Perspective Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Synthetic Biology Approaches in Immunology Niema Binth Mohammad,† Candice Chee Ka Lam,† and Kevin Truong*,†,‡ †
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Institute of Biomaterials and Biomedical Engineering, University of Toronto, 164 College Street, Toronto, Ontario M5S 3G9, Canada ‡ Edward S. Rogers, Sr. Department of Electrical and Computer Engineering, University of Toronto, 10 King’s College Circle, Toronto, Ontario M5S 3G4, Canada ABSTRACT: Breakthroughs in gene synthesis has allowed synthetic biologists the ability to design any DNA sequence of interest, enabling the possibility to create complex systems inside cells with novel functions to tackle problems in immunology. Synthetic immunology of mammalian cells expressing natural or synthetic genes can guide and induce immune responses in patients. Through recent developments in engineering chimeric receptors, it is now feasible to customize control over engineered cells to target the disease sites with specificity. These cells can avoid immune rejection if derived from expandable cell types (e.g., stem cells or T cells) and then can be grown in abundance before implantation. However, safety concerns of engineered cells in circulation necessitates the development of a wide range of mechanisms to kill cells after their therapeutic life ends. This therapeutic effect is still predominantly the secretion of therapeutic proteins, but novel therapeutic interventions have been explored by synthetic biologists. In the pursuit of engineering new cell functions for synthetic immunology, it is possible that many problems previously thought intractable may actually be possible.
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lymphocyte-associated antigen 4 (CTLA-4) and functions to facilitate the stimulation of the T-cell immune response.4 It is an approved drug for treatment of metastatic melanomas. Patients treated with Ipilimumab were found to have a significantly increased overall survival rate from 25.3% to 45.6%, when compared to previously popular gp100 antigen treatment.4 Furthermore, mAbs have been significantly improved as a form of cancer treatment over the years, with most recent developments focusing on making current mAbs proteinase-resistant and creating new antihinge antibodies, such as c7E3 IgG, capable of counteracting the IgGinactivating microenvironments of tumors.5 An emerging and alternative class of biomaterials for synthetic immunology is engineered cells expressing novel natural or synthetic genes that guide and induce an immune response in patients. Engineered mammalian cells have several desirable properties as a therapeutic platform including the ability to support large networks of transgenes,6 the potential to be drawn from the patient themselves,7 and the inherent ability to perform useful functions such as migration, secretion, membrane fusion, and target-cell lysis. Some general limitations of engineered mammalian cells as a therapeutic platform are their complexity and heterogeneity, making their
ynthetic biology began with the curiosity of whether basic electrical circuitry could be recreated inside of a living cell.1 Since then, it has expanded following this bottom-up, forward-engineering approach to be a much broader field that explores the potential of manipulating basic biological components from the level of DNA to alter protein expression, cellular behavior, and multicellular structures for a variety of applications. Breakthroughs made in molecular biology technologies such as DNA synthesis, editing and assembly, characterization of “BioBricks”, and high-throughput screening have made it possible to create complex, fully self-contained systems inside cells with novel functions that address issues in medical diagnostics and therapeutics. The first success in bioengineering a therapeutic was made by Anderson et al. in 2006, where bacteria were edited to encode for invasin protein under quorum control, allowing it to invade tumor cells in an environmentally dependent manner.1,2 Major advances in mammalian cell engineering gene therapy followed with biosensing abilities that control artificial insemination, glucose homeostasis, and gout treatment.3 A frontier area of application for biologically engineered therapeutics is synthetic immunology, which aims to harness the potential of the immune system to help in the healing process, typically, by introducing monoclonal antibodies. The ability of monoclonal antibodies (mAbs) to bring potential therapeutics to specific immune system targets when administered in patients has made it a central agent for immunotherapy. For example, ipilimumab is a monoclonal antibody against immune check point molecule cytotoxic T© XXXX American Chemical Society
Special Issue: The Chemistry of Synthetic Biology Received: October 10, 2018 Revised: November 24, 2018 Published: November 27, 2018 A
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Figure 1. General schematic of an engineered mammalian cell (center) depicting each of the four major components required to confer therapeutic abilities during clinical in vivo treatment. Components are encoded within transgenes, blue-green DNA strands, which are transformed and integrated into the host’s genome. Targetability (top left) is achieved through expression of surface receptors/antibodies specific to identifying ligands found on target cells, pictured as red to represent a diseased tumor cell, and is usually linked with activation of therapeutic effects (top right). Therapeutic effects include secretion of cytokines and/or proteins and changes in cell behavior, such as increased cell proliferation or change in transcription profile, or changes in cell structure, such as triggering of cell fusion (not pictured). Immunocloaking (bottom right) through constitutively expressed surface ligands and secreted molecules that repel, kill, and suppress host’s immune cell function; the light blue cell pictured represents a white blood cell T lymphocyte. Kill switches (bottom left) are modular surface receptors allowing controlled activation of a suicide transgene (red), leading to death.
