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Overview Intrexon’s RheoSwitch® Precise Gene Expression Technology RheoSwitch® technology is a novel transcriptional regulator platform based upon the highly specific interaction between small molecule ligand inducers and receptor proteins that control the timing and level of transgene expression in eukaryotic cells. In its simplest form, the switch is a three-‐part system, comprising a ligand, a receptor heterodimer, and an inducible promoter. When the ligand binds to the receptor, specific gene expression is activated in a controlled manner from the inducible promoter. RheoSwitch® gene regulation delivers precise rheostatic control and optimizes performance in multiple biological systems (human, animal, yeast, and plant) for a diverse range of applications, including biotherapeutics, gene therapy, and agriculture. The RheoSwitch® technology offers several advantages over traditional gene regulation tools. Key attributes include: •
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Dose-‐dependent response for adjustable target gene expression levels Precise dosing for gene therapy -‐ right place, right time, right level of expression Transcriptional control of a wide variety of therapeutic genes Regulatable system for producing difficult-‐to-‐express and/or cytotoxic proteins Low potential for pleiotropic or nonspecific effects from receptors or inducers Superior control with no irreversible loss or gain of gene function Library of pharmacologically safe activator ligands suitable for acute and chronic dosing RheoPlex® multigene regulation technology providing independent control over multiple genes per cell RheoSwitch® technology provides a key platform for Intrexon’s gene regulation systems. To enable inducible control over cellular function and optimized biological activity, the RheoSwitch® technology can be fully integrated into Intrexon’s UltraVector® platform, an operating system comprising advanced DNA construction technologies, cellular and protein engineering, computational models and statistical methods which facilitate the rapid design, testing and production of complex biological systems. Three Essential Elements Inducible expression systems need three basic components: an inducible promoter, a receptor protein to regulate the inducible promoter, and a ligand to activate the receptor protein. The RheoSwitch® system is a transcriptional regulator defined by these three core elements: 1) Switch Heterodimers: a Co-‐Activation Partner (CAP), which consists of a fusion between an engineered retinoid X receptor (RXR) and a transcription activator (VP16); and a Ligand-‐controllable Transcription Factor (LTF), which is a fusion protein comprising a GAL4 DNA binding domain fused to a portion of the ecdysone receptor (EcR), a member of the nuclear receptor family. The expression of the receptor proteins themselves can be rendered constitutive or inducible. 2) Activator Ligand: an ecdysone agonist (a non-‐steroid small molecule), which serves as the activator for the entire system. 3) Inducible Promoter: Customizable promoter which binds the LTF and to which basal transcription proteins are recruited to initiate gene expression. © 2015 Intrexon Corporation. All rights reserved. Intrexon’s RheoSwitch® technology enables expression of target genes only when the small molecule activator ligand combines with the switch components (CAP and LTF) and native cellular transcription factors, activating gene transcription from an inducible promoter, resulting in expression of desired proteins (Figure 1). The timing, location, and level of gene expression can be regulated in a dose dependent manner with RheoSwitch® technology and activator ligand. ®
Figure 1. RheoSwitch Components The LTF binds to the inducible promoter but does not form a stable interaction in the absence of ligand. In presence of the ligand, a conformational change in the LTF leads to a stable, high affinity interaction with the CAP at the inducible promoter. With this new conformation, ®
