Theranostic cells: emerging clinical applications of synthetic biology

Abstract

Synthetic biology seeks to redesign biological systems to perform novel functions in a predictable manner. Recent advances in bacterial and mammalian cell engineering include the development of cells that function in biological samples or within the body as minimally invasive diagnostics or theranostics for the real-time regulation of complex diseased states. Ex vivo and in vivo cell-based biosensors and therapeutics have been developed to target a wide range of diseases including cancer, microbiome dysbiosis and autoimmune and metabolic diseases. While probiotic therapies have advanced to clinical trials, chimeric antigen receptor (CAR) T cell therapies have received regulatory approval, exemplifying the clinical potential of cellular therapies. This Review discusses preclinical and clinical applications of bacterial and mammalian sensing and drug delivery platforms as well as the underlying biological designs that could enable new classes of cell diagnostics and therapeutics. Additionally, we describe challenges that must be overcome for more rapid and safer clinical use of engineered systems.

Fig. 1. Synthetic biology shows promise for use in diagnostics and therapeutics.

A | Clinical applications of synthetic biology. Microbial sensors can be used as diagnostics ex vivo, reporting on biomarker levels through easily detectable colour changes. Microorganisms can also report on in vivo biomarker levels: ingested bacterial sensors can report on biomarker levels in real time (through incorporation with biocompatible electronic systems) or through subsequent analysis of stool samples. Therapeutic cells can be used to target diseased states. Immune cells can be engineered to specifically target and kill cancer cells or to differentially modulate the immune system, and bacterial cells can be used to control the microbial and metabolic composition of the gut. B | Selection of circuit elements used to engineer cells. Ba | Transcription factors control cell output. A repressor binds to its cognate promoter to block expression of the gene of interest. Conversely, an activator binds to its cognate promoter to turn on expression of the gene of interest. Bb | A toggle switch uses two repressors to stably turn on and off gene expression. Each repressor binds to its cognate promoter, and inducers control the effective state of the cell. Upon addition of Inducer 2, Repressor 2 no longer inhibits expression from its cognate promoter (P_Rep,2), and Repressor 1 and the gene of interest are transcribed. Even if Inducer 2 is removed, the cell will stay in this state, until Inducer 1 is added to ‘switch’ the cells to an off state by turning on expression of Repressor 2 and, thus, repressing expression of the gene of interest. Bc | Example of an AND-gated genetic circuit. The circuit requires two activators (A and B) to be turned on. The gene of interest is only transcribed when both activators are present. Bd | Example of an OR-gated genetic circuit. The gene of interest will be transcribed when either activator A or activator B is present. To create different logic gates, various transcription factors can be used, and the promoter architecture can be altered by changing the layout of the transcription factor binding sites.

Fig. 2. Bacterial diagnostics report on internal inflammatory markers.

A | Ex vivo diagnostics. Aa | Bacterial diagnostics for ex vivo diagnosis of zinc deficiency. Production of different pigments (lycopene, violacein and β-carotene) is controlled by zinc-responsive transcription factors. Cells can be lyophilized, rehydrated with serum and, after incubation at body temperature, turn different visible colours. Ab | Cell-free systems for ex vivo diagnosis of target nucleic acid sequences. Reactions consist of bacterial proteins, added reagents and the sensor plasmid. Upon addition of a sample, the reactions turn purple to indicate the presence of the target sequence. B | In vivo diagnostics. Ba | Toggle switch for analysis of gut inflammation. In the absence of an inflammatory stimulus (pink), the circuit is ‘off’ and no β-galactosidase (lacZ) is expressed from P_R, a promoter that is repressed by cI. Upon exposure to an inflammatory stimulus (green), the protein Cro is expressed from an inflammatory-responsive promoter (P_inflam.). Cro represses cI expression from the promoter P_RM and ‘flips’ the genetic switch into the ‘on’ state, leading to expression of β-galactosidase. A positive feedback loop ensures that the circuit stays in the ‘on’ state, even if the inflammatory stimulus is removed. The engineered bacteria can be orally administered to mice, and stool analysis reveals whether the mice have internal inflammation. Bb | Real-time monitoring of gut activity through bacterial-electronic systems. Bacteria are engineered to sense some internal signal (that is, blood) and then produce luciferase from a haem-responsive promoter (P_haem). These bacteria are embedded in a small electronic device that can be orally delivered to large mammals. The electronic device detects luciferase produced by the sensor bacteria and transmits the signal in real time via radio waves to electronic devices outside the body.

