Externally-Controlled Systems for Immunotherapy: From Bench to Bedside

REVIEW published: 04 September 2020 doi: 10.3389/fimmu.2020.02044 Frontiers in Immunology | www.frontiersin.org 1September 2020 | Volume 11 | Article 2044 Edited by: Jose A. Garcia-Sanz, Consejo Superior de Investigaciones Científicas (CSIC), Spain Reviewed by: Stephen Gottschalk, St. Jude Children’s Research Hospital, United States Ana Gutierrez Del Arroyo, Queen Mary University of London, United Kingdom *Correspondence: Francisco Martin [email protected] †These authors have contributed equally to this work Specialty section: This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology Received: 25 May 2020 Accepted: 28 July 2020 Published: 04 September 2020 Citation: Tristán-Manzano M, Justicia-Lirio P, Maldonado-Pérez N, Cortijo-Gutiérrez M, Benabdellah K and Martin F (2020) Externally-Controlled Systems for Immunotherapy: From Bench to Bedside. Front. Immunol. 11:2044. doi: 10.3389/fimmu.2020.02044 Externally-Controlled Systems for Immunotherapy: From Bench to Bedside María Tristán-Manzano1†, Pedro Justicia-Lirio 1,2†, Noelia Maldonado-Pérez1, Marina Cortijo-Gutiérrez1, Karim Benabdellah1and Francisco Martin1* 1Gene and Cell Therapy Unit, Genomic Medicine Department, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain, 2LentiStem Biotech, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (GENYO), Granada, Spain Immunotherapy is a very promising therapeutic approach against cancer that is particularly effective when combined with gene therapy. Immuno-gene therapy approaches have led to the approval of four advanced therapy medicinal products (ATMPs) for the treatment of p53-deficient tumors (Gendicine and Imlygic), refractory acute lymphoblastic leukemia (Kymriah) and large B-cell lymphomas (Yescarta). In spite of these remarkable successes, immunotherapy is still associated with severe side effects for CD19+malignancies and is inefficient for solid tumors. Controlling transgene expression through an externally administered inductor is envisioned as a potent strategy to improve safety and efficacy of immunotherapy. The aim is to develop smart immunogene therapy-based-ATMPs, which can be controlled by the addition of innocuous drugs or agents, allowing the clinicians to manage the intensity and durability of the therapy. In the present manuscript, we will review the different inducible, versatile and externally controlled gene delivery systems that have been developed and their applications to the field of immunotherapy. We will highlight the advantages and disadvantages of each system and their potential applications in clinics. Keywords: immunotherapy, gene therapy, externally controlled, inducible, ATMPs, transgene expression, cancer, autoimmunity INTRODUCTION Immunotherapy has drastically evolved since the past 30 years, providing diverse approaches for boosting the intrinsic power of the host’s immune system to target different diseases, especially cancer. This field includes a broad spectrum of strategies that includes the administration of cytokines, chemokines, monoclonal antibodies, cell lysates, and living cells (1–7) to directly or indirectly boost the immune system to fight cancer or to defuse it for mitigating transplant rejection (8), autoimmune diseases (9), or chronic inflammation (10). Immunotherapeutic molecules can be delivered systemically or locally into the patients through different systems such as non-viral or viral strategies that can be administered through in vivo or ex vivo strategies (11). Immuno-gene therapy is a new strategy of immunotherapy that involves genetic modification of cells in order to control immune responses. Some of the most successful immuno-gene therapy applications target tumor cells (12–14) and reduce autoimmune/inflammatory disorders (8,9,15). Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy The re-administration of T cells that are genetically modified to recognize and kill specific cell types (Chimeric Antigen Receptor, CAR-T cells) are particularly successful immunotherapeutic lines to fight refractory tumors (7,16). Nowadays, Kymriah (Tisagenlecleucel) and Yescarta (Axicabtageneciloleucel, Axi-cel) CAR-T cells became the first two advanced therapy medicinal products (ATMPs) approved in 2017 for the treatment of refractory CD19+acute lymphoblastic leukemia and aggressive B-cell lymphomas, respectively (17). A third potential ATMP, JCAR017 (Liso-cel) has received the Food and Drug Administration (FDA) breakthrough designation and priority access to medicine program by the European Medicine Agency (EMA) for Relapsed/Refractory Large B-cell Lymphoma (16) and expected to be clinically approved in 2020 (18). Besides the excellent clinical outcome reported for several immuno-gene therapy approaches, the continuous expression and secretion of potent active molecules [such as IL-12, interferons (IFNs)] can generate adverse clinical events that can lead to life-threatening organ damage and death. This toxicity also limits efficacy, due to the impossibility to reach the appropriate concentrations in target organs. There is therefore a clear necessity to develop fine-tune strategies capable of modulating immune cell activity in order to improve safety and effectiveness of immunotherapies. In this sense, gene therapy field has developed multiple strategies to control the potency and duration of the immune responses by controlling transgene expression. Several autonomous and externally-control strategies for regulating activity in immuno-gene therapy have been developed [reviewed in (19–21)] (Figure 1). First autonomous systems are self-regulated and respond to signals such as stress, inflammation, cytokines, or endogenous hormones. However, those strategies do not allow clinicians to control the intensity and durability of the therapy. On the contrary, remote-controlled systems allow the modulation of activity and associated side effects. Those approaches rely on the co-administration of an inductor, which should fulfill certain characteristics in terms of pharmacokinetics, tolerability and biodistribution (Table 1). There are various systems for controlling gene expression or managing toxicities at different levels (Figure 2). For example, inducible suicide herpes simplex virus tyrosine kinase (HSV-TK) or human thymidylate kinase (TMPK) systems trigger cell death upon a small molecule administration [reviewed in (27)] but are irreversible systems. On the other hand, several systems have been developed to control CAR-T activity (28–31). Despite their clinical potential, they are CAR-specific and not able to control other immuno-gene therapy strategies. In this review, we will focus on externally controlled, reversible (on/off switchable) and versatile inducible systems which can constitute potential tools for improving immunotherapeutic application. We will discuss the benefits and weaknesses of every emerging approach regarding their state of development, safety, on/off dynamics, inductor properties and closeness to