EMIM 2018 ControlCenter

Online Program Overview Session: ES-03

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CRISPR IMAGES ‐ Synthetic Biology meets Molecular Imaging

Session chair: Gil Westmeyer - Munich, Germany
 
Shortcut: ES-03
Date: Tuesday, 20 March, 2018, 2:30 PM
Room: Lecture Room 03 | level -1
Session type: Educational Session

REGISTRATION REQUIRED!

TOPICS COVERED:

1. Delivered vs. home-brewed - the advantages and challenges of genetically controlled contrast agents

2. What can reporter genes not report on? - from cell type to cell function

3. How would nature make a reporter protein? - directed evolution vs. ‘rational' design

4. How smart are contrast agents compared to cells? - from promoters and logic circuits

5. Outnumbered by consortia of smart bacteria? - Bacterial imaging from infectious diseases to cancer

6. Gene and/or cell therapy and the urgent need for molecular imaging

7. Seeing what you are doing - reading out and talking back with cellular precision

LEVEL: 4 (scale: 1-10)

TARGET GROUP: Researchers who are interested in learning more about how biology can be altered to ‘report' to our imaging instruments in order to guide tissue engineering, as well as future gene and cell therapy.

PREVIOUS KNOWLEDGE: Basics of molecular biology

Abstract

Click on an contribution preview the abstract content.

2:30 PM ES-03-1

Watching synthetic biology at work (#596)

G. G. Westmeyer1, 2

1 Technical University of Munich, Nuclear Medicine, Munich, Bavaria, Germany
2 Helmholtz Zentrum Munich, Institute for Biological and Medical Imaging, Neuherberg, Bavaria, Germany

Objectives

As indicated in the 'Topics Covered,' this educational talk will introduce fundamental principles and concepts of synthetic biology and the capabilities that arise from them for molecular imaging across different scales and organ systems - to complement the expert talks by Drs. Jerala and Witney on designer proteins and genetic circuits and translational gene reporter imaging, respectively.

We especially invite students and postdocs to join us in this educational session and the following interest group meeting so we can connect the next generation of multidisciplinary molecular imagers.

Content

Reporter genes such as GFP are indispensable for biological research, and targeted modifications of this protein have famously lead to variants with all colors of the rainbow.

As our understanding of biomolecules and fundamental cellular processes grows, the capabilities of synthetic biology expand far beyond reporter genes. Bottom-up biological engineering can construct designer DNA, RNA, proteins, protein assemblies, cellular compartments, …, to control cellular machinery and, e.g., reprogram input/output behavior of cells or install new cellular and tissue functions.

In a positive feedback loop, new biosynthetic sensors will further increase the capabilities of molecular imaging to quantify cellular parameters and reverse-engineer cell and tissue functions. Furthermore, molecular imaging will be vital to spatiotemporally control molecular interventions that can be exerted by bioengineered actuators such as optogenetic tools. 

Importantly, because of the enormous success of cellular therapies such as those based on CAR T-cells, there is a growing need to monitor - and in the future control - the function of genetically modified cells in the body of patients. These exciting biomedical advances call for the combined expertise and joint efforts of synthetic biologists and molecular imagers as we will outline in this educational session.

Relevant Publications

Kitada, T., DiAndreth, B., Teague, B., Weiss, R., 2018. Programming gene and engineered-cell therapies with synthetic biology. Science 359, eaad1067.

Huang, P.-S., Boyken, S.E., Baker, D., 2016. The coming of age of de novo protein design. Nature 537, 320–327. doi:10.1038/nature19946

Sigmund, F., Massner, C., Erdmann, P., Stelzl, A., Rolbieski, H., Fuchs, H., de Angelis, M.H., Desai, M., Bricault, S., Jasanoff, A., Ntziachristos, V., Plitzko, J., Westmeyer, G.G., 2017. Eukaryotically expressed encapsulins as orthogonal compartments for multiscale molecular imaging. bioRxiv. doi:10.1101/222083

Massner, C., Sigmund, F., Pettinger, S., Seeger, M., Hartmann, C., Ivleva N., Niessner, R., Fuchs H., Hrabé de Angelis, M., Stelzl, A., Koonakampully, N., Rolbieski, H., Wiedwald, U., Spasova, M., Wurst, W., Ntziachristos, V., Winklhofer, M., Westmeyer, G.G. Genetically controlled lysosomal entrapment of superparamagnetic ferritin for multimodal and multiscale imaging and actuation with low tissue attenuation. Advanced Functional Materials, in press

Westmeyer, G.G., Jasanoff, A., 2007. Genetically controlled MRI contrast mechanisms and their prospects in systems neuroscience research. Magnetic Resonance Imaging 25, 1004–1010. doi:10.1016/j.mri.2006.11.027

Acknowledgement

Funding by the ERC-SG ‘Magnetogenetics’, the DFG priority program SPP 1665, and the Bavarian Research Network for Molecular Biosystems is gratefully acknowledged.

Biological Engineering of cell therapies

Genetically modified therapeutic cell running a therapeutic program.

The figure is taken from: Kitada, T., DiAndreth, B., Teague, B. & Weiss, R. Programming gene and engineered-cell therapies with synthetic biology. Science 359, eaad1067 (2018). Rights for reuse were obtained from the publisher. 

