Engineering the next generation of cell-based therapeutics

Abstract

Cell-based therapeutics are an emerging modality with the potential to treat many currently intractable diseases through uniquely powerful modes of action. Despite notable recent clinical and commercial successes, cell-based therapies continue to face numerous challenges that limit their widespread translation and commercialization, including identification of the appropriate cell source, generation of a sufficiently viable, potent and safe product that meets patient- and disease-specific needs, and the development of scalable manufacturing processes. These hurdles are being addressed through the use of cutting-edge basic research driven by next-generation engineering approaches, including genome and epigenome editing, synthetic biology and the use of biomaterials.

Fig. 1. A cellular therapy process flow.

Options and considerations that go into developing each step of a new cell therapy process can lead to challenges at each stage. The cell source is the starting point, whether allogeneic or autologous in nature, but these are often modified into bespoke cell therapies. These must then be manufactured at scale, which is currently a tremendous bottleneck in the industry. Finally, testing, distribution and delivery for clinical application are less challenging than some of the earlier production processes. aAPC, artificial antigen presenting cell; GMP, good manufacturing practice; IP, intellectual property.

Fig. 2. Leveraging CRISPR–Cas-mediated genome and epigenome editing for improved cell-based therapeutics.

a | Genome editing can be applied to correct monogenic diseases. Double strand breaks (DSBs) resulting from programmed genome editing outcomes generally resolve via non-homologous end joining (NHEJ) or homology-directed repair (HDR) repair mechanisms in human cells. NHEJ typically results in insertions or deletions (indels) near the targeted genomic site, which can be leveraged for programmable endogenous genetic disruption. By contrast, in the presence of a donor DNA template, HDR can permit precision replacement of genomic DNA, including donor templated to correct DNA associated with pathology or to incorporate clinically important transgenic payloads. b | CRISPR–Cas-based genome editing technologies are highly amenable to multiplexing, which can be used to improve cell-based therapeutics, including chimeric antigen receptor T cell (CAR-T) therapies. Multiplexed CRISPR–Cas9-based genome editing (shown here simultaneously targeting the genes encoding human β₂-microglobulin (β₂m), PD1 and endogenous T cell receptor (TCR)) in combination with a lentivirally delivered CAR can be used to generate CAR-T cells with improved function and safety profiles. c | CRISPR–Cas systems with inactivated nuclease activity do not result in DSBs, but can still precisely target genomic DNA. These CRISPR–Cas-based ‘epigenome editing’ platforms enable robust activation or repression of transcription (CRISPR activation (CRISPRa) or CRISPR interference (CRISPRi), respectively) or tailored control over epigenetic modifications within human cells, which can be used to shape gene regulation and cell functions. d | The convergence of these transformative technologies can be used to engineer favourable properties within cell-based therapeutics, for example, by disrupting loci that elicit immunological recognition in therapeutic-grade induced pluripotent stem cells (iPSCs), overcoming limitations to therapeutic efficacy and natural potency by harmonizing integrated payloads with natural genetic regulatory programmes (that is, expressing a CAR-T receptor from a locus (TRAC) that naturally drives TCR expression) or overexpressing beneficial endogenous molecules, and by remodelling chromatin signatures to more efficiently reprogramme cellular lineage commitment, for instance, improving the derivation of iPSCs from fibroblasts. gRNA, guide RNA; PAM, protospacer adjacent motif; Transcript., transcriptional.

Fig. 3. Using synthetic biology approaches to endow therapeutic cells with enhanced functional properties.

a | Making new synthetic regulatory connections. Engineered regulatory circuits can be introduced into therapeutic cells to create artificial input–output relationships. This can connect external user control or disease-associated molecular cues to diverse therapeutic outputs. b | Outstanding challenges for synthetic biology in engineering new cell-based therapy applications. Future developments should include developing human-derived components, developing larger capacity vectors to accommodate larger, more sophisticated circuitry and using synthetic circuits to guide cell differentiation (for example, from induced pluripotent stem cells to immune effector cells).

Fig. 4. Using synthetic circuits to enhance therapeutic function.

a | Synthetic regulatory circuits have been developed that improve chimeric antigen receptor T cell (CAR-T) therapy by enabling small-molecule remote control over CAR activity and enhancing target cell specificity (synNotch). The remote-control circuit features a split CAR that can be used to reconstitute CAR activity through administration of a small-molecule dimerizer, enabling exogenous control over T cell antitumour function. SynNotch is a programmable receptor that can sense cell surface ligands and respond by activating gene expression. This response can be coupled to production of a CAR, which is then able to recognize a second ligand, thereby enabling Boolean AND-gate function. b | Synthetic sense-and-respond programmes have been engineered that can autonomously treat diseased tissue. In one set of example applications, systems have been developed in which the sensing of inflammatory cytokines is coupled to secretion of those that are anti-inflammatory. TA, transcriptional activator.

Fig. 5. Strategies to overcome immune rejection for allogeneic cell therapy.

Schematic representation of current approaches being investigated to overcome immune mechanisms that underlie the rejection of transplanted allogeneic cells. (1) Systemic immunosuppression is the only clinically approved approach, but it results in compromised immunity and risk of malignancy. Several drug regimens and combinations of approaches including treatments with rapamycin and/or glucocorticoids, cyclosporine A and/or cyclophosphamide, cytokine blockade and/or JAK–STAT inhibitors, and B cell depletion with antibodies, are available to enable cell and organ transplantation. (2) Cell encapsulation using biocompatible polymers provides a physical barrier that limits cell–cell contact required for activation and functional lysis by T cells and natural killer (NK) cells. (3) Tolerance induction through direct overexpression of ligands for inhibitory pathways in transplanted cells leads to induction of tolerance. (4) Hypoimmunogenic cells (that is, ‘universal’ stem cells) generated through CRISPR-mediated deletion of major histocompatibility complex (MHC) molecules and overexpression of CD47 limit T and NK cell-mediated cell killing and limit macrophage-mediated phagocytosis. ADCC, antibody-dependent cell-mediated cytotoxicity; FC, fragment crystallizable region; IFN, interferon; MAC, membrane attack complex; MHC, major histocompatibility complex; TCR, T cell receptor.