression unit.
Another widely used class of transcriptional control circuits is based on the dimerization of split components to reconstitute effective modules, induced by small molecules.In these circuits, ligand-binding induces interactions between domains such as DNA-binding domains and effector modules (trans-activators/repressors).These interactions ultimately lead to the tran-scription or repression of the desired gene.An example is the ABA-responsive domain (ABI/PYL1), which mediates the binding of a dCas9 or TetR DNA-binding domain to a designed effector module (VP16), thereby triggering gene expression in human cells upon administration of ABA. [37,38]mall molecules can also directly regulate the binding activity of a transactivator and repressor, providing another well-known class of circuits for controlling transcription in engineered cells. [3]The Tet system (Tet-Off), for instance, includes a tTA transcription factor that binds to the corresponding sequence on a synthetic expression unit and transcribes the desired gene in the absence of tetracycline or doxycycline; in the presence of the drug, the interaction between DNA and tTA is disrupted. [75,76]An engineered version of the Tet system (Tet-On) enables the expression of a gene of interest exclusively in the presence of tetracycline by modulating the binding of rtTA to the targeted sequence on the expression unit. [77,78]

Small-molecule regulation at the post-transcriptional level
Many of the systems described for regulating gene circuits at the DNA and RNA synthesis levels rely on regulatory proteins, which may impose a metabolic burden on host cells through their production or may increase the risk of immunogenicity when used in cell and gene therapy applications. [55]In contrast, regulation at the post-transcriptional level is often mediated by the endogenous protein-regulatory machinery, and so does not require external regulatory proteins.Therefore, these circuits are more compact, being suitable candidates for well-known viral vectors with limited cargo capacity, such as rAAV (recombinant adeno-associated virus). [85]ost-transcriptional control can be achieved by regulating the maturation or stability of synthesized RNA.Aptamers, short single-stranded RNA molecules, can bind to specific target molecules with high affinity and specificity, [76] and implementing small-molecule-responsive aptamers within an RNA structure allows the regulation of a desired conformational change in the secondary structure of engineered RNA (Figure 3j).For example, tetracycline-responsive aptamers have been designed to control alternative splicing in a synthetic construct. [86]In this case, the presence of tetracycline rescues splicing between an exon from the desired gene and an engineered exon, often referred to as a "suicide exon," containing a stop codon.In the absence of tetracycline, this alternative splicing does not occur, and the immature and improperly folded protein will be synthesized, which is then subjected to degradation by the proteasome.
Another example of gene regulation at the post-transcriptional level through alternative splicing is the Xon system (Figure 3k). [55]This system employs a synthetic short exon containing a start codon (ATG), flanked by two exons from the SMN2 gene.In the uninduced condition, the synthetic exon is excluded from the transcript, resulting in mature mRNA lacking the start codon (ATG) for protein expression.However, the FDAapproved small molecule Branaplam (LMI070) induces the desired splicing between the exons, resulting in the synthetic exon being included in the mature mRNA, which now contains ATG in the transcript, and can produce the desired protein.
Alternatively, an aptamer-based RNA circuit was designed to control the expression of a desired gene from an RNA molecule containing multiple payloads. [87]In this case, RNA-binding proteins (RBPs) were engineered to suppress gene expression from the engineered RNA, while small molecules were used to regulate the half-life of the RBPs fused to degrons.
By introducing a member of the class of tetracyclineresponsive RNA molecules with catalytic activity, known as aptazymes, in the 3' untranslated region (UTR) of transcribed RNA, the RNA is made stable only in the presence of tetracycline (Figure 3l). [85]This riboswitch system exhibited a significant fold induction of the desired gene in a mouse model when the animals were exposed to tetracycline.