overall behaviors often difficult to predict, and stringent handling requirements when compared to nanoparticles, viruses, or small-molecule drugs. Notwithstanding these limitations, there are several recent reports in the literature of mammalian cells being reprogrammed by the delivery of transgenes to perform therapeutically valuable functions in vivo, including secreting inflammatory cytokines from tumor cells,8 targeting and destruction of chronic lymphocytic leukemia cells by host T cells,7 and expression of RNA-based anti-HIV moieties from hematopoietic stem cells.9 Advancements in the tools used in synthetic immunology are rapid, and these tools have been the focus of prior commentaries and reviews.10−12 In this Perspective, we focus on key advancements made in recent years encompassing four main characteristics that allow an engineered cell to facilitate immunotherapy: targetability, the ability to find the disease site; immunocloaking, the ability to evade the immune system; kill switch, the ability to commit suicide after performing its therapeutic function; and therapeutic effect, the ability to perform functions that help the healing process (Figure 1).
the therapy, allowing fewer cells to be used in the treatment dose. In order to move the engineered cell to the appropriate target, the specificity and modularity of existing cell surface receptors has been utilized to create chimeras with extracellular domain targeting ability and intracellular domain signaling effectors (Figure 2). CAR T cells are patient T cells that have been engineered to express chimeric antigen receptors (CARs) to enhance their targeting abilities. The extracellular domains of these CARs recognize specific antigens expressed on cancerous cells to redirect the patient’s T cells toward tumors. Upon binding, CARs are activated and the intracellular domain activates signaling cascades that trigger an enhanced immune response by increasing T-cell proliferation and cytokine secretion. CAR T-cell therapy was effective in clinical studies and successfully redirected patient’s T cells to tumors and boosted immune response to eliminate cancerous cells. Numerous successful clinical trials have been conducted with CD19-targeted CAR T cells for the treatments of CD19-positive hematological diseases such as B-cell acute lymphoblastic leukemia (BALL). One of the first clinical studies with second-generation CD19-specific CAR T cells was published by researchers at New York’s Memorial Sloan Kettering Cancer Centre. T cells were genetically modified using γ retroviral vectors to express CAR with anti-CD19 scFv linked to CD28 and CD3ζ signaling domains. Five relapsed B-ALL patients were treated with these genetically modified autologous T cells named 19−28z. The
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ENGINEERING TARGETABILITY Following administration into the patient, the first, and arguably most important, step of therapy is the localization of the engineered cells to the diseased cells or target environment. This ensures that healthy native cells are not disrupted by off-target effects and increases the effectiveness of B
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Figure 2. Chimeric receptors for customized targetability of engineered mammalian cells. (A) CAR T-cell receptor binds to target antigens with using single-chain antibodies that subsequently trigger cell proliferation and cytokine secretion. (B) synNotch receptors bind to target antigens using single domain or chain antibody fragments that subsequently triggers proteolytic cleavage of fused transcription factors. (C) DREADDs are evolved to bind synthetic molecules that subsequently trigger G-protein-coupled pathways.