the RheoSwitch receptor proteins recruit transcription factors a nd drive expression of the target gene. Extensive Library of Chemically Diverse Inducer Ligands Inducibility of Intrexon’s RheoSwitch® system is dependent on the small molecule ligands which trigger the conformational changes required to activate gene transcription. Intrexon’s synthetic diacylhydrazine molecule veledimex (Figure 2) is the Company’s first clinically validated oral ligand, which has been shown to demonstrate a favorable safety profile with a wide therapeutic window and no without dose-‐limiting toxicities in vivo. Additionally, its short half-‐life (2.5-‐5 hours) offers a desirable pharmacokinetic profile, and enables tight control for turning off gene expression by removal of the ligand. Veledimex also has a broad biodistribution across multiple tissue types. Whether they are diacylhydrazine based inducer ligands, such as veledimex, or alternate chemotypes, Intrexon’s medicinal chemistry expertise enables engineering of the most appropriate chemistry to activate the switch in a physiologically relevant manner for varied applications. Intrexon has a large, chemically diverse library of inducer ligands that bind with high H
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specificity to RheoSwitch® receptors. These ligands include veledimex, other N
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N
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diacylhydrazine analogs, as well as proprietary novel chemotypes, which have a range of O
physical and EcR-‐related pharmacological properties. Potency expressed as EC50 extends Figure 2. Chemical as low as 100 pM for some activator ligand molecules, whereas solubility for others ranges Structure of Veledimex as high as 2 mg/mL while maintaining permeability. The structural diversity of RheoSwitch® inducers yields an array of ligand/receptor affinities. Extensive toxicology testing on the diacylhydrazine chemical family has demonstrated exceptional safety in mammals, since the chemical class is designed to activate non-‐mammalian EcR rather than mammalian targets. Studies have demonstrated that the physiochemical characteristics of the inducers can be modulated to optimize drug metabolism and pharmacokinetics for specific therapeutic indications and to address other biopharmaceutical and agricultural biotech applications. Intrexon has also demonstrated that corresponding alterations to the ligand binding portions of the receptor proteins can be made to optimize the ligand/receptor interactions.1 Therefore, the use of multiple types of RheoSwitch® ligands and variants of the receptor proteins provide further opportunities to tailor gene expression yielding desired biological effects. © 2015 Intrexon Corporation. All rights reserved. 2 RheoSwitch® Platform’s Broad Range -‐ From Basic Scientific Research to Clinical Applications 1. Dose-‐dependent Control of Expression Levels ®
The RheoSwitch® system responds rapidly when ligands are introduced to provide robust induction of protein synthesis and depletion of protein levels when withdrawn. Induction can be detected within 1 hour of ligand addition and most transcription activity stops within 24 hours after withdrawal. Importantly, ligand concentration modulates the protein expression levels and allows for repeated inducibility with on/off/on regulation. This is shown in Figure 4 with the addition and withdrawal of the RSL1 diacylhydrazine activator ligand. Luciferase AcIvity (RLU) 9.E+04 5.E+04 8.E+04 4.E+04 7.E+04 4.E+04 6.E+04 3.E+04 5.E+04 3.E+04 4.E+04 2.E+04 3.E+04 2.E+04 2.E+04 1.E+04 1.E+04 5.E+03 0.E+00 0.E+00 0 800 pM M 20 nM 100 nM 5 500 nM 0 0 .8nM 4 4n
nM 20nM 1 00nM 00nM Veledimex Dose ®
Figure 4. RheoSwitch Ligand Induced Expression Luciferase RLU (mg cellular protein) 2. Repeated Inducibility in Cultured Cells Figure 3. RheoSwitch Dose-‐Dependent Response Time Addition of activator ligand r control solvent + a +
ddition of activator ligand ( ) o r c oontrol solvent ( ) -‐ w
of activator ligand ( ) o r ontrol solvent ( -‐ithdrawal Withdrawal of activator ligand ocr control solvent ) 3. RheoSwitch® Deployment Mediates Inducible Expression of Proteins Research demonstrated that in neuronal cell lines, the RheoSwitch® system can mediate conditional expression of mutant huntingtin protein in the development of an in vitro Huntington’s disease model.3 The ability to control expression enabled evaluation of the mutant protein’s effect on cellular function and provides a mechanism by which to investigate the time-‐ and concentration-‐dependent effects of the mutant protein on cell behavior. Through RheoSwitch® application, the engineered cell lines represent a cellular model of Huntington’s disease with broad utility as a high-‐throughput screening tool for potential use in therapeutic drug discovery and for evaluating the disease biology. 4. Controlled Expression of Bioactive RNAs Intrexon’s RheoSwitch® platform, is not only a valuable tool to modulate protein expression, but can also be utilized for the expression of bioactive RNAs. Studies have demonstrated that primary microRNA expression can be regulated in a dose-‐dependent manner and yield functionally active miRNAs.4 The rheostatic nature of the switch allows for successive on/off cycles of ligand dosing/removal to fine-‐tune the expression of miRNAs for potential use in research applications and therapeutic development. 