Small-molecule regulation of protein modification and trafficking
Small-molecule regulators designed to control a gene circuit at the post-translational level can provide rapid responses. [88]rotein synthesis can be controlled by designing RNA structures that inhibit ribosome-binding unless small-molecule triggers are present to release the ribosome binding site, allowing the ribosome to initiate translation. [89]Additionally, designing a premature termination codon (PTC) in the mRNA sequence can target the prematurely terminated protein for degradation.Small molecules, such as PTC124, can enable PTC read-through, allowing full translation of the mRNA transcript. [90,91]ost-translational controls take place after the production of mature proteins and their release from the ribosome.Many small-molecule-switchable degrons can regulate the function of the targeted protein by lengthening or shortening its half-life in response to the appropriate small molecules. [48]An example is the Split Ubiquitin for the Rescue of Function (SURF) system, which induces protein stability by removing a tagged degron (Figure 4a). [92]In this system, reconstitution of the ubiquitin domain is regulated by the rapamycin-induced dimerization of split domain components.The reconstructed ubiquitin domain is then cleaved by endogenous proteasome-associated deubiquitinating enzymes (DUBs), releasing the protein of interest from its degron-tagged format.Conversely, the Small Molecule-Associated Shutoff (SMASh) system is an example of a smallmolecule destabilizing system (Figure 4b). [93]In this case, the protein of interest is fused to a degron domain separated by NS3 protease.Under unstimulated conditions, NS3 cleaves the cleavage site located between the protein of interest and NS3, leading to protein stabilization.Protease inhibitors, on the other hand, block the protease activity, resulting in the degradation of the entire fusion protein by the proteasome.
Another example of small-molecule-mediated post-transcriptional control involves engineering a protein circuit that programs the trafficking of pre-synthesized proteins.We and others have developed a small-molecule-induced protease complementation protein circuit strategy that enables the release of desired proteins from the endoplasmic reticulum (ER) (Figure 4c). [17,94,95]In an experimental mouse model of type I diabetes, this system released insulin within 2-4 hours upon ABA administration. [17]In addition, a single-component fragment has been engineered to aggregate and accumulate in the trans-ER.In this case, small molecules induce monomerization and subsequent trafficking of the targeted protein from the ER to the Golgi, leading to secretion into the extracellular environment (Figure 4d). [96]

