Synthetic biology in mammalian cells: next generation research tools and therapeutics

Abstract

Recent progress in DNA manipulation and gene circuit engineering has greatly improved our ability to programme and probe mammalian cell behaviour. These advances have led to a new generation of synthetic biology research tools and potential therapeutic applications. Programmable DNA-binding domains and RNA regulators are leading to unprecedented control of gene expression and elucidation of gene function. Rebuilding complex biological circuits such as T cell receptor signalling in isolation from their natural context has deepened our understanding of network motifs and signalling pathways. Synthetic biology is also leading to innovative therapeutic interventions based on cell-based therapies, protein drugs, vaccines and gene therapies.

Box 1

Figure 1. Tools used in mammalian synthetic biology

a | Tools for transcriptional control. DNA-binding domains from transcription factors (TFs) such as LacI, TetR or GAL4 are fused to protein domains that activate or repress transcription in mammalian cells^, . These artificial transcription factors regulate genes that contain their target-binding sites. Synthetic transcription factors based on zinc-fingers (ZFs), transcription activator-like effectors (TALEs)^, and clustered regularly interspaced short palindromic repeats (CRISPR)^– can be used to target any endogenous genomic region. b | Tools for RNA control. Aptamers can bind to proteins or small molecules. Addition of a protein-binding aptamer to an intron has been used to control the exclusion of an alternatively spliced exon (Ex2). When combined with ribozymes, aptamers can degrade the RNAs in which they reside. Similarly, aptamers placed in 5′ untranslated regions (UTRs) can regulate translation. mRNA translation can also be controlled by placing combinations of sequences complementary to endogenous microRNAs (miRNAs) in the mRNA 3′ UTR. c | Tools for protein turnover regulation. Ligand-induced protein degradation has been achieved by fusing a degradation recognition site (degron) to the target protein. In the presence of a ligand, the degron is bound by an E3 ubiquitin ligase complex. Alternatively, a ligand-induced degradation (LID) domain can be fused to the protein of interest. The LID domain mediates ligand-dependent degradation. This approach can also stabilize the target protein, by using a modified degradation domain (DD), the activity of which is blocked by a ligand. d | Tools for signalling pathway engineering. Rerouting the signalling of cell surface receptors can be achieved by engineering their ligand specificity. Intracellular receptor domains have been modified by fusing the intracellular receptor domains with a tobacco etch virus (TEV) protease cleavage peptide followed by an artificial transcription factor^, . In the shown example, the TEV peptide was fused to a SRC-homology 2 (SH2) protein signalling domain, which leads to its recruitment to the receptor upon activation and subsequent release of the transcription factor. Chimeric antigen receptors (CARs) are synthetic T cell receptors that enable the retargeting of T cell activity towards cells with the targeted surface antigen. Rerouting of intracellular signalling proteins has been achieved by reengineering their interaction domain such that it only recognizes an engineered binding partner but not the natural occurring counterpart. Alternatively, proteins have been brought into close proximity to each other by fusing them to protein dimerization domains, which are either constitutively active or induced by small molecules or light^{, , ,} . Inteins and split (trans-acting) inteins are proteins that can self-excise and ligate the peptides fused to them. e | Tools for genome engineering. Recombinases catalyse the recombination of a pair of short target sequences (triangles), which are pre-integrated into the genome. Depending on the target sequence configuration, DNA elements can be inserted, deleted, inverted or replaced. Nucleases fused to DNA-binding factors such as zinc-finger nucleases (ZFNs)^, or TALE nucleases (TALENs), as well as CRISPR-based systems^– have been used to induce a double-strand break at any given DNA locus. Upon double-strand break formation, the non-homologous end joining (NHEJ) or homologous recombination pathway induces a mutation or insertion of sequence fragments, respectively. GPCR, G protein-coupled receptor; ORF, open reading frame; RTK, receptor Tyr kinase.