clinics. PRINCIPLE OF EXTERNALLY CONTROLLED SYSTEMS Gene therapy provides us a robust, safe and heterogeneous platform of gene transfer for clinical applications. This field has generated a wide range of long-term, stable (or transient, if required) tools, with reduced immunogenicity for modifying immune cells by using non-viral and viral delivery systems. Although multiple inducible systems have been developed, we will focus on those that are externally controlled, are able to regulate any transgene and are potentially applicable to humans. In order to compare the different versatile available systems for clinical applications, several characteristics must be considered including the inducer properties, vector architecture, and origin, single or dual systems, promoters, target cells, leaking (basal expression in absence of the inducer) and potential risks parameters. For clarity, we will classify the different externally controlled systems on (1) those based on the administration of drugs and (2) those based on the application of physical inductors (light, ultrasounds or irradiation). Drug-Inducible Systems Inducible systems controlled through the administration of drugs are designed to trigger conformational changes on target proteins so they induce (ON-systems) or block (OFF-systems) transcription of the desired transgenes. OFF-systems have the disadvantage of continuous administration of inductor, necessary for silencing transgene expression. Permanent-high levels of antibiotics, for example, can lead to several complications for the patients and have therefore very limited applications in clinics. In this review, we will focus on the ON-systems (Figure 2). These systems require, in general, two key components: (1) a chimeric transcription factor which contains a DNA-binding domain and a drug-binding domain; and (2) a regulated minimal promoter, with very low basal activity, followed by the gene of interest. This promoter includes several copies of a non-natural DNAbinding site in which the chimeric transcription factor binds in the presence of the drug. Tetracycline-Regulated Expression Systems Tetracycline (Tet)-based gene expression control systems have been established as the systems par excellence for gene induction due to ease of handling, high efficiency and minimal side effects. This system has been designed to have three different configurations: (1) the system based on the original tetracycline repressor, TetR (Tet-ON) (32–34). In these configurations, TetR-binding sites (Tet operator-tetO) are inserted between a constitutive promoter and the gene of interest blocking its activity. The addition of tetracycline or its derivatives [such as doxycycline (Dox)] promotes a conformational change in the TetR that makes it incapable of tetO binding, allowing transcription to proceed. (2) tTA-based systems (Tet-OFF) (35). These systems are based on a chimeric protein formed by the fusion of TetR and a domain of VP16 (derived from herpes simplex virus type 1). Contrary to the TetR-based system, here the inducible transgene is placed downstream Frontiers in Immunology | www.frontiersin.org 2September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy FIGURE 1 | Gene therapy strategies to control immunogene therapy using inducible systems. Externally controlled systems (left) require the addition of an external stimuli (chemical or physical) to modulate the expression of the desire transgene. Autonomous systems (right) are designed to control the expression of the transgene in function of different cellular situations such as inflammation, cytokines, hypoxia, or pH. Figures were created with BioRender.com. TABLE 1 | Characteristics of Dox-inducible Tet-On CARs. System Target Delivery Population DosesaIn vivo induction? Leaking rtTA? ClinicalStage Ref Tet-On 3G (TaKaRa Bio) CD19 Single Selected 100 ng/ml Yes (pre-induced) Yes Yes Pre-clinical (22) Tet-On (Sangon Biotech) CD19 Single Bulk 4 g/ml No Yes Yes Pre-clinical (23) Tet-On 3G(Clontech) CD38 Dual Selected 1,000 ng/µl No No Yes Pre-clinical (24) Tet-On 3G (TaKaRa Bio) CD147 Single Bulk 1,000 ng/ml Yes(pre-induced) Yes Yes Pre-clinical (25,26) aDoses in vitro; Ref, reference. of a minimal (inactive) promoter harboring tetO sequences. Only if tTA bind to the tetO sequences, will the promoter be active through the activity of the VP16 domain, and will express the transgene. In this configuration, the addition of tetracycline also makes the tTA unable to bind to tetO and transcription stop. (3) rtTA-based systems (Tet-ON) (Figure 2, bottom-right). As mentioned before, Tet-OFF systems have limited applications in clinics. Different groups have therefore designed Tet-On systems based on mutational modifications of tTA in order to allow its binding to the tetO only in the presence of tetracycline. In these new systems, transcription requires the presence of tetracycline, becoming a Tet-ON system. The first Tet-On system (36) based on the rtTA had high leaking, but new developments improved the control of the expression (37–39). However, these systems, as we will discuss in detail, still have important drawbacks for clinical applications due to the presence of transactivators. In this direction, new developments of the original TetR systems have managed to control transgene expression in the absence of transactivators in most cell types analyzed (33,34), including primary T cells (40). These developments could open new opportunities in the field of immunotherapy. Frontiers in Immunology | www.frontiersin.org 3September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy FIGURE 2 | Externally-controlled inducible systems applied to immunogene therapy applications. Two main groups can be established: (1) Drug-based systems (Gray circle in the middle) which include ecdysone, mifepristone, tetracycline, and rapamycin systems. In these systems, clinicians could control the activity of the immunogene therapy through the administration of a drug that will, usually, activate the expression of the desired transgenes. (2) Physical-based systems include two light-based (melanopsin-based and LINTAD), one radiation-based (TNFerade) and one ultrasound-based (FUS-CAR). White area of the each dashed-line square shows the rational of each approach to achieve externally-controlled transgene expression. Key molecular players (inductors and regulatory-proteins) in each system are also indicated. Gray area inside the dashed-line squares indicate published immunogene therapy approaches for each system and the current status; In vitro studies (indicated by the absence of a mice or human drawing), in vivo studies (indicated by the presence of a mice) and clinical trials (indicated by a drawing of human figure). In addition, the target disease, therapeutic gene, modified cells and vector type are also shown. The legend at the bottom of the figure illustrate the meaning of the different symbols used in the figure. Figures were created with BioRender.com. An important advantage of these systems is that tetracycline and its