first clinical trials showed MRD (minimum residual disease negative) complete remission after 19−28z CAR T-cell infusion.13 Subsequent studies suggested a 91% complete remission rate in 32 patients with relapsed or refractory BALL.14 IL-6R inhibitors and corticosteroids were used when severe cytokine release syndrome (sCRS) was demonstrated in 7 patients. The recently published longitudinal follow-up shows long-term complete remission in 83% of the patients who received infusion of autologous CAR T cells.15 A greater incidence of reported toxicities, including sCRS, B-cell aplasia, and neurological toxicities, were observed in patients with a higher burden of disease. For all of the 53 adult patients in the study, the median survival rate was 12.9 months with a median survival rate of 20.1 months in the case of low disease burden.15 Another engineered receptor for targetability is the synthetic notch (synNotch) receptors, which are derived from the transmembrane Notch protein that mediates juxtacrine signaling. Endogenous Notch signaling is particularly important for cellular differentiation, migration, and division during development. Two successive proteolytic cleavages in the Notch intramembrane domain following ligand binding to the extracellular domain lead to the release of the intracellular domain transcriptional regulator.16 SynNotch receptors take advantage of this mechanism as a chimera of an extracellular recognition domain, specific for user-defined cell contact, and a unique intracellular transcriptional regulator released to drive the expression of defined target genes. Highly diverse forms of synNotch receptors have been engineered to detect different contact ligands and produce a wide variety of cellular
responses.16 Since they require direct cell−cell contact, precise spatial control can be achieved with synNotch receptors making them particularly useful for engineering cell targetability. A recent study showed that expression of synNotch receptors can modify cadherin cell adhesion and result in self-organization of cells into robust multidomain structures.17 By only using two different synNotch receptor−ligand pairs for cell−cell communication, a CD19 ligand that binds to an antiCD19 single-chain antibody (scFv) receptor and a surface green fluorescent protein (GFP) ligand that binds to an antiGFP nanobody receptor, the study showed how minimal intercellular communication can generate complex selforganizing multicellular structures. This extends the application of synNotch receptors from localized contact-dependent therapy to also being a tool for specifying formation of synthetic tissues with novel functions. Signal-induced spatial reorganization can modify and increase complexity of local signals received by target cells, which has useful implications in medicine. The modular nature of these receptors make them capable of combinatorial integration of different extracellular cues, giving them extraordinary flexibility in many gene therapy applications.16 SynNotch receptors have been used to improve on the safety and efficacy of CAR T-cell therapy by driving user-defined antigen-induced transcription to sculpt the immune cell response.18 SynNotch receptors can drive customized T-cell cytokine secretion profiles, skew T-cell differentiation, deliver specific therapeutics, and boost the production of antibodies, BiTEs, and adjuvants.18 Customizing T-cell sensing and C
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Frey threshold at 6 h (P < 0.001) when compared to control rats. Stably transfected BV-2 microglia cells with Gi and Gq DREADDs allowed the comprehension of mechanisms underlying hM4Di inhibition and hM3Dq stimulation. GiDREADDs attenuated inflammatory signaling,and GqDREADDs induced pro-inflammatory mediators in vitro. DREADDs targeted microglia using a CD68 promoter, and CNO was used to specifically activate the DREADDs.21 The temporal control through CNO and selectivity of DREADDs make them a very useful tool to conclusively demonstrate the role of microglia in pain as neuronal off-target effects are not present here as in regular glia inhibitory drugs. Alternatively, engineering targetability of therapeutic cells using receptors can be accomplished via Ca2+ rewiring systems that consist of, first, a chimeric receptor that generates a Ca2+ signal upon a ligand and, second, a chimeric RhoA protein that activates cell migration in response to a Ca2+ signal. Such chimeric receptors that generate Ca2+ signals can be created by fusing a ligand-binding extracellular domain with the intracellular domain of vascular endothelial growth factor receptor 2 (VEGFR2).19,20 The only requirement is that binding of the target ligand on the extracellular domain of the natural receptor induces oligomerization because this causes the corresponding oligomerization of the fused intracellular domain of VEGFR2 that in turn recruits phospholipase C to initiate IP3-mediated Ca2+ release from the endoplasmic reticulum (ER). In this way, chimeric receptors were created for tumor necrosis factor α (TNFα),22 colony-stimulating factor 1 (CSF1),23 interleukin-6 (IL6),24 shedded CD14,25 green fluorescent protein,25 mCherry,25 granulocyte-macrophage colony-stimulating factor (GM-CSF),26 rapamycin,27 extracellular Ca2+ changes,27 and monoclonal antibodies.28 The Ca2+ signals generated by the chimeric receptors are rewired to cell migration toward their respective ligands by coexpressing an engineered Ca2+ sensitive RhoA protein.29−32 These VEGFR2-based chimeric receptors are robust in rewiring cell migration because of the adaptivity in Ca2+ signal generation.25,27 In other words, stable concentrations of the ligands do not generate Ca2+ signals and thus no RhoA activation, whereas increasing concentrations of ligands (i.e., gradients from point sources) generate more Ca2+ signals and thus more RhoA activation.