Figure 2. Studying chromatin and gene regulation

A | Testing libraries of gene regulatory regions. A large number of synthetic variants of endogenous promoters or sequences containing combinations of transcription factor (TF)-binding motifs are cloned upstream of a minimal promoter driving a reporter gene. The constructs also contain a short variable nucleotide sequence that serves as a ‘barcode’ and can be associated with the inserted promoter sequence by high-throughput sequences of the library. The library is injected into mice or transfected into cultured cells, and its activity is measured by RNA sequencing. B | Recruitment of chromatin-modifying enzymes by zinc-fingers (ZFs) or transcription activator-like effectors (TALEs). Site-specific targeting of the histone demethylase LSD1 has been used to study the interplay between histone marks found at enhancers and nearby genes (Ba). Reversible recruitment of chromatin-modifying enzymes. Rapamycin-inducible dimerization with the transcription factor GAL4 has been used to recruit heterochromatin-binding protein 1α (HP1α), a component of repressive chromatin, to GAL4-binding sites integrated at the Oct4 promoter. The histone H3 Lys9 methylation mark, indirectly induced by HP1α, has been shown to be epigenetically transmitted after washout of rapamycin and loss of HP1α binding (Bb). Light-inducible transcriptional effectors (LITEs). Light stimulation induces dimerization of the cryptochrome protein CRY2 and CIB1 (Ca2+- and integrin-binding protein 1), leading to the recruitment of a chromatin-modifying enzyme (shown in light blue) to the target promoter (Bc).

Figure 3. Studying gene and signalling networks

a | Schematic of reverse engineering with synthetic circuits. A benchmark gene circuit is integrated into a mammalian cell line. Different reverse engineering algorithms are used to generate a network model from perturbation and gene expression analysis. The quality of reverse engineering algorithms is assessed and improved by comparing the resulting models to the actual circuit architecture in an iterative cycle. b | Studying gene network architectures. Synthetic gene circuits enable the study of network modules in isolation from their natural context in which they may be interconnected with other endogenous signalling pathways (shown in blue). The schematic shows the design of a type I incoherent feedforward loop circuit, consisting of an input (shown in brown) that activates both an output (shown in red) and an heterologous reconstitution. A mammalian MAPK pathway, consisting of an estradiol-inducible RAF1 protein that activates MEK1, which in turn activates ERK2, has been expressed in yeast. Increased concentration of each protein at each step of the cascade has been shown to augment the degree of input ultrasensitivity. ER, oestrogen receptor.

Figure 4. Chimeric antigen receptor therapy

A | The classic chimeric antigen receptor (CAR) therapy (CAR-T) approach. T cells are engineered ex vivo to express a CAR and then transferred into the original donor patient, where they destroy cells that display the target antigen. B | AND gate CAR therapy. T cells are engineered to express two CARs, one with a weakened single chain variable fragment (scFv) domain and one that contains co-stimulatory domains in its intracellular domain. These engineered T cells have been shown to preferentially target cells that display two antigens together. C | Gene circuits for controlling CAR therapy activity. A RNA device enables the control of T cell proliferation. The device stabilizes the expression of secreted interleukin-15 (IL-15), a proliferation-inducing cytokine, in the presence of the small-molecule drug Theophylline (Ca). The amplitude limiter device uses a promoter that is activated upon T cell signalling and induces the expression of the bacterial virulence protein OspF, which in turn irreversibly inactivates T cell signalling. This negative feedback loop has been shown to dampen the amplitude of T cell activation (Cb). The pause switch device consists of a construct that induces OspF expression and thereby inhibits T cell activation in response to doxycycline (Cc). LAT, linker activator for T cells; TCR, T cell receptor; ZAP70, ζ-chain-associated protein kinase 70.

Figure 5. Prosthetic networks and protein-based therapies

a | Prosthetic networks. Implantation of genetically engineered cells that are encapsulated in a semi-permeable membrane enables diffusion of small molecules and proteins and at the same time acts as a barrier to the immune system. Cells implemented in such systems have been engineered to release small effector proteins (the ‘output’) in response to specific molecular inputs (shown in blue and orange)^{, , ,} . b | Protein-based therapies. When fused to antibodies or receptor ligands, therapeutic proteins such as cytokines can be targeted to cells that express the corresponding antigen or receptor, respectively. In the case of chimeric activators, the therapeutic protein has been mutated to have low affinity for its receptor, resulting in a reduced response in cells that only bind the therapeutic protein but are not bound by the targeting element^, . Fusion to proteins that carry a high positive surface charge has been shown to facilitate intracellular delivery of proteins^–. GLP1, glucagon-like peptide 1.