derivatives, such as doxycycline, have been widely used as antibiotics in humans for decades and have been very well-characterized clinically (41). With 93% of oral absorption efficient, 14–22 h of half-life and deep tissue penetration, including blood-brain barrier (BBB) (Table 2), they are ideal inducing agents for a rapid increase in expression, long-term and rapid decrease of the desired transgene. Immunotherapeutic application CAR-T cells. A number of studies utilizing Tet-regulatory systems to regulate CAR expression have been carried out. CART therapy is a promising approach in antitumor therapy, with remarkable results obtained so far in hematological diseases. However, there are important limitations due to uncontrolled responses as a consequence of constitutive expression of the CAR molecules on the surface of T cells. For this reason, a temporary and reversible CAR expression, in which CAR-T cells response can be turned on/off, would be a convenient and eligible solution. Sakemura et al. (22) used the all-in-one pRetroX-TetOne-3G vector in which the CD19CAR-tEGFR sequence was expressed in an inducible manner in primary CD8+T cells using the rtTA system. CAR+cells were first selected for obtaining an almost 93% pure population. Maximal CAR expression in SUP-T1 cells was achieved with 100 ng/ml Dox and the expression went down after 20 h of Dox removal, although it did not reach zero in the absence of Dox. Clear differences regarding antitumor efficacy In vitro between (Dox+) Tet-CD19 CAR-T cells and (Dox-) Tet-CD19 CAR-T cells were found, but the system exhibited a significant CAR expression in the absence of Dox. For in vivo experiments, only Tet-CD19CAR-T cells incubated with Dox prior to inoculation suppressed tumor growth. Following a similar strategy, Gu et al. (23) generated an all-in-one vector expressing the rtTA2S-M2 protein (an improved version of the rtTA) and CD19-CAR (23). In this case, a concentration of 4µg/ml of Dox was necessary to induce CAR expression 5-fold. Frontiers in Immunology | www.frontiersin.org 4September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy TABLE 2 | Pharmacokinetics of the small molecules used as inductors for inducible immunotherapy. Inductor Type FDA-approved FDA-DoseaOral Tmax T1/2 BBB Ref Doxycycline Antibiotic Tetracycline Yes, for bacterial infections 200 mg/day Yes 1–3 h 18–22 h Yes (41) Veledimex Diacylhydrazine Investigational, Fast Track-FDA, as Inductor 10–20 mg/ml Yes 2.5–5.5 h 18–27.5 h Yes (42) Mifepristone Progestational and glucocorticoid antagonist Yes, abortive, contraceptive 4.5 mg/kg Yes 1–2 h 15–30 h Yes (43) Rapamycin (Sicrolimus) Antibiotic macrolide Yes, as immunosuppressant 2–5 mg/day Yes 1–6 h 57–68 h Yes (44) Rimiducid (AP1903) Antibiotic macrolide Investigational, Orphan-Drug designation 0.4 mg/kg Yes N.D 5 h Yes (45) Tmax, peak in blood after administration; T1/2, elimination half-life of the drug; BBB, blood-brain barrier; N.D, non-determined; Ref, reference. aFDA approved or used in the current clinical trial in adults. Those inducible CAR-T cells also presented better killing of tumor cells in the presence of Dox, although they produced killing also in its absence. The efficiency of the TetOn system has been also tested for multiple myeloma (MM), using CD38 antigen as the target of Dox-regulated CAR T cells (24). Here, the authors used two vectors, the pRetroX-TRE3G vector to control the expression of the CD38-CAR and the pRetroX-TET-On 3G for expression of the rtTA transactivator. CAR-expressing cells were selected by puromycin to obtain a pure population. Maximal tumor lysis In vitro was assessed with 1,000 ng/ml and the prompt reversion of the CAR activity was better achieved after a short exposition (24 h) with 10 ng/ml Dox. There are also pre-clinical assays using the Tet-On 3G system in solid tumors, specifically for hepatocellular carcinoma (HCC) treatment (26). Zhang and co-workers constructed the Tet-CD147-CAR lentiviral vector to generate Tet-CD147-CART cells. With a Dox concentration of 1,000 ng/ml CAR expression reached the peak at 24 h and returned to baseline level at 48 h after removal of Dox, but expression never reached zero. CART cells exhibited higher lytic activity in the presence of Dox, but residual lysis as a consequence of CAR leaking was observed. In an HCC mouse model, mice treated with pre-induced (Dox+) Tet-CD147-CART significantly reduced tumor volume and weight compared with those of mice that received (Dox-) Tet-CD147-CART (26), but in vivo leaking was noticeable. Others immune-gene therapy approaches. Tet-On systems have also been applied to control cytokine expression in order to boost or control immune responses in a doxycycline-dependent manner. One of the first demonstrations of the potential of this strategy used two adeno-associated vectors (AAVs), one AAV harboring the Tet-responsive promoter driving the expression of interleukin-10 (IL-10) and the other expressing rtTA (46). The authors showed therapeutic efficiency over In vitro human rheumatoid synovium from rheumatoid arthritis patients as well as in vivo mice model, after intramuscular injection of both AAVs. In another approach, the group of Dr. Castro developed a combined strategy that used Adenoviral vectors (AdV) to express HSV-TK constitutively and FLT3L in a Dox-dependent manner (47) for the treatment of Glioblastoma multiforme (GBM), a primary malignant brain cancer with very poor prognosis. This strategy aims to induce apoptosis in dividing cells in the presence of ganciclovir, and to stimulate the recruitment of DCs to the site of HSV-TK-mediated tumor killing through Doxinduction of FMS-like tyrosine kinase three ligand (Flt3L). The authors observed significant therapeutic benefits in rat models of GBM after intracranial inoculation of the AdV vectors and after treatment with Dox. Of note, a dose of 300 mg/day Dox was more effective than 200 mg/day equivalent, showing the high Dox concentrations required in this Tet-On system. Interestingly, rats were able to generate adaptive immune responses against the implanted tumors (48). Based on these studies, a clinical trial was approved in 2013 (ClinicalTrials.gov Identifier: NCT01811992) and currently ongoing Phase I (updated on April 2020). Ecdysone-Regulated Expression System Another interesting system to control gene expression in a rapid, robust, precise, and reversible way are based on the use of steroids-based regulatory domains from insects. Steroids present very interesting properties as inducers of externallycontrolled systems: they can penetrate all tissues and are quickly metabolized. The group of R.M. Evans developed the first regulated system based on the ecdysone receptor of Drosophila melanogaster to regulate transgene expression on mammalian cells (49). Different versions of these systems have been published since with different success in different cell types and tissues (50,51). Of all the systems, the RheoSwitch (52) has been the most successful, with applications even in clinical trials. The