Fig. 3. Bacterial therapeutics for treating diseases in vivo.

a | Facultative anaerobic bacteria have been engineered to colonize tumour environments, which may include tumour-specific microbiome communities (represented as blue and green rectangles), by sensing various tumour-specific signals, such as hypoxia or decreasing glucose concentrations. This triggers activation of host immune defences, which facilitate destruction of tumour cells (step 1). Additional circuits have been employed to specifically trigger autolysis when a certain bacterial density is reached (step 2). This allows constitutively expressed effector molecules to be delivered within the tumour microenvironment. b | As a proposed treatment for phenylketonuria disorders caused by defects in phenylalanine-metabolizing enzymes, Escherichia coli has been metabolically engineered to increase assimilation of l-phenylalanine (l-Phe) to form trans-cinnamate, lowering blood l-Phe levels in mouse models. Expression of the periplasm-associated enzyme L-amino acid deaminase also lowers blood L-Phe levels by converting L-Phe into phenylpyruvate. This provided the basis of a clinical trial that investigated the application of using engineered bacteria to treat phenylketonuria disorders in patients with defects in L-Phe-metabolizing enzymes. c | E. coli Nissle was demonstrated to decrease viability of pathogenic Pseudomonas aeruginosa in a co-culture experiment and to impair pathogenic P. aeruginosa colonization in the mouse gut. In the presence of the target-derived quorum-sensing molecules, this ‘sense-and-kill’ strain expresses several effector genes placed under the control of a P_luxR promoter. Specifically, DspB disrupts the target biofilm. The antibacterial agent pyocin S5 is produced and released into the environment after the E7 lysis proteins lyse the host cell. For biocontainment, the engineered strain requires exogenous d-alanine for growth owing to deletions of the alanine racemase genes alr and dadX. d | E. coli was engineered to deliver a plasmid encoding RNA-guide nuclease to cleave an antibiotic-resistance gene in enterohaemorrhagic E. coli. In this particular study, a type II CRISPR–Cas system was used to cleave the target DNA sequence. AHL, acyl-homoserine lactone.

Fig. 4. Anatomy of a CAR.

A | Chimeric antigen receptors (CARs) consist of four domains: the antigen-recognition domain (single-chain variable fragment (scFv)), the spacer domain (hinge), the transmembrane domain and the intracellular signalling domain. There are four generations of CAR designs that differ by the number and type of intracellular signalling domains. First-generation CARs contain one signalling domain for T cell activation, usually the CD3ζ chain of the endogenous T cell receptor (TCR). Second-generation and third-generation CARs contain one or two additional co-stimulatory domains, for example, CD28, 4-1BB, OX40 or CD27, which improve cytokine production, proliferation and persistence of CAR T cells^,. Currently approved CAR T cells are second-generation designs directed towards the B cell antigen CD19. Fourth-generation CAR T cells (also known as T cells redirected for universal cytokine-mediated killing (TRUCKs)) are designed to deliver a transgenic protein (such as cytokines) upon CAR signalling, which improves persistence relative to earlier generations. B | Recent synthetic biology innovations to improve the safety and efficacy of CAR T cells seek to better regulate CAR T cell activation in vivo compared with older generations. Ba | Suicide genes cause CAR T cell apoptosis in the presence of a small-molecule drug. Bb | Antigen-specific inhibitory CARs (iCARs) prevent CAR T cell activity against healthy cells. Bc | A ligand-induced degradation (LID) domain allows the selective degradation of CAR surface molecules in a small-molecule dose-dependent manner. Bd | SynNotch-gated CARs require recognition of two antigens in a sequential fashion. After recognition of the first antigen, an intracellular transcription factor is cleaved, translocates to the nucleus and triggers transcription of the second CAR. Be | Split, universal and programmable (SUPRA) CARs are designed to be modular and can be controlled through injection of different protein fusions that target different antigens. Bf | AvidCARs are lower-affinity, single antigen-binding domain CARs that must bind antigen and dimerize to activate T cell effector function. HSV-TK, herpes simplex virus thymidine kinase; iCasp9, inducible caspase 9.

Fig. 5. Theranostic cells for non-cancer applications.

a | Cytokine converter cells sense the presence of two inflammatory cytokines expressed in psoriasis, TNF and IL-22, and express anti-inflammatory cytokines, IL-4 and IL-10, in response. The endogenous TNF receptor (TNFR)/NF-κB signalling pathway of HEK293 cells is rewired to activate expression of IL-22 receptor-α (IL-22RA) in the presence of TNF. When IL-22RA is expressed and IL-22 is present, IL-22RA and endogenous IL-10RB dimerize and activate the endogenous JAK–STAT pathway. STAT3 signalling is rewired to activate expression of anti-inflammatory cytokines IL-4 and IL-10 that can improve the psoriatic phenotype by calming inflammation. b | Highlighted strategies for engineering native therapeutic pathways in mesenchymal stem cells include expression of receptor intracellular domains to induce a pro-erythropoietic phenotype (step 1), transgenic expression of vascular endothelial growth factor (VEGF) (step 2) and upregulation of the native isoforms of VEGF to promote angiogenesis (step 3).