5. Plant Specific RheoSwitch® Application Intrexon carries a portfolio of RheoSwitch® receptor protein variants and promoters to control receptor and target gene(s) that can be combined to facilitate targeted expression in plant systems. RheoSwitch® driven gene expression is active in multiple plant tissues, including flowers and leaves, and can be utilized to © 2015 Intrexon Corporation. All rights reserved. 3 Fold InducIon The RheoSwitch® system allows for a tight and potent dose-‐
dependent response to the ligand, enabling titration of recombinant protein production by adjusting ligand concentration.2 For example, response of the RheoSwitch® system was demonstrated using a luciferase reporter gene (Figure 3). As the concentration of the ligand veledimex is increased, the amount of luciferase produced increased. The RheoSwitch® system has the ability to fine-‐tune expression through the dosing levels of the ligand, an important consideration in gene-‐based therapies. optimize biological solutions for agriculture and environmental applications. As shown in Figure 5, RheoSwitch® designs were introduced into Camelina sativa leaves by Agrobacterium infiltration. The activator ligand was applied by root drench, leading to controlled gene expression as visualized in the leaves. ®
Figure 5. RheoSwitch Mediated Expression in Camelina Sativa Leaves Constitutive Promoter
Uninduced
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RheoSwitch Promoter
Induced
High-‐level of expression
6. Cell Type and State Specific Spatial Regulation The RheoSwitch® system can be tailored to provide desired spatial and temporal expression properties for genes of interest. For some applications, it may be important to restrict the activation of the switch to specific cell or tissue types (e.g., liver or brain tissue) and/or physiological or disease-‐related states (e.g., hypoxia). Intrexon has deployed RheoSwitch® system with conditional promoters that drive expression of the RheoSwitch® receptor proteins, limiting expression to defined cellular environments and biological conditions (Figure 6). ®
Figure 6. RheoSwitch Cell Type Specific Conditional Regulation 7. Therapeutic Indications RheoSwitch® technology has been applied toward the development of oncology therapeutics. Intratumoral administration of adenoviral constructs containing immunomodulatory cytokines interleukin 12 (IL-‐12) and interferon alpha (IFN-‐α) under RheoSwitch® control exhibited both in vitro and in vivo regulated production of IL-‐12 and IFN-‐α in murine lung and breast cancer models and were shown to mediate enhanced anti-‐
tumor activity.5 © 2015 Intrexon Corporation. All rights reserved. 4 Additionally, RheoSwitch® controlled in vivo expression is delivery modality agnostic and can be used for viral DNA-‐, plasmid DNA-‐, or cell-‐based therapies. Intrexon has demonstrated that intramuscular electroporation of an optimized RheoSwitch® regulated IFN-‐α plasmid transgene showed long term persistence in vivo and supports that the switch can be utilized with different gene delivery methods.6 Further, the ability to express multiple therapeutic effectors under RheoSwitch® control, has potential for multi-‐faceted treatment regimens for cancer and other complex diseases. Using the immunostimulatory and immunomodulatory proteins IL-‐12, IFN-‐α, and a CTLA-‐4 decoy as exemplars, Intrexon has examined the pharmacodynamics and functionality of RheoSwitch® regulated expression following intramuscular electroporation in mice (Figure 7).7 Experimental data indicates that RheoSwitch® technology can be used to conditionally deliver distinct immune effectors from a single multigenic construct following veledimex ligand administration. RheoSwitch® technology also holds the potential to provide optimized expression levels and ideal biological activity for targeted, tissue-‐specific, inducible in vivo protein expression. Studies have demonstrated that RheoSwitch® inducibility may have utility in treatment for intervertebral disc degeneration.8 Adeno-‐