response independently using a single synNotch receptor overcomes the constraints placed on CAR T-cell therapy by the endogenous system. Modified T cells with synNotch receptors have a wide variety of application in diverse diseases due to their ability to rewire cellular response programs and create specific highly localized signals in very complex microenvironments. A recent study focused on using synNotch receptors to enhance CAR T-cell specificity and drive antigen-dependent delivery of therapeutic payloads to tumors.19 Unlike previous studies that have only looked at anti-CD19, GFP, or Her2 synNotch receptors for CAR T cells, this study focused on developing a humanized Axl single-chain variable fragment to create an anti-Axl CAR and synNotch receptors. Axl was chosen as a suitable marker because it is a tyrosine kinase receptor found to be overexpressed in many different types of cancers. Anti-Axl CAR T cells caused cytokine production and induced cell death in Axl-expressing human primary T cells, and simultaneously, anti-Axl synNotch receptors were activated to trigger a therapeutic response.19 This study demonstrates the potential of Axl scFv as a target for cancer therapy and the possibility of combining synNotch and CAR T cells for cancer therapy. While CARs and synNotch systems are utilizing the modularity of receptors to harness new therapeutic potential with chimeras, mutations to receptors have also been made to change a receptor’s activating ligand. This allows for specific selectivity and spatiotemporal control over targeting in a manner dependent on synthetic ligand availability. These engineered receptors have been genetically modified to be desensitized to their endogenous ligand and receptive to physiologically inert synthetic molecules. They have been referred to as designer receptors exclusively activated by designer drugs (DREADDs) or receptors activated solely by synthetic ligands (RASSLs). DREADDs are widely used in behavioral neuroscience to study a receptor’s role in cell signaling. They offer an advantage to traditional knockout models, as direct identification of cellular responses to receptor activation are more accurate than physiological changes or responses upon loss-of-function mutations or knockouts. A recent experiment characterized Gq- and Gi-coupled DREADDs that can selectively stimulate or inhibit microglia.20 This offered major contributions to the understanding of how microglia behave in vivo. Neuropathic pain was reversed by ̈ rats through intrathecal inhibiting spinal microglia in naive CD68-hM4Di transfection using an adeno-associated virus serotype 9 (AAV9) vector. Grace et al. found that an intrathecal doze of clozapine-N-oxide (CNO), the synthetic ligand that this particular DREADD responds to, reversed allodynia in a selective manner in hM4Di-expressing rats when compared to the control.21 Von Frey thresholds were determined prior to chronic constriction injury surgery, 2 weeks after surgery prior to CNO administration, and 24 h after CNO treatment to detect allodynia. Rapid reversal of allodynia was observed within 2 h post CNO treatment (P < 0.001), persisted through 6 h post treatment (P < 0.001), and returned within 24 h. Inhibition of spinal microglia through CD68-hM4Di also resulted in CD11b upregulation in rats treated with CNO for 3 days compared to the control (P < 0.001). Allodynia was induced in rats using mM3Dg stimulation of microglia. When CNO was administered to rats expressing the Gq-coupled DREADDs, allodynia developed over a period of 4 h (P < 0.05) and had a maximum Von
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ENGINEERING IMMUNOCLOAKING The application of engineered mammalian cells for therapy by the use of virus-derived vectors can trigger an immune response in the patient. Even though the recombinant virion in adeno-associated virus and lentivirus vectors are very different from the wild-type virus, it is critical to understand how the immune system will respond to these vectors and manage the immune response in patients to achieve clinical translation. CAR T cells are derived from the host, and this facilitates evasion of the patient’s immune response along with the use of surface mAbs, which “cloaks” the cell. As mentioned, conventional CAR T-cell therapy has been used widely in cancer research to induce immune responses at tumor sites to eliminate the cancerous cells. Recent advances show that the use of regulatory T cells with a chimeric antigen receptor that binds mAbs can encourage immune tolerance. Graft-versus-host-disease was mitigated in allograft transplantation procedures with transient genetic expression of mAb-directed CAR in regulatory T cells.33 FITC-H-2DdmAbCAR Tregs demonstrated enhanced localization to D
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mCherry and CD genes downstream of the promoters and observed mCherry expression. The genetically engineered cells that were transfected with pU5BI-mCherry-CD and pGAVPO LightOn system plasmids underwent apoptosis when exposed to blue light, whereas the control cells continued to grow rapidly, thereby showing the potential of suicide genes in photodynamic gene therapy. Another potential kill switch was originally designed to prevent the escape of laboratory strains of E. coli into the environment. The bacterium was engineered to have dependency on a noncanonical alanine amino acid called bipA.42 Synthetic auxotrophs have essential genes (e.g., tyrosyl-tRNA synthetase) that were computationally design to preferentially incorporate bipA into the protein product. Since a natural environment does not have bipA, any autotrophs that are accidentally released should not survive. This system can be broadened and adopted into a conditional kill switch for therapeutic cells. Evolutionary instability of kill switches limit the application of genetic circuits in synthetic biology. Most of the kill switches that currently exist lose their functionality in a few days.43 A recent study designed a toxin/antitoxin tittering approach to screen potential kill switches with evolutionary stability.44 The two constructs, the “essentializer” and “cryodeath” kill switch circuits, were shown to be stable for at least 140 generations of growth. This system can be used in a diverse range of genetic engineering applications to develop effective kill switch designs.