RheoSwitch Therapeutic System R (RTS) consists of a series of inter-dependent functional components for gene induction (Figure 2, top-right): (1) two transcription factors (VP16-RXR and Gal4-EcR), (2) an inducible promoter and (3) an activating small molecule ligand. The first factor arises from the fusion between the ligand binding domains of a chimeric RXR and the transcriptional activation domain of VP16 of HSV1, that acts as a co-activation partner. The second consists of a DNA-binding domain of the yeast transcription factor Gal4 fused with the hinge domains of the mutated ecdysone receptor (EcR) of the Spruce budworm (Choristoneurafumiferana), where the Frontiers in Immunology | www.frontiersin.org 5September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy ligand is bound. To achieve regulation, both proteins must be constitutively expressed. The addition of the ligand promotes the stabilization of the heterodimeric complex which binds the responsive-promoter through Gal4 and leads to transcriptional activation thanks to the VP16 domain. In the absence of an inducer, the complex is destabilized and transcription is blocked. The RTS system has been clinically validated for the control of IL-12 expression through clinical trials (53). Previous studies using IL-12 were based on the use of strong constitutive promoters, such as CMV or EF1-αto achieve high expression levels. However, IL-12 plays crucial roles in naive T cells differentiation into cytotoxic T-lymphocytes (CTLs) via IFN-γ production. It does need therefore a clear control in order to achieve the desired therapeutic benefits minimizing side effects. Different groups have also investigated the most appropriate ligand to be used in clinical settings (54). Ecdysteroids are contained in vegetables thus its safety for humans is well-proven. Veledimex is a synthetic analog of ecdysone used as the ligand of the RheoSwitch system and is currently under investigational in the Fast-track line of FDA due to its pharmacokinetics features (42,55) (Table 2). Immunotherapeutic applications The VP16-RXR and Gal4-EcR sequences was adapted into an AdV vector to express IL-12 under the control of the RTS [reviewed in (56)]. Using this rationale, two strategies were followed: (1) to transduce dendritic cells (DCs) ex vivo and introduce them into the patients, and (2) to introduce the Ad-vector in vivo (56). In the first strategy, a complete tumor regression was reached in a subcutaneous B16F0 melanoma model by delivering mIL-12-DCs intratumorally (57,58). Using the second strategy, between 73 and 90% of tumor regression was obtained using the melanoma model and tested successfully against other tumoral models (56,59). In all the cases, IL-12 increased DCs life, generated a high infiltration into the tumors of cytotoxic CD4+and CD8+T cells producing high levels ofIFNγ. Based on these data, the first-in-human clinical trial was approved that used externallyregulated gene therapy intervention (NCT00815607). This first study aimed to analyze safety, regulation of the IL-12, tolerance, response rate, and immunological effects. Patients enrolled received 5 ×107DCs transduced with Ad-RTS-hIL12 and oral administration of inducer ranging from 0.6 to 200 mg. A second phase I clinical trial was also approved using the second strategy. Patients were injected with 1 ×1012 AdRTS-IL-12 particles into accessible lesions in combination with oral inducer administration. Patients included had stage IIIIVmelanoma (NCT01397708) and metastatic breast cancer (NCT01703754, NCT02423902). Although only a minority of the patients achieved a partial regression, a veledimex dosedependent increment of mRNA IL-12 intratumorally as well as serum IFNγlevels were manifested (60). Unfortunately, several patients experienced serious toxic effects but were rapidly solved after veledimex discontinuation (60,61). In another phaseI study targeting Glioblastoma (NCT02026271), the authors showed a significant improvement in patient’s survival (55). In this study, several patients experienced severe adverse events (CRS or neurological-related) that were quickly controlled after suspension of veledimex uptake. Today, there are four open clinical trials to evaluate the intratumoral injection of Ad-RTShIl-12 and activated with oral veledimex (20 mg/day during 15 days) as a therapy to treat patients with recurrent or progressive glioblastoma (alone or combined with anti-PDL1 monoclonal antibody (mAb), NCT04006119, NCT03679754, NCT03330197, NCT03636477). Mifepristone-Regulated Expression System The first development of Mifepristone (MFP)-based systems (62) took advantage of the modular nature of functional domains of steroid receptors. The authors generated a Mifepristoneresponsive regulator (pGL-VP) by fusing the ligand-binding domain of the human progesterone receptor, the DNAbinding domain of yeast GAL4 protein and the VP16 transactivation domain of the HSV protein. They showed that this chimeric protein was able to promote transcription of minimal promoters containing GAL4-binding sites after administration of MFP In vitro and in vivo. Importantly for these systems, the MFP (RU486) concentration required for transgene activation is lower than that required for antagonizing progesterone action. A later development consisted in a chimeric regulator, GLp65 composed of a mutated ligand-binding domain (LBD) of the human progesterone receptor, the DNA-binding domain of yeast GAL4 protein and the activator domain (AD) from the human p65 protein, part of the nuclear factor kappa B complex (63). This system was commercially named as the GeneSwitchTM (GS) platform (64). GS needs two expression cassettes: the first one expressed constitutively (normally through the CMV promoter) the GLp65 transactivator protein, and the second cassette includes the inducible promoter, which contains at least four sequences for GAL4 binding, and the gene of interest. When MFP is present and binds to the LBD, a conformational change allows the GLp65 transactivator to dimerize and binds to the GAL4-promoter, activating transcription through the p65 domain (Figure 2, middle-right). The elimination of the VP16domain from the system reduced expression levels but also reduced leaking, improved safety and reduced immunogenicity. MFP is a clinically approved drug, with anti-progestin and anti-glucocorticoid properties (65). The long-term use of this drug in both females and males is under current investigation in phase III for psychotic depression (66) but MFP appeared safe and well-tolerated at the doses required to activate transcription (Table 2). However, the potential side effects as a glucocorticoid antagonist should be further characterized. Its progesterone antagonistic activity could be a problem on human T cells, which present progesterone receptors in the membrane, and T cell proliferation was inhibited after 5 mM of MPF (43, 67). Altogether, suggest that dose-escalation for future clinical application should be carefully validated. Different GS system has been developed