Conclusion and future opportunities
Small molecules have significantly contributed to the programming of synthetic biology circuits in mammalian cells. [15]The applications of synthetic biology are vast, ranging from in vitro screening and biomolecule production to clinical use in patients. [2,3]In contrast to other chemicals, such as peptides or antibodies, small molecules are generally inexpensive, easy to use, and scalable, [18] though when utilizing small molecules as triggers for gene circuits, parameters such as efficiency, specificity, orthogonality, and safety must be carefully considered. [2,16]he concentration of the drug used to trigger the gene circuit, as well as its biodistribution and bioavailability in different tissues, can significantly influence efficiency.Small molecules for triggering gene circuits in cell and gene therapy should be effective at low doses and have the ability to penetrate target tissues or cells.Furthermore, they should exhibit high specificity for the targeted circuit (on-target) while exhibiting minimal interactions with other biological components.The interaction of small molecules with non-intended pharmacological targets is referred to as off-target, and it has been demonstrated that small molecules, on average, bind to a minimum of 6-11 off-target sites. [97,98]Although off-target interactions generally exhibit weaker affinity than those with the intended pharmacological target, they may become relevant in cases of high cellular expression of the off-target or high systemic exposure. [99]Therefore, the potential off-target interactions and associated outcomes for small molecules need to be considered during cell and gene therapy programs.Potential off-target interactions can be investigated, for example, through computational approaches known as Off-Target Safety Assessment (OTSA).These approaches predict safetyrelevant interactions not covered by normal drug discovery assays. [19]In addition, structural bioinformatics algorithms can be utilized to predict side effects of small molecules, offering an opportunity to improve the probability of successful development by reducing preclinical safety-related attrition rates. [100]mall molecules that exclusively target the intended gene circuit without affecting other sites can be considered orthogonal compounds. [15,92]afety considerations, which include specificity, orthogonality, and toxicity, also play a crucial role in selecting small molecules for synthetic biology applications.Often, small molecules derived from plants or food additives may be safer options.However, the dosage required for circuit activation can be critical, as the persistence of these compounds in the daily diet could unintentionally trigger the gene circuit.In-vitrodeveloped binary molecules, composed of two small molecules connected through a chemical linker, can show enhanced specificity, but may exhibit lower membrane permeability, potentially impacting biodistribution.
Another challenge that could impact the applicability of small molecules is their ability to reach the desired tissue while maintaining a high on-target effect.For example, the bloodbrain barrier (BBB) serves as a protective barrier that regulates the passage of substances from the blood into the brain, and it can limit the access of small molecules to the central nervous system (CNS). [101]However, a variety of small molecules can penetrate the BBB through passive diffusion or carrier-mediated mechanisms, allowing them to act on the brain.The effects of small molecules on the nervous system can be diverse, ranging from potential therapeutic benefits to adverse outcomes.Small molecules that successfully penetrate the BBB may interact with various targets in the CNS, potentially influencing neuronal function, neurotransmission, and other neurological processes. [102]These effects can be context-dependent and may contribute to the pharmacological actions of CNS-targeted drugs.Therefore, minimization of off-target effects while maintaining favorable pharmacokinetic and safety profiles of small molecules that cross the BBB is critical.This can be achieved, for example, by implementing strategies for structural modification of small molecules or through advances in drug delivery technologies, such as nanotechnology and targeted delivery systems. [103,104]The development of a modified pro-drug (e. g., 5-fluorocytosine; 5'FC) designed to be converted into an active form (in this case, 5-fluorouridine monophosphate; FUMP) exclusively in the desired location (in cells expressing uracil phosphoribosyltransferase; UPRTase) serves as an example of a strategy implemented to minimize off-target effects in other parts of the body. [105]or the future, we believe that the development of a toolbox of efficient, specific, and safe small molecules capable of triggering appropriate gene circuits within engineered cells has the potential to transform the future of synthetic biologyinspired cell and gene therapies, providing high precision, The protein of interest is fused to an FRP degron and a split Ub peptide.Rapamycin-responsive domains (FKBP and FRP) control the reconstruction of the Ub peptide, which can be recognized by a DUB that cuts the desired protein from the degron in the presence of rapamycin.b.SMASh system: A designed protease placed between the protein of interest (POI) and the degron controls the half-life of the target protein.A protease inhibitor induces protein degradation, while in the absence of the drug, the protein is rescued from the degron by the protease.c-d.Control of trafficking of a translated protein within intracellular organelles.c.POSH system: A protease system is reconstructed in the presence of small molecules (e. g., ABA) and removes the ER retention signal from the target protein, allowing it to be trafficked to the Golgi apparatus and subsequently secreted outside the engineered cell.d.RAPID system: A chimeric protein containing a dimerization domain aggregates and accumulates in the Golgi apparatus.Small molecules bind to the dimerization domain and release protein monomers, which are trafficked to the trans-Golgi, where furin removes the dimerization domains.Eventually, the desired protein is secreted into the extracellular environment.
efficiency, and safety.Emerging artificial intelligence (AI) and deep learning models can be expected to aid in the design of suitable molecules.

Martin
Fussenegger is Professor of Biotechnology and Bioengineering at the ETH Zurich and the University of Basel and is widely considered as one of the cofounders of the field of synthetic biology.He has received the Gaden Award, the Merck Cell Culture Engineering Award, the Medal of the European Society for Animal Cell Technology (ESACT), the Gutenberg Chair Excellence Award, two European Research Council (ERC) Advanced Grant Awards and the James Bailey Award.He is an expert panel member of the Swiss Innovation Agency (Innosuisse), the Swiss Agency for Therapeutic Products (Swissmedic) and the Global Forum on Technology (GFTech) of the Organisation for Economic Cooperation and Development (OECD).Martin is a member of the Swiss Academy of Engineering Sciences (SATW), the American Institute for Medical and Biological Engineering (AIBME), the German National Academy of Science and Engineering (ACATECH), the European Molecular Biology Organization (EMBO), the Academia Europaea (AE) and the US National Academy of Engineering (NAE).