allogeneic pancreatic islet grafts as observed through bioluminescence imaging. Mice with engineered H-2Dd-mAbCAR Tregs displayed a significant difference in islet survival (P = 0.002) after transplantation when compared to mice without genetically modified Tregs. Genetic CAR modification of Tregs solves many of the complications associated with current polyclonal Treg immunotherapy as it provides a method to target cells and affords a temporary immune protection that is antigen-specific. Aside from engineering cells beginning with patient-derived cells and the expression of certain surface ligands known to aid immune evasion, there are few strategies known that accomplish immunocloaking. To produce therapeutic cells in a patient-dependent manner would be time-consuming and inefficient. Furthermore, there has recently been a case of a patient experiencing leukemia relapse following this procedure due to the risk of collecting cancerous cells during the harvesting of patient T cells.34 By transfecting the leukemic cell with the CAR machinery, it was able to evade host T cells and engineered CAR T cells and ultimately lead to the death of the patient. Although this is a rare case (only 1 in 369 patients), it illustrates the need for further development of new cloaking strategies without harvest patient cells as well as kill switches.
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ENGINEERING A KILL SWITCH Cells engineered with CAR and transgenic T-cell receptor have shown to have many applications in translational medicine, especially as a cancer therapeutic. However, severe unpredictable toxicities have been observed in some cases due to the prolonged persistence of these engineered T cells in the patient after adoptive transfer.35,36 A kill switch that allows for selective destruction of these engineered cells in the case of unexpected toxicity is desirable in T-cell therapies. Clinical studies with the herpes simplex virus thymidine kinase (HSVTK) suicide gene result in a strong in vivo immune response due to the foreign nature of HSV-TK.37 Therefore, adoptively transferred HSV-TK-modified donor T cells require severe immunosuppression to be used in clinical applications. Previous studies have shown the potential of inducible caspase 9, a fusion of self-proteins FKBP12 and caspase 9 catalytic domain, in adoptive transfer, but the lack of a small chemical inducer molecule that is pharmacologically inert has resulted in limitations to the use of iCasp9.38,39 A recent study shows the effectiveness of a rapamycin-activated caspase 9 system (rapaCasp9) as a suicide gene that functions similar to iCasp9 but uses off-the-shelf pharmacological rapamycin to be activated.40 Rapamycin heterodimerizes the FKBP12-rapamycin binding domain (FRB) fragment with FKBP12, and when fused to the caspase 9 catalytic domain, this system was shown to have the best configuration to effectively activate caspase 9. The study demonstrated the effectiveness of the rapaCasp9 system in vitro and in vivo in CD19 CAR T cells. The use of rapamycin as a switch in a short dosing schedule to activate a suicide cascade results in a very minimal pharmacological activity, thereby making this a convenient system for adoptive transfer of T cells. Another kill switch developed by Chen et al. consists of a bidirectional expression module based on the LightOn system using GAVPO, a light-switchable transcription factor that homodimerizes under blue light, and a GAVPO responsive promoter that activates transcription.41 Cytosine deaminase (CD) converts the nontoxic 5-fluorocytosine (5-FC) to the cytotoxic component 5- FU. Here, the investigators placed
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ENGINEERING THERAPEUTIC EFFECTS While the therapeutic potential that can be encoded within a cell can be quite diverse, existing engineered cells being used in clinical trials generally function by secreting therapeutic proteins that help treat diseased cells directly or indirectly by influencing other host cells. Cells have been genetically modified to secrete functionally active therapeutic proteins that exert different remedial biological effects in various carcinomas and injuries. For example, to treat hepatocellular carcinoma using antiangiogenesis, recent studies have genetically engineered mesenchymal stem cells (MSCs) to secrete soluble fms-like tyrosine kinase-1 (sFlt-1)45 that has been shown to exhibit antiangiogenesis effects by free VEGF ligand sequestration and inactive heterodimer formation with VEGFR2. These cells secreted sFlt-1, demonstrated antiangiogenesis by showing a lower microvessel density, and inhibited tumor growth following intravenous injection of lentiviralinfected sFlt-1-MSCs in the mouse model.45 Likewise, genetic engineering of MSCs allow for the secretion of particular proteins that can promote cellular survival post traumatic injury. A recent study showed an increased density of GAP-43positive axons in rats when MSCs that overexpressed stromal derived factor-1 (SDF-1) were injected into the lesion epicenter 9 days post spinal cord injury (SCI).46 Moreover, following transplantation, rats treated with SDF-1-MSCs demonstrated reduced cavitation around the SCI graft site. This study shows the potential of using engineered cells for secretion of SDF-1 to promote axonal regrowth and regeneration after injury in patients with SCI. Genetic modification through gene delivery improves the therapeutic potential of MSCs and allows for treatment of many different diseases and regeneration of diverse tissues post injury. Cells have been engineered to kill target cells in several ways. One approach involves using a chimeric recombinant protein E