for inducible gene therapy approaches to fight liver cancer (68–70), as well as for the treatment of neurological diseases (71,72). Another important aspect to consider of inducible systems is the alterations provoked by the constitutive expression of Frontiers in Immunology | www.frontiersin.org 6September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy the chimeric regulators. Reboredo et al. (73) analyzed the effect of GLp65 and rtTA2(S)-M2 in the liver’s transcriptomics. They found that while rtTA2 expression induced alterations in 69 genes, GLp65 caused an altered expression of 1,059, although functional analysis showed only mild alterations. Immunotherapeutic applications GS system has also been applied to regulate the expression of potent cytokines such as IL-12 with the aim to control its activity while keeping their therapeutic potential. In an elegant study, Wang et al. developed a strategy to achieve hepaticspecific expression of IL-12 that also responded to the control of MFP. The authors developed an AdV harboring the sequences for GAL4 binding into a hepatic-specific promoter driving the expression of IL-12 (6). Direct administration of these AdVs enabled controlled hIL-12 expression in the liver for more than 48 weeks when MFP was administered every 24 h. In addition, this system achieved complete tumor regression in an aggressive model of liver metastases in vivo. Whereas, using a specific-liver promoter seems to be useful for preventing immunogenicity, the IL-12 production in the liver was associated with a moderate inflammatory reaction opens the possibility that higher doses of AdV-MFP could induce-IL-12-related severe inflammation. In a different approach, MFP-GS was used to express IFNbeta for the treatment of a murine model of multiple sclerosis, experimental autoinflammatory disease (EAE) (74). In that model, a single intramuscular administration of the inducible mIFNβvector delivered as DNA plasmid was sufficient to decrease significantly the onset of disease. The procedure was well-tolerated and the overall severity of the disease scores was reduced in the presence of MFP (74). Rapamycin-Regulated Expression System Rapamycin-regulated system is a human platform designed by Rivera et al. (75,76) that is based on the interaction between two cytosolic proteins that only dimerizes in the presence of rapamycin. FK506 binding protein (FKBP12) is a 12 kDa cytosolic protein and FKBP12-rapamycin-binding protein (FRB) is a 11 kDa domain derived from mammalian target of rapamycin (mTOR). The original system contained three copies of FKB12 fused to a DNA-binding domain (zinc finger homeodomain transcriptional factor 1, ZFHD1) composing the DBD and FRB was fused to the DNA activation domain (AD) of Nuclear Factor Kappa B p65 subunit, driven expression of the gene of interest in a three-plasmid system. Transgene expression was induced after a 24 h incubation with 10 nM of rapamycin (75) but induction failed when DBD was incorporated in a retroviral vector (77). A new and more potent AD domain, called SH3, containing sequences from human heat shock factor one (HSF1) and p65, overcomes that problem even with only one copy of FKB12 in a single vector and placing the target gene cassette in reverse orientation achieves no leaking. In this case, 1µM rapamycin or analog AP1903 was necessary for maximal induction (Figure 2, bottom-middle). Rapamycin (Sirolimus) is a macrolide antibiotic with potent immunosuppressant activity used for allograft rejection in renal and cardiac transplantation (45). This immunosuppressive action occurs by targeting calcineurin and IL-2 production in T cells by Rapamacyin-FKB12 and inhibiting mTOR, thus affecting cell proliferation and metabolism via Rapamycin-FBR. In order to improve safety, a mutation in the FKBP domain was generated (FKBP12-F36V) (78) to allow the design of novel rapalogs (AP1903/AP20187) that bind the mutated but not the wild-type FKBP protein. Therefore, AP1903 (Rimiducid) is a safe and welltolerated drug that can be administered up to 1 mg/kg (44,79) (Table 2). Immunotherapeutic applications Since these systems are based in human-derived components, they present minimal immunogenicity and have been efficiently adapted to immunotherapy (Table 3). In addition, the inductor is able to cross the BBB (84) and required low concentrations (78,84) (Table 2). One of the most important uses of this system has been adapted to induce the activation of the proapoptotic enzyme caspase 9 (85), initially adapted to kill tumor cells, it did translate soon for suicide and irreversible T-cell depletion (86) to treat GVHD (BPX-015, Phase I/II). iCasp9 or CaspaCIDE is based on the homodimerization of mutated FKB12 fused to the signaling domain of caspase 9 after the treatment of AP1903/Rimiducid. This system can eliminate 85 to 95% of circulating CD3+T cells within 30 min [NCT01494103 (84)]. A phase I trial had demonstrated long-term-persistence of transduced T cells (up to 3.6 years) without compromising proliferation. However, a single clone of iCasp9-transduced T cells caused a delayed CRS in one patient that developed de novo Epstein–Barr virus-associated post-transplant lymphoproliferative disease (EBV-PTLD), being unresponsiveness to Rimiducid (87). iCasp9 have been also included in TCR-restricted (88) and CAR-T cell therapies as a safety measure (89). iCasp9 showed efficient clearance in anti-CD19 CAR-T cells co-expressing IL15 (90) and in third generation anti-CD20 CAR-T cells, where the 90% of engineered T cells were depleted in vivo in only 12 h (91). GD2-specific and iCasp9-expressing CAR (GD2-iCAR) T cells have reached clinical trials against advanced melanoma (CARPETS, ACTRN12613000198729), neuroblastoma (GRAIN, NCT01822652), sarcoma (VEGAS, NCT01953900), and other GD2+solid tumors (NCT02107963). Of note, while iCasp9 can rapidly reverse toxicity, sacrifices the long-term antitumor efficacy. Stavruo et al. (78) have exploited the same Caspase 9 strategy in CAR19-T cells but using original FRB/FKBP system to generate homodimers of Casp9 after rapamycin addition (RapaCasp9), which is indeed a clinically-drug approved, exhibiting a similar response of rapaCasp9 to iCasp9 at 1 nM (78). Another elegant FKBP/FRBmut system specific for CARs, are the “ON-switch” CARs (28), where the CAR structure is split into two chimeric polypeptides: CAR-I encloses the antigen recognition domain, transmembrane and 4-1BB costimulatory domain and CAR-II harbors the main CD3zeta-ITAMS signaling domain. Both chimeric proteins are fused to intracellular FKBP/FRBmut. Only when AP21967 was administered, an antitumoral effect was observed in mice, but due to its shorter life, another heterodimer system would be desirable for a moresuitable future clinical application. Frontiers in Immunology | www.frontiersin.org 7September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy TABLE 3 | Systems for controlling transgene expression applied to immunotherapy. System Inductor Gene Model/Disease Product Administration Clinical