associated viral vector transduced intervertebral disc cells showed dose-‐ and time-‐dependent reporter protein expression with activator ligand administration. When applied to intervertebral discs in vivo in rabbits, no toxicity or non-‐specific expression in adjacent tissues was observed. These results highlight the potential of the RheoSwitch® platform for therapeutic use by offering tightly controlled, localized modulation of protein expression, independent of titrating viral doses. Uninduced Induced Uninduced Induced Uninduced Induced Figure 7. Concomitant In Vivo Expression of hIL-‐12, hIFNα, and CTLA-‐4 DCY Following Intramuscular Electroporation Plasmids were administered via a single IM injection and electroporation. T hree days post IM/EP and daily oral treatment with veledimex, animals were bled and sera were evaluated by ELISA for the single or concomitant expression of the encoded GOIs. Expression of hIL-‐
12, hIFNα, and the CTLA-‐4 DCY was detected only in the serum of mice that r eceived the single or multi-‐
effector plasmids in combination with oral veledimex. Differences in expression between the single and multieffector plasmids could be due to variations in plasmid size, and consequently, transgene copy 7
number. Mean ± SEM shown. Clinically Evaluated Gene Switch -‐ Expression Control for Therapeutic Safety In order to meet standards of safety and specificity for clinical applications, gene therapy systems should be highly targeted with dose-‐dependent inducible regulation and favorable drug metabolism for the inducer compound. To date, the RheoSwitch® platform has been shown to function as a regulatable switch in an array of cell types, as well as in vivo, for multiple proteins and bioactive RNAs. An in-‐depth independent study on the RheoSwitch® system showed the receptors and ligand have no adverse effects on cell function.2 Microarray analysis demonstrated that the RheoSwitch® receptor proteins and ligand have minimal effects on endogenous gene expression and normal cellular processes. The RheoSwitch® system responds rapidly and in a dose-‐
dependent manner when the ligand is introduced or withdrawn. © 2015 Intrexon Corporation. All rights reserved. 5 Intrexon's proprietary RheoSwitch® system is uniquely positioned as the first clinically-‐evaluated gene switch with in vivo data showing the ability to control gene expression with a broad dynamic range. Human therapeutic product candidates using RheoSwitch® technology (referred to as RheoSwitch Therapeutic System® or RTS®) are currently in phase I/II clinical trials for melanoma and breast cancer. Ad-‐RTS-‐IL-‐12 is a DNA-‐based therapeutic delivered to tumor sites via an adenoviral vector with RTS® enabled finely controlled expression of IL-‐12 by dosing with the ligand. RTS® technology maintains the gene program in an inactive state within transduced cells until the patient takes a capsule containing orally available veledimex; which has been shown to be well tolerated and to induce clinical activity in subjects.9 Application of RheoSwitch® technology allows Intrexon and its partners the ability to control the timing and expression levels of intracellular and secreted therapeutic effectors by regulating the timing and dosing of ligand. Use of Intrexon’s switch with orally available activator ligand avoids toxicities caused by bolus dosing, as well as the difficulties associated with intravenous administration. The RheoSwitch® platform further provides a mechanism for titrating therapeutic effects on a patient-‐specific and predictable basis, as well as a safety switch to rapidly turn off gene-‐expression. Importantly, the ability to administer or withdraw veledimex to sustain continued treatment cycles is a key benefit of such a switch technology. RheoPlex® Multi-‐gene Regulation The ability to modify both inducers and receptors simultaneously gives Intrexon the capability to create proprietary RheoPlex® systems, which may advance therapeutic technologies by enabling the independent regulation of multiple genes. RheoPlex® systems are unique, different inducer/receptor pairs that independently regulate spatial and temporal gene expression of multiple genes in the same cell, or in different cell types in the same organism. For example, differential expression of β-‐galactosidase and luciferase genes was achieved by placing β-‐galactosidase under the control of a ligand responsive switch (Receptor 1) and luciferase under the control of a second (Receptor 2), independent switch in the same cell (Figure 8). The ligand/receptor pairs induce strong transcription with little to no off-‐target activation. With RheoPlex® systems it is possible to have inducer concentration selected wherein only one gene or both genes can be activated by inducer treatment. Other orthogonal or non-‐cross reactive switches are currently in development. ®