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Biochemistry vector47 that targets the prostate-specific membrane antigen, found primarily in prostate cancer cells. The protein vector then delivers dsRNA and polyinosinic/polycytidylic acid to activate apoptosis pathways that killed cancer cells in monolayer and 3D cultures. Neighboring nontargeted cancer cells were also eradicated since the vector can harness the immune system and activate immune cells against the entire tumor through bystander effects. Another approach involves a fuse and die mechanism by expressing vesicular stomatitis virus glycoprotein (VSVG) and herpes simplex virus type 1 thymidine kinase (TK).21,23−26,28 Engineered cells expressing VSVG undergo membrane fusion with adjacent target cells to form syncytia (i.e., multinucleated cells) at a low pH (∼6) to deliver the TK suicide gene. Upon addition of the ganciclovir (GCV) antiviral drug, TK-modified GCV is incorporated into genomic DNA where it causes DNA instability, cell cycle arrest, and ultimately caspase-9-dependent apoptosis.48 The safety of any engineered cells can be enhanced by regulated expression of therapeutic proteins through conditional switches. There are many methods of conditional control in bioengineering, and many are similar to those used in in vivo spatial-temporal knockout experiments aimed to study protein function. A recent development is the use of a GFP nanobody fused with auxin-inducible degron (AID) to specifically degrade GFP-tagged proteins. Only upon addition of auxin does the AID nanobody recruit SCF complex to trigger proteasomal degradation in vitro as well as in an in vivo zebrafish vertebrate model.49 This robust system is unrestricted by the target protein’s cellular localization, as it can function cotranslationally, and allows for reversibility by removal of auxin. There is potential for this system to be incorporated into engineering of therapeutic cells. Alternatively, for engineered cells employing type I or II CRISPR-Cas9 for therapy, antiCRISPR (Acr), discovered in phages as a method of combating bacteria immunity, can be adapted as a conditional control. For example, AcrIIA4 and AcrF2 were found to inhibit CRISPR activity by functioning as dsDNA mimics that block Cas9’s PAM recognition site.50,51 Introduction of Acr to human cells effectively controlled therapy treatment time and offered a way to reduce off-target editing.50 While these are examples of switching genes off upon intervention, there are many cases where it is desirable to have therapeutics off as default and only expressed when a stimulus is added. This is ideal for treatments that do not require constant activity of the engineered cells. An example of this is readily reversible CRISPR-Cas9 modulation, which has also been achieved through fusion of estrogen receptor binding domains ERT2 to be flanking Cas9.52 This variant of Cas9 quickly becomes activated only in the presence of 4hydroxytamoxifen (4-HT). Further work needs to be done to find optimal 4-HT treatment to balance efficient targeted gene editing and excessive off-target modifications.52
engineered cells used in therapy, however, are still derived from the patient’s own cells to avoid the issues with immune rejection, but this strategy suffers from challenges in scalability. A synthetic biology approach is needed to immunocloak any cell to allow the creation of a universal cell for therapeutics that will be better quality controlled and scalable. Such a universal cell, however, poses safety concerns if they cannot be eliminated with near certainty. Recent developments in creating kill switches have expanded the repertoire of possible mechanisms to kill cells, but kill switches are difficult to maintain in the genome because of the strong forces of natural selection acting on cells as they undergo many divisions during cell cultures. It may be necessary to use multiple kill switches or couple the kill switch to cell survival processes that cannot be mutated away in order to reach the requisite level of safety. While the therapeutic effect is still predominantly secretion of therapeutic proteins, cells can deliver a wide variety of biomaterials including RNA and DNA. Engineered cells perhaps hold the key to in vivo delivery of CRISPr genome editing tools that require the delivery of both proteins and RNA.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: 416-978-7772. Fax: 416-978-4317-164. ORCID
Kevin Truong: 0000-0002-9520-2144 Notes
The authors declare no competing financial interest.