stage Ref Tet-On 3G (TaKaRa Bio) Dox CAR-CD19 CD19+Raji cells (Burkitt’s lymphoma) All-in-one RV-T cells Cellsa: intravenously Inductor: pre-induced ex vivo+oral Pre-clinical (22) Tet-On (Sangon Biotech) Dox CAR-CD19 CD19+Raji cells (Burkitt’s lymphoma) All-in-one LV-T cells In vivo experiments were not conducted In vitro (23) Tet-On 3G (Clontech) Dox CAR-CD38 CD38+cell lines (Multiple myeloma) Dual system RV-T cells in vivo experiments were not conducted In vitro (24) Tet-On 3G (TaKaRa Bio) Dox CAR-CD147 CD147+cells (Hepatocellular carcinoma) All-in-one LV-T cells Cellsa: intratumoral Inductor: pre-induced ex vivo Pre-clinical (26) Tet-On Dox IL-10 DBA1 mice (Rheumatoidarthritis) All-in-one AAV vp Vectorb: intramuscularly Inductor: oral Pre-clinical (46) Tet-On Dox FLT3L Glioblastoma multiforme All-in-one Ad Vectorb: intracranial Inductor: oral Phase I (47) RheoSwitch (RTS) Veledimex IL-12 Stage III or IV melanoma All-in-one Ad-DCs Cellsa: intratumoral Inductor: oral Phase I (57) RheoSwitch (RTS) Veledimex IL-12 Stage III-IV melanoma Metastatic breast cancer All-in-one AdV Vectorb: accessible lesions Inductor: oral Phase I/II (56) Gene Switch MFP IL-12 MC-38 mice (Livermetastases) All-in-one AdV Vectorb: intravenous Inductor: intraperitoneal Pre-clinical (68) Gene Switch MFP IFN-βEAE mice (multiple sclerosis) DNA plasmid Plasmidc: intramuscular Inductor: subcutaneous Pre-clinical (74) LightpNFAT Blue light IL-2, IL-15, TNF-α SK-HEP-1 mouse (Hepatocellular carcinoma) All-in-one LV-T cells Cellsa: subcutaneous Inductor: externally applied Pre-clinical (80) LINTAD Blue light CAR-CD19 CD19+Nalm6+mice (B-lymphoblastic leukemia) Dual system LV-T cells Cellsa: subcutaneous Inductor: externally applied Pre-clinical (81) TNFerade Radiation TFN-a Metastatic pancreatic cancer All-in-one Deficient AdV Vectorb: intratumor Inductor: externally applied Phase III (82) FUS-CAR Ultra-sounds CAR-CD19 CD19+Nalm6+cells (B-lymphoblastic leukemia) PC3 cells (Prostatecancer) Dual system LV-T cells Cellsa: subcutaneous Inductor: externally applied Pre-clinical (83) aEx vivo transduction. bin vivo transduction. cDirect plasmid injection Dox, doxycycline; MFP, mifepristone; CAR, chimeric antigen receptor; IL-10, interleukin 10; FLT3L, FMS-like tyrosine kinase 3 ligand; IL-12, interleukin 12; IFN-β, interferonβ; IL-2, interleukin 2; IL-15, interleukin 15; TNF-α, tumor necrosis factor α; EAE, experimental autoimmune encephalomyelitis; RV, retroviral vector; LV, lentiviral vector; AAV, Adeno-associated vector; vp, viral particles; Ad, adenoviral vector; Ref, reference. In a different configuration but with a similar idea, the dimerizing agent–regulated immunoreceptor complex (DARIC)- T cells is also composed of two CARs (92): the CAR-I is composed by the ScFv-FRB-TM domains and the CAR-II by the FKPBTM-41BB-CD3ζdomains. In both CARs, the FKBP/FRBmut domains are located extracellularly. DARIC-T cells also allow the application of a plugin for targeting another antigen (a subunit of the ScFv and FRB). In addition, Tacrolimus, which has a high affinity for FKBP12, can be used as a rapamycin competitor, which could be interesting as a safe method for reducing severe CRS or persistent neurotoxicity (92). Controlling the ScFv presentation at the cell surface after AP21967 addition is another FKB16/FRBmut design, where the FRB and FKBP12 domains were placed between the CD8a hinge and the scFv domains, modulating the cytotoxic properties of this “transient” CAR-T cell (93). Rapamycin-based systems have also been developed to activate immune cells such as dendritic cells (DC) or T cells, regulating the synergy of TLR/IL1R (through MyD88) and CD40 signaling within the context of an immunological synapse. In this case, rimiducid-inducible MyD88 and CD40 (iMC) system is composed by aTIR domain-deleted version of MyD88 fused to tandem copies of the modified FKBP12V36 and a myristylationtargeting sequence for membrane anchoring, whereas the same fusion structure was used for the cytosolic domain of CD40. Autologous iMCs-DCs showed a strong antitumoral effect in vivo (94,95). More recently, this iMC strategy has been applied for the CAR-T field against HER2+solid tumors. In the presence of Rimiducid, T cells expressing HER2–CARζand this FKBP12 iMyD88/CD40/FKBP12 system exhibited potent antitumor activity in pre-clinical models, allowing their remote-control post-infusion (96), becoming a very promising platform. Duong et al. (97) engineered CAR-T cells with this Rimiducid iMCsignaling system for CAR T cell activation in combination with the rapamycin-induced caspase-9-based safety switch (iRC9) for controlling potential risks. This dual-switch (DS) system generated higher CAR-T cell expansion in a drug-dependent Frontiers in Immunology | www.frontiersin.org 8September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy manner while triggering apoptosis to avoid severe toxicities if required (97). However, escalation doses of Rim to in vivo/clinical application should be carefully evaluated since 100x more of the Rim dose for activate iMC counterpart (1 nM), could also trigger the iRC9 system in vitro. Future Drug-Inducible Systems for Immunogene Therapy Applications Here, we will briefly describe systems that fulfill the above criteria of versatile, reversible and inducible system but have not been used yet for immunotherapeutic applications. Antibiotics Other antibiotic-based systems found in different bacterial strains have been modified and adapted to control gene expression in mammalian cells such as streptogramin (PipOFF/PipON) and macrolide (EON/EOFF) based gene regulation systems (98). Those four systems have been tested In vitro using different cell lines and different transgenes, obtaining a fast (<24 h) and great induction (until 100-fold) of transgene and low leaking (98,99). Both the macrolide and the streptogramin antibiotic families present interesting clinical properties, such as excellent bioavailability, optimal pharmacokinetics, and human compatibility (100,101). Another system that has not been applied yet to immunotherapy approaches is the system based on the original tetracycline repressor, TetR (32–34). These systems have several advantages over the traditional Dox-based system that use transactivators such as the absence of toxicity, the low leaking, and the low Dox requirements. Quorum Sensing A chimeric transcription factor controlled by an acylated homoserine lactone (AHL), getting up to 1000-fold induction and low basal transcription in different human cell lines have been developed (102). However, AHL signaling molecules can influence the behavior of eukaryotic cells and tissues and it is unknown its pharmacodynamics in vivo (103,104). Looking ahead, it is possible to engineer other transcription factors from different bacterial species and develop inducer compounds with improved characteristics. Physically-Induced Systems Light-Based Systems (Optogenetics-Based) Optogenetics rely on light-sensitive proteins that have a physiological role of regulating the behavior