Figure 2 .
Figure 2. Small-Molecule-Mediated Molecular Modifications of DNA.a.The ObLiGaRe system is a Tet-On system in which doxycycline regulates the expression of spCas9 causing permanent indels (knockout) in desired cell types.b.Split Cas9 is reconstituted in the presence of a small molecule to induce indels in a targeted locus.c.An estrogen receptor fused to the Cre enzyme (CreER) is translocated to the nucleus in the presence of tamoxifen and mediates interaction between LoxP sites to remove a designed DNA sequence harboring a stop codon.d.Cell consortia: L-Trp induces the expression of B3 recombinase, which eventually mediates the interaction between recombination sites flanking a stop codon, leading to the expression of the desired gene.Dox, doxycycline; indel, insertion-deletions; Cas9, CRISPR-associated protein 9; GOI, gene of interest.

Figure 3 .
Figure 3. Small-molecule-mediated gene circuits controlling transcription and post-transcriptional modification.a. GEM platform: A synthetic receptor platform dimerizes in the presence of a small molecule, and the resulting phosphorylated intracellular domains recruit endogenous transcription factors (e. g., STAT3) to the nucleus, initiating the expression of a desired gene located on a synthetic expression unit with a landing site for the activated transcription factor.b.Inducible CAR: An AND-gated CAR system is activated in the presence of the targeted antigen as well as rapamycin, which triggers CAR signaling.c.SNIP CAR: A designed protease cuts the internal domain in CAR unless the system is activated by a protease inhibitor.d.Tango system: A Chimeric GPCR is Cterminally fused to a transactivator (TA), and in the presence of the inducer drug, chimeric beta-arrestin, fused to a protease, binds to GPCR.The protease cuts the recognition site introduced between GPCR and TA, releasing TA.TA is then translocated to the nucleus and initiates transcription of a transgene from a provided synthetic expression unit.e. POST system: Phosphorylated POST sensor domains in the presence of a small molecule (e. g., caffeine) transfer the phosphate to a bacterial TA, DcuR, which is subsequently translocated to the nucleus and expresses the desired gene from an expression unit.f-g.Transcriptional control mediated by HCV (f) and coronavirus (g) proteases that control the function of an engineered TA (e. g., VP16).h.A small molecule controls the interaction of a split transactivator that initiates the transcription of a transgene in the presence of the inducer.i. Tet-On system: Dox binds to rtTA and mediates the interaction of rtTA with its DNA-binding site downstream of the transgene.j.Small-molecule-responsive aptamer mediating alternative splicing between a suicide exon (in the absence of a small molecule) or the regular exon (in the presence of a drug).k.Xon system: Branaplam mediates splicing to include a synthetic exon containing ATG, which eventually enables proper translation of the desired gene.l.Tetracycline-responsive ribozyme designed in the 3'UTR of a transgene, regulating the stability of synthesized mRNA.

Figure 4 .
Figure 4. Small-Molecule Regulation of Gene Circuits Controlling Protein Synthesis and Trafficking.a-b, Degron-based systems that control the half-lives of proteins of interest.a. SURF system:The protein of interest is fused to an FRP degron and a split Ub peptide.Rapamycin-responsive domains (FKBP and FRP) control the reconstruction of the Ub peptide, which can be recognized by a DUB that cuts the desired protein from the degron in the presence of rapamycin.b.SMASh system: A designed protease placed between the protein of interest (POI) and the degron controls the half-life of the target protein.A protease inhibitor induces protein degradation, while in the absence of the drug, the protein is rescued from the degron by the protease.c-d.Control of trafficking of a translated protein within intracellular organelles.c.POSH system: A protease system is reconstructed in the presence of small molecules (e. g., ABA) and removes the ER retention signal from the target protein, allowing it to be trafficked to the Golgi apparatus and subsequently secreted outside the engineered cell.d.RAPID system: A chimeric protein containing a dimerization domain aggregates and accumulates in the Golgi apparatus.Small molecules bind to the dimerization domain and release protein monomers, which are trafficked to the trans-Golgi, where furin removes the dimerization domains.Eventually, the desired protein is secreted into the extracellular environment.

Table 1 .
Examples of small-molecule-responsive transcriptional control systems.