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REFERENCES
(1) Cameron, D. E., Bashor, C. J., and Collins, J. J. (2014) A brief history of synthetic biology. Nat. Rev. Microbiol. 12, 381−390. (2) Anderson, J. C., Clarke, E. J., Arkin, A. P., and Voigt, C. A. (2006) Environmentally controlled invasion of cancer cells by engineered bacteria. J. Mol. Biol. 355, 619−627. (3) Karlsson, M., and Weber, W. (2012) Therapeutic synthetic gene networks. Curr. Opin. Biotechnol. 23, 703−711. (4) Hodi, F. S., O’Day, S. J., McDermott, D. F., Weber, R. W., Sosman, J. A., Haanen, J. B., Gonzalez, R., Robert, C., Schadendorf, D., Hassel, J. C., Akerley, W., van den Eertwegh, A. J., Lutzky, J., Lorigan, P., Vaubel, J. M., Linette, G. P., Hogg, D., Ottensmeier, C. H., Lebbe, C., Peschel, C., Quirt, I., Clark, J. I., Wolchok, J. D., Weber, J. S., Tian, J., Yellin, M. J., Nichol, G. M., Hoos, A., and Urba, W. J. (2010) Improved survival with ipilimumab in patients with metastatic melanoma. N. Engl. J. Med. 363, 711−723. (5) Jordan, R. E., Fan, X., Salazar, G., Zhang, N., and An, Z. (2018) Proteinase-nicked IgGs: an unanticipated target for tumor immunotherapy. Oncoimmunology 7, e1480300. (6) Weber, W., and Fussenegger, M. (2009) Engineering of synthetic mammalian gene networks. Chem. Biol. 16, 287−297. (7) Kalos, M., Levine, B. L., Porter, D. L., Katz, S., Grupp, S. A., Bagg, A., and June, C. H. (2011) T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced Leukemia. Sci. Transl. Med. 3, 95ra73. (8) Breitbach, C. J., Burke, J., Jonker, D., Stephenson, J., Haas, A. R., Chow, L. Q., Nieva, J., Hwang, T. H., Moon, A., Patt, R., Pelusio, A., Le Boeuf, F., Burns, J., Evgin, L., De Silva, N., Cvancic, S., Robertson, T., Je, J. E., Lee, Y. S., Parato, K., Diallo, J. S., Fenster, A., Daneshmand, M., Bell, J. C., and Kirn, D. H. (2011) Intravenous delivery of a multi-mechanistic cancer-targeted oncolytic poxvirus in humans. Nature 477, 99−102. (9) Kamata, M., Liu, S., Liang, M., Nagaoka, Y., and Chen, I. S. (2010) Generation of human induced pluripotent stem cells bearing
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CONCLUSIONS AND FUTURE DIRECTIONS The application of synthetic biology to immunology will allow the creation of the next generation of cell-based therapeutics that are aware of disease conditions and respond locally and appropriately to reduce side effects, just like the immune system when it functions at its best. Already, recent advances in the engineering of chimeric receptors, particularly with the use of antibodies fragments, have allowed unprecedented control over the targetability of cells to disease sites with clearly definable markers such as the various types of cancers. Most F
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