of living cells. Most optogenetic tools are based on light-sensitive ion channels, but there are also other types of molecules able to respond to light, such as enzymes and protein interaction modules (105–110). This variety of tools has opened the opportunity of modulating gene transcription and to use it for gene therapy applications (111–113). Transgene induciblesystems based in Optogenetic are a very promising approach due to their high spatial-temporal control capacity (114) compared to other systems and because light can be applied locally without affecting other organs. However, the low penetrance of blue light may be a limiting factor for future clinical application. Two main optogenetic strategies have been used in immunotherapy: Melanopsin-based (calcineurinNFAT-based) and biLINuS-based (nuclear translocation induced by light). The Melanopsin-based system (Figure 2, top-middle) relies on the ability of this protein to induce calcium influx under blue light illumination. Intracellular Ca++ increment activates calcineurin that triggers the nuclear translocation of NFAT (111), a transcription factor involved in the expression of multiple genes related to effective immune responses (115). For the system to achieve light-response into target cells we need to express the melanopsin and introduce an expression cassette harboring an NFAT-responsive promoter (Figure 2) (111). Once all the components are into the target cell, light will activate Ca++ influx through the melanopsin that is expressed in target cells. This Ca++ influx initiates a signaling cascade that leads to NFAT-nuclear translocation, activation of NFAT-promoter and expression of the desired genes. In the biLINuS system (Figure 2, top-left), the light will expose the NLS motif to cause nuclear translocation of the complex (generally harboring transcriptional activators) required for transcriptional activation. These systems are based in the light-inducible nuclear localization signal (LINuS) from the LOV2 domain of Avena sativa phototropin 1 (ASP-1), a small tag that can be added to different proteins and cell types (116). In particular, the LINuS system has been used in combination with the blue light–based CRY2-CIB1 (used for blue lightdependent transgene expression) (117) but that had a high background. Huang et al. (81) developed the LINTAD gene activation system that rely in two chimeric proteins and a light responsive promoter (Figure 2): (1) The LexA-CIB1-biLINuS (LCB) protein combines the CRY2-CIB1 pair with the LOV2 domain reducing non-specific CRY2/CIB1 dimerization, (2) the CV protein contains the NLS from CRY2PHR (Arabidopsis CRY2 photolase homology region) and a strong VPR transcription activator (a tripartite VP64-p65-Rta), and (3) the light-inducible promoter harbors several LexA-binding sequence (LexA BS) and a minimal promoter that require the presence of transactivators to be active. The LCB remains in the cytoplasm, while the CV remains in the nucleus. When stimulated by blue light, biLINuS in the LCB is activated, exposing the NLS motif to cause nuclear translocation of the SCB. At the same time, the CRY2PHR domain in CV is activated by blue light and can bind to the CIB1 domain of LCB with high affinity. Therefore, the LCB-CV complex is directed to the LexA BS in the reporter cassette so that the VPR is very close to the minimal promoter, which triggers transcription of the target reporter gene. This would generate a strong activation of the gene by stimulating blue light with a high signal-to-noise ratio. Immunotherapeutic applications Following the two main strategies described above, optogenetics have pursued two main strategies for immunotherapy: to increase the expression of NFAT-targeted genes (mainly cytokines), key regulators of T cell function (80,112,115,118) and to induce CAR expression through the biLINuS system (80,116). Frontiers in Immunology | www.frontiersin.org 9September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy interactions mediated by the extended synaptotagmins. Cell. (2013) 153:1494. doi: 10.1016/j.cell.2013.05.026 107. Xu B, Sun Y, Guo Y, Cao Y, Yu T. Synthesis and biological evaluation of N4-(hetero)arylsulfonylquinoxalinones as HIV1 reverse transcriptase inhibitors. Bioorg Med Chem. (2009) 17:2767–74. doi: 10.1016/j.bmc.2009.02.039 108. Repina NA, Rosenbloom A, Mukherjee A, Schaffer DV, Kane RS. At light speed: advances in optogenetic systems for regulating cell signaling and behavior. Annu Rev Chem Biomol Eng. (2017) 8:13– 39. doi: 10.1146/annurev-chembioeng-060816-101254 109. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, et al. Optical control of mammalian endogenous transcription and epigenetic states. Nature. (2013) 500:472–6. doi: 10.1038/nature12466 110. Motta-Mena LB, Reade A, Mallory MJ, Glantz S, Weiner OD, Lynch KW, et al. An optogenetic gene expression system with rapid activation and deactivation kinetics. Nat Chem Biol. (2014) 10:196–202. doi: 10.1038/nchembio.1430 111. Ye H, El Baba MD, Peng RW, Fussenegger M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science. (2011) 332:1565–8. doi: 10.1126/science.1203535 112. Kim K-D, Bae S, Capece T, Nedelkovska H, De Rubio RG, Smrcka AV, et al. Targeted calcium influx boosts cytotoxic T lymphocyte function in the tumour microenvironment. Nat Commun. (2017) 8:15365. doi: 10.1038/ncomms15365 113. Tan P, He L, Han G, Zhou Y. Optogenetic immunomodulation: shedding light on antitumor immunity. Trends Biotechnol. (2017) 35:215– 26. doi: 10.1016/j.tibtech.2016.09.002 114. Yamada M, Suzuki Y, Nagasaki SC, Okuno H, Imayoshi I. Light control of the tet gene expression system in mammalian cells. Cell Rep. (2018) 25:487–500.e6. doi: 10.1016/j.celrep.2018.09.026 115. Macian F. NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol. (2005) 5:472–84. doi: 10.1038/nri1632 116. Niopek D, Benzinger D, Roensch J, Draebing T, Wehler P, Eils R, et al. Engineering light-inducible nuclear localization signals for precise spatiotemporal control of protein dynamics in living cells. Nat Commun. (2014) 5:4404. doi: 10.1038/ncomms5404 117. Taslimi A, Zoltowski B, Miranda JG, Pathak GP, Hughes RM, Tucker CL. Optimized second-generation CRY2-CIB dimerizers and photoactivatable Cre recombinase. Nat Chem Biol. (2016) 12:425–30. doi: 10.1038/nchembio.2063 118. He L, Zhang Y, Ma G, Tan P, Li Z, Zang S, et al. Near-infrared photoactivatable control of Ca2+signaling and optogenetic immunomodulation. eLife. (2015) 4:e10024. doi: 10.7554/eLife.10024 119. Hallahan DE, Mauceri HJ, Seung LP, Dunphy EJ, Wayne JD, Hanna NN, et al. Spatial and temporal control of gene therapy using ionizing radiation. Nat Med. (1995) 1:786–91. doi: 10.1038/nm0895-786 120. Morii A, Ogawa R, Watanabe A, Kakutani S, Zhao QL, Kume K, et al. Regulation of gene expression in prostate cancer cells with an artificially constructed promoter responsive to radiation. Gene Ther. (2012) 19:219– 27. doi: 10.1038/gt.2011.89 121. Ogawa R, Morii A, Watanabe A, Cui ZG, Kagiya G, Fukuda S, et al. Development of a therapeutically important radiation induced promoter. Bioengineered. (2013) 4:44. doi: 10.4161/bioe.21965 122. Radiotherapy Dose Fractionation. 