Figure 8. RheoPlex Independent Regulation of Multiple Genes Orthogonality Cells were transiently simultaneously transfected with two plasmids for each β-‐galactosidase and lucerifase under RheoSwitch® control, ligands added individually, and cells assayed for F-‐luc and B-‐GLO activity © 2015 Intrexon Corporation. All rights reserved. RheoPlex® Demonstration in a Single Cell .0025 .025 .25 2.5 25 250 2500 Ligand Concentration (nM) 6 RheoSwitch® Expression in Targeted Genomic Regions via AttSite® recombinases In addition to regulating of the level and timing of gene expression, the RheoSwitch® system can be used in conjunction with Intrexon’s proprietary AttSite® recombinases to direct gene expression to targeted genomic regions. AttSite® recombinases break and rejoin DNA at specific sequences to mediate integration of a transgene into a host cell genome, as well as to control gene excision. In carrying out unidirectional, irreversible recombination, AttSite® technology provides stable and efficient gene exchange in a range of hosts, including human cells, rodent stem cells, and plant cells. The RheoSwitch® system can be used to modulate the timing and localization of recombinase expression in cells and tissues. Conversely, AttSite® recombinases may also be utilized to deliver gene programs under RheoSwitch® control to defined sites within target cells, permitting specific gene transfers in a reliable and repeatable fashion. This reduces the time and costs associated with developing engineered cell lines for bioproduction or therapeutic applications. Targeted genomic modification with the AttSite® platform may increase the speed and reliability of generating transgenic cells and organisms that meet specific criteria, as well as improve the safety profile of gene therapies. Summary Intrexon’s exclusive biologic RheoSwitch® technology can be used to precisely regulate the timing and concentration of gene expression with on/off/on kinetics, and allows for tailored production of proteins and bioactive RNAs both in vitro and in vivo. Intrexon’s large, chemically diverse library of inducer ligands, including veledimex, other diacylhydrazine analogs and proprietary novel chemotypes, bind with high specificity to RheoSwitch® receptors thereby yielding a wide range of dynamic systems for gene control. RheoSwitch® driven inducible expression can provide physiological levels of targeted gene expression spanning a broad range of utilities, from scientific basic research to clinical applications, and has the potential to enable development of safe, next generation biotherapeutics, as well as agriculture and environmental solutions. References 1.
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Lapenna et al. (2009) Semi-‐synthetic ecdysteroids as gene-‐switch actuators: synthesis, structure-‐activity relationships, and prospective ADME properties. ChemMedChem 4: 55-‐68. PMID: 19065574. Lessard et al. (2007) Characterization of the RSL1-‐dependent conditional expression system in LNCaP prostate cancer cells and development of a single vector format. Prostate 67: 808-‐819. PMID: 17373718. Weiss et al. (2009) Inducible mutant huntingtin expression in HN10 cells reproduces Huntington's disease-‐like neuronal dysfunction. Molecular Neurodegeneration 4: 11. PMID: 19203385. Shea et al. (2010) Controlled expression of functional miR-‐122 with a ligand inducible expression system. BMC Biotechnology 10: 76. PMID: 20961424. Murugesan et al. (2012) Combined direct intratumoral adenoviral delivery and production of RheoSwitch®-‐regulated mIL-‐12 and mIFNα enhances antitumor activity in lung and breast cancer models. American Association for Cancer Research Annual Meeting. Agarwal et al. (2012) Intramuscular electroporation of an optimized RheoSwitch® regulated interferonα plasmid transgene shows long term persistence in vivo: implications for therapy of cancer. AACR-‐NCI-‐EORTC International Conference on Molecular Targets and Cancer Therapeutics. ®
Agarwal et al. (2013) Pharmacodynamics and functionality of RheoSwitch regulated immunomodulatory proteins, expressed from a multigenic embedded cellular bioreactor following intramuscular electroporation in mice. AACR-‐NCI-‐EORTC International Conference on Molecular Targets and Cancer Therapeutics. Sowa et al. (2011) In vitro and in vivo testing of a novel regulatory system for gene therapy for intervertebral disc degeneration. Spine 36: E623-‐628. PMID: 21224765. Linette et al. (2013) A Phase I open-‐label study of Ad-‐RTS-‐hIL-‐12, an adenoviral vector engineered to express hIL-‐12, in combination with an oral activator ligand in subjects with unresectable stage III/IV melanoma. American Society of Clinical Oncology Annual Meeting. © 2015 Intrexon Corporation. All rights reserved. 7