Engineering precision therapies: lessons and motivations from the clinic

Abstract

In the past decade, gene- and cell-based therapies have been at the forefront of the biomedical revolution. Synthetic biology, the engineering discipline of building sophisticated 'genetic software' to enable precise regulation of gene activities in living cells, has been a decisive success factor of these new therapies. Here, we discuss the core technologies and treatment strategies that have already gained approval for therapeutic applications in humans. We also review promising preclinical work that could either enhance the efficacy of existing treatment strategies or pave the way for new precision medicines to treat currently intractable human conditions.

Keywords: ATMP; cell-based therapies; gene therapy; synthetic biology; translational medicine.

Figure 1.

Treatment strategies and molecular targets of ATMPs. (A) Endogenous (im)balances of immune tolerance exemplified by (i) autoimmune diseases and (ii) cancer progression. (B) Consequences of different therapeutic interventions for immune tolerance, including (i) cellular adoptive immunotherapies, (ii) transgenic ATMPs and (iii) treatments based on implantation of encapsulated cells. Left: molecular mechanisms stimulating immune tolerance (avoiding immune clearance). Right: molecular mechanisms stimulating immune clearance (suppressing immune tolerance).

Figure 2.

Cell therapy and gene therapy products using ATMPs. Cell and gene therapy approaches either use non-viral materials (naked plasmids, oligonucleotides or proteins or materials formulated in cationic polymer shells or lipid particles) or viral transgene carriers (non-integrative DNA viruses such as adenoviruses or AAV or integrative RNA viruses such as lentivirus or retrovirus) to integrate one or multiple therapeutic transgenes into host cells. In gene therapy approaches, this integration occurs directly in the patient following injection of the appropriate transgene carrier into the tissue. In cell therapy approaches, this integration occurs ex vivo in cells isolated from the patient (autologous) or derived from a donor (allogeneic). Therapeutic cells (genetically modified cells or somatic cell therapies) can be directly injected into the patient or encapsulated into a semipermeable biocompatible device to provide immune-isolation and nutrient exchange. While retroviruses and transposases trigger random transgene integration (into multiple and different genomic loci), designer nucleases such as ZFN/TALEN/Cas9 enable targeted transgene integration (into known genomic loci). For safety reasons, gene therapy approaches should prefer the use of non-integrative transgene carriers. To facilitate commercialization, therapeutically active batches of cell therapy products should be compatible with cryopreservation methods.

Figure 3.

Clinically eligible cell therapy approaches. (A) Evolution of CARs. Most CAR-transgenic T-cell lines (CAR-T) were produced by retro/lentiviral transduction. First-generation CARs (1G) consist of an scFv-antibody fused to the native TCR signaling domain CD3ζ, and therefore require costimulatory domains such as CD28, CD137 (4-1BB) or IL15Rα on other receptors for full activation. Second-generation CARs (2G) integrate a costimulatory domain into the receptor architecture, while third-generation CARs comprise two costimulatory domains. The scFv-domain can also be replaced with antigens such as CD16 (to design universal CAR-Ts), ligands such as IL13 (to target cognate cell-surface receptors), DARPins (to target almost any protein of interest) or PD-1-receptors (to create converter CARs that transform the inhibitory effect of PD-1 into activating signals). Using the synNotch concept, scFv binding can be synchronized with expression of any therapeutic transgene (see Figure 4C). Current CAR-T research primarily focuses on (i) reduction of adverse effects related to on-target off-tumor toxicity, (ii) creation of allogeneic and/or universal CAR-T products to avoid patient- and/or tumor-specific re-engineering of new CAR-T cells, (iii) increasing CAR-T efficacy in terms of killing capacity and migration efficiency and (iv) broadening of the portfolio and types of possible CAR-T targets. (B) Blood replacement therapies (prosthetic blood). Defective blood cells caused by inherited genetic diseases can be ‘washed away’ by infusions of ex vivo-corrected CD34⁺ HSCs that can differentiate into the appropriate cell type in vivo. (i) Currently approved gene correction strategies use retroviral vectors to restore the expression of missing genes. (ii) In the future, gene-editing technologies based on designer nucleases could directly correct genomic sequences through site-specific DNA repair. (C) Cell-based prostheses. Prostheses complement or restore defective body functions in a seamless and automated manner. Physical prostheses are implanted at the appropriate body site to restore mechanical processes. Solid prostheses are based on the injection of cells (such as MSC) at body sites where specific cell–cell contact is missing. By constantly coordinating diagnosis (detection of disease markers) with treatment (synchronized production and secretion of therapeutic proteins), encapsulated cells also provide prosthetic functions by restoring metabolic balances of nutrients and hormones. For the treatment of diabetes, impaired metabolic function of glucose-dependent insulin production can be restored with stem-cell-derived β cells or β-cell-mimetic designer cells. Because designer cells use gene circuits to program any type of sense-and-respond behavior, this strategy can be applied in principle to any metabolic disease. (D) Safety switches in ATMP therapies and synthetic biology. Safety switches allow orthogonal control compounds to abort or resume the activity of transgenic cells at any point in time and operate in parallel with the therapeutic core program. Early generations of safety switches are based on drug-induced apoptosis to eliminate genetically modified cells in vivo on demand. Current and future generations of safety switches are based on trigger-inducible cell activity using soluble compounds (such as antibody mixes in SUPRA CAR; see A) or traceless remote signals such as ultrasound and light. Optogenetics involves the regulation of cell activities with light.

Figure 4.

Gene therapies and future prospects for ATMPs. (A) Gene correction. Inherited genetic disorders in somatic tissues can be corrected by ectopic overexpression of the missing gene or by transient knock-down of pathologic genes through RNAi or antisense mRNAs. Non-integrative overexpression is based on episomal expression from viral (adenovirus or AAV; stronger) or non-viral transgene carriers (plasmid DNA; weaker), which transiently override defective or deficient genomic expression. Integrative approaches enabling genome correction using designer nucleases are also possible but must overcome off-target editing events. (B) Oncolytic virotherapy. Adenoviruses lyse the infected host cell upon replication. Engineering oncolytic adenoviruses for tumor-specific replication (through E1A expression) enables tumor-selective re-infection and killing. Oncolytic HSV and poxviruses are DNA viruses with higher packaging capacities for therapeutic transgenes. Poxviruses and the RNA virus Toca511 are suitable for systemic administration. Lentiviruses can also be used for the local delivery of oncolytic transgenes into tumors. (C) Receptor-mediated transcription. Activation of SynNotch receptors triggers proteolytic cleavage of the transmembrane domain, resulting in the nuclear translocation of synthetic transcription factors and the initiation of transgene expression from cognate-specific promoters. Transcription of therapeutic transgenes can also be synchronized with endogenous signaling pathways such as JAK/STAT and MAPK. Stimulation of these pathways by custom-designed cell-surface receptor actions activates synthetic promoters engineered to contain signaling-specific response elements. (D) Therapeutic bacteria. Lactobacteria can be engineered to sense infection markers in the gastrointestinal system. Upon oral ingestion, genetically engineered bacteria self-sufficiently migrate to the gut and produce specific reporter signals that can be measured in feces. Some bacterial strains such as E. coli or Salmonella preferentially colonize and proliferate in hypoxic and immune-privileged tumor microenvironments upon systemic administration. Engineering these bacteria to carry gene circuits that trigger population-density-dependent cell lysis and drug release is a promising alternative for cancer therapy.