3rd ed. The Royal College of Radiologists. Available online at: https://www.rcr.ac.uk/publication/radiotherapy-dosefractionation-third-edition (accessed May 23, 2020). 123. Herman JM, Wild AT, Wang H, Tran PT, Chang KJ, Taylor GE, et al. Randomized phase iii multi-institutional study of tnferade biologic with fluorouracil and radiotherapy for locally advanced pancreatic cancer: final results. J Clin Oncol. (2013) 31:886–94. doi: 10.1200/JCO.2012.44.7516 124. Wang S, Olumolade OO, Sun T, Samiotaki G, Konofagou EE. Noninvasive, neuron-specific gene therapy can be facilitated by focused ultrasound and recombinant adeno-associated virus. Gene Ther. (2015) 22:104– 10. doi: 10.1038/gt.2014.91 125. Skalina KA, Singh S, Chavez CG, Macian F, Guha C. Low intensity focused ultrasound (LOFU)-mediated acoustic immune priming and ablative radiation therapy for in situ tumor vaccines. Sci Rep. (2019) 9:15516. doi: 10.1038/s41598-019-51332-4 126. Moonen CTW. Spatio-temporal control of gene expression and cancer treatment using magnetic resonance imaging-guided focused ultrasound. Clin Cancer Res. (2007) 13:3482–9. doi: 10.1158/1078-0432.CCR-07-0204 127. Norelli M, Camisa B, Barbiera G, Falcone L, Purevdorj A, Genua M, et al. Monocyte-derived IL-1 and IL-6 are differentially required for cytokinerelease syndrome and neurotoxicity due to CAR T cells. Nat Med. (2018) 24:739–48. doi: 10.1038/s41591-018-0036-4 128. Zhang Q, Ping J, Huang Z, Zhang X, Zhou J, Wang G, et al. CAR-T cell therapy in cancer: tribulations and road ahead. J Immunol Res. (2020) 2020:1924379. doi: 10.1155/2020/1924379 129. Chmielewski M, Abken H. CAR T cells releasing IL-18 convert to T-bethigh FoxO1low effectors that exhibit augmented activity against advanced solid tumors. Cell Rep. (2017) 21:3205–19. doi: 10.1016/j.celrep.2017.11.063 130. Kueberuwa G, Kalaitsidou M, Cheadle E, Hawkins RE, Gilham DE. CD19 CAR T cells expressing IL-12 eradicate lymphoma in fully lymphoreplete mice through induction of host immunity. Mol Ther Oncolytics. (2018) 8:41–51. doi: 10.1016/j.omto.2017.12.003 131. Xie YJ, Dougan M, Ingram JR, Pishesha N, Fang T, Momin N, et al. Improved antitumor efficacy of chimeric antigen receptor T cells that secrete single-domain antibody fragments. Cancer Immunol Res. (2020) 8:518– 29. doi: 10.1158/2326-6066.CIR-19-0734 132. Rafiq S, Yeku OO, Jackson HJ, Purdon TJ, van Leeuwen DG, Drakes DJ, et al. Targeted delivery of a PD-1-blocking scFV by CAR-T cells enhances anti-tumor efficacy in vivo.Nat Biotechnol. (2018) 36:847– 58. doi: 10.1038/nbt.4195 133. Lin JT, Lineberry NB, Kattah MG, Su LL, Utz PJ, Fathman CG, et al. Naive CD4 T cell proliferation is controlled by mammalian target of rapamycin regulation of grail expression. J Immunol. (2009) 182:5919– 28. doi: 10.4049/jimmunol.0803986 134. Alexander-Savino CV, Hayden MS, Richardson C, Zhao J, Poligone B. Doxycycline is an NF-κB inhibitor that induces apoptotic cell death in malignant T-cells. Oncotarget. (2016) 7:75954–67. doi: 10.18632/oncotarget.12488 135. Hall DB, Struhl K. The VP16 activation domain interacts with multiple transcriptional components as determined by protein-protein cross-linking in vivo.J Biol Chem. (2002) 277:46043–50. doi: 10.1074/jbc.M2089 11200 136. Hackl H, Rommer A, Konrad TA, Nassimbeni C, Wieser R. Tetracycline regulator expression alters the transcriptional program of mammalian cells. PLoS ONE. (2010) 5:e13013. doi: 10.1371/journal.pone.0013013 137. Han HJ, Allen CC, Buchovecky CM, Yetman MJ, Born HA, Marin MA, Rodgers SP, et al. Strain background influences neurotoxicity and behavioral abnormalities in mice expressing the tetracycline transactivator. J Neurosci. (2012) 32:10574–86. doi: 10.1523/JNEUROSCI.0893-12.2012 138. Judelson HS, Narayan R, Ah Fong AMV, Tani S, Kim KS. Performance of a tetracycline-responsive transactivator system for regulating transgenes in the oomycete Phytophthora infestans.Curr Genet. (2007) 51:297– 307. doi: 10.1007/s00294-007-0125-2 139. Sisson TH, Hansen JM, Shah M, Hanson KE, Du M, Ling T, et al. Expression of the reverse tetracycline-transactivator gene causes emphysema-like changes in mice. Am J Respir Cell Mol Biol. (2006) 34:552–60. doi: 10.1165/rcmb.2005-0378OC 140. Perl AK, Zhang L, Whitsett JA. Conditional expression of genes in the respiratory epithelium in transgenic mice cautionary notes and toward building a better mouse trap. Am J Respir Cell Mol Biol. (2009) 40:1– 3. doi: 10.1165/rcmb.2008-0011ED 141. Moreau A, Vandamme C, Segovia M, Devaux M, Guilbaud M, Tilly G, et al. Generation and in vivo evaluation of IL10-treated dendritic cells in a nonhuman primate model of AAV-based gene transfer. Mol Ther Methods Clin Dev. (2014) 1:14028. doi: 10.1038/mtm.2014.28 142. Le Guiner C, Stieger K, Toromanoff A, Guilbaud M, Mendes-Madeira A, Devaux M, et al. Transgene regulation using the tetracycline-inducible TetRKRAB system after AAV-mediated gene transfer in rodents and nonhuman primates. PLoS ONE. (2014) 9:e102538. doi: 10.1371/journal.pone.01 02538 143. Favre D, Blouin V, Provost N, Spisek R, Porrot F, Bohl D, et al. Lack of an immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated Frontiers in Immunology | www.frontiersin.org 16 September 2020 | Volume 11 | Article 2044 Tristán-Manzano et al. Externally-Controlled Systems for Immunogene Therapy transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus. J Virol. (2002) 76:11605–11. doi: 10.1128/JVI.76.22.11605-11611.2002 144. Latta-Mahieu M, Rolland M, Caillet C, Wang M, Kennel P, Mahfouz I, et al. Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther. (2002) 13:1611– 20. doi: 10.1089/10430340260201707 145. Chenuaud P, Larcher T, Rabinowitz JE, Provost N, Joussemet B, Bujard H, et al. Optimal design of a single recombinant adeno-associated virus derived from serotypes 1 and 2 to achieve more tightly regulated transgene expression from nonhuman primate muscle. Mol Ther. (2004) 9:410– 8. doi: 10.1016/j.ymthe.2003.12.015 146. Zaldumbide A, Alkemade G, Carlotti F, Nikolic T, Abreu JRF, Engelse MA, et al. Genetically engineered human islets protected from CD8mediated autoimmune destruction in vivo.Mol Ther. (2013) 21:1592– 601. doi: 10.1038/mt.2013.105 147. Guilbaud M, Devaux M, Couzinié C, Le Duff J, Toromanoff A, Vandamme C, et al. Five years of successful inducible transgene expression following locoregional adeno-associated virus delivery in nonhuman primates with no detectable immunity. Hum Gene Ther. (2019) 30:802– 13. doi: 10.1089/hum.2018.234 Conflict of Interest: PJ-L was contractually linked to LentiStem Biotech, a Spin Off company that have the license for Lent-On-plus technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Copyright © 2020 Tristán-Manzano, Justicia-Lirio, Maldonado-Pérez, CortijoGutiérrez, Benabdellah and Martin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Frontiers in Immunology | www.frontiersin.org 17 September 2020 | Volume 11 | Article 2044