Keywords: synthetic biology, protein engineering, biological engineering, designer proteins, de novo proteins, directed evolution, reporter genes, genetic circuits, molecular sensors, molecular actuators, optogenetics, magnetogenetics, bacterial imaging, whole-cell sensors, theranostics, T-cell Therapy, CAR T-cells, image guided therapies
3:30 PM ES-03-2

Synthetic biology design from new protein folds to cellular circuits (#590)

R. Jerala1

1 National institute of chemistry, Synthetic biology and immunology, Ljubljana, Slovenia

Objectives

Two areas of synthetic biology - design of transcriptional genetic circuits and designed proteins will be presented.

Content

Synthetic biology introduces engineering principles into biological or biomimetc systems, combining features of both approaches. Construction of devices for a complex cellular environment requires orthogonal building elements, which may be difficult to harvest from nature. Nucleotide sequence provides a large and easily accessible combinatorial diversity that can be used to program biological systems. Designable DNA-binding TALE domains can be used to construct an almost limitless number of artificial transcriptional regulators enabling construction of orthogonal genetic logic NOR gates. This allows construction of complex logic functions, which was demonstrated by all 16 two-input logic gates in mammalian cells. Construction of genetic bistable switches based on designable DNA binding modules required introduction of additional feedback loops and competition. An even more fundamental challenge for the synthetic biology is construction of new protein folds instead of modifying or combining the existing protein domains. We decided to use a modular engineering approach based on designed orthogonal coiled-coil building elements. This principle allows the design of completely new protein folds, composed of a single polypeptide chain of concatenated coiled-coil building elements called coiled-coil protein origami (CCPO). CCPO represents a new type of protein folds not found in the nature, where the structure is defined by the order of interacting segments defining the final topology rather than by compact hydrophobic core as natural proteins. Second generation CCPOs demonstrated that designed protein polyhedra can self-assemble bacterial or mammalian cells and may be applied to design intracellular scaffolds of molecular machines.

Relevant Publications

  1. Gradišar H, Božič S, Doles T, Vengust D, Hafner-Bratkovič I, Mertelj A, Webb B, Šali A, Klavžar S, Jerala R. (2013) Nat Chem Biol. 6:362-6.
  2. 2. Kočar V, Schreck JS, Čeru S, Gradišar H, Bašić N, Pisanski T, Doye JP, Jerala R. Nat Commun. (2016) 7:10803.
  3. Drobnak I, Gradišar H, Ljubetič A, Merljak E, Jerala, R. (2017) Modulation of Coiled-Coil Dimer Stability through Surface Residues while Preserving Pairing Specificity. J.Am.Chem.Soc.139: 8229-8236.
  4. Ljubetič, A., Lapenta, F., Gradišar, H., Drobnak, I., Aupič, J., Strmšek, Ž., Lainšček, D., Hafner-Bratkovič, I.,  Majerle, A., Krivec, N.,  Benčina, M., Pisanski, T., Ćirković Veličković, T., Round, A., Carazo, J.M., Melero, R. and Jerala, R., (2017) Design of in vivo self-assembling coiled-coil protein origami. Nat Biotech, 35:1094-1101.

Acknowledgement

Funded by the Slovenian Research Agency and ERANET Synthetic biology project Bioorigami. Contribution of the members of the Department of synthetic biology and immunology at NIC and slovenian iGEM teams to research results is acknowledged.

Example of designed antiinflammatory cellular device.
Design of coiled-coil protein origami cages.
Keywords: synthetic biology, designed bionanostructures, designed genetic circuits
4:30 PM ES-03-3

BREAK

5:00 PM ES-03-4

Reporter gene imaging of CAR-engineered T-cells in patients: a path for synthetic biology into clinical practice (#589)

T. H. Witney1

1 University College London's Centre for Advanced Biomedical Imaging, Division of Medicine, London, United Kingdom

Objectives

  • To understand the role of non-invasive imaging in the tracking of therapeutic cells in the body.
  • To be able to differentiate between direct and indirect cell labelling strategies, along with their advantages and disadvantages.
  • How HSV1-tk reporter gene expression can be used to track CAR-engineered T-cells in humans, along with its inherent limitations.
  • That quantification of reporter gene tracking is non-trivial.

Content

Immunotherapy holds great potential for the treatment and management of cancer patients with advanced disease (1). Through numerous divergent mechanisms, the body’s adaptive immune system can be primed to target malignancies normally recognized as ‘self’. While the success of recent Phase III trials have validated the principle that immunotherapy can sometimes extend cancer patient survival (2), tumour cells are known to escape immune surveillance and develop resistance to immunotherapy (3). Given the variable success of immunotherapy in the clinic, there is an urgent need to design non-invasive techniques that could give early indications of response to treatment and help predict patient outcome.

Through imaging it is possible to monitor the viability, biodistribution and trafficking of therapeutic cells to the site of the tumour. Two main approaches have been undertaken for cell tracking: direct and indirect labelling methods (4, 5). Direct labelling of cells involves incubation and retention of a contrast agent by the therapeutic cells, which are injected into the subject. Whilst relatively cheap and easy to perform, these methods are hampered by potential toxicity to the therapeutic cells (6). Moreover, the contrast agent becomes diluted upon cell division, and is lost from the cells upon cell death, making image analysis difficult to interpret. In order to circumvent these issues, indirect imaging reporter strategies have been developed to successfully track these cells. These methods enable imaging over the entire lifetime of the cell, with signal maintained following cell division, and provide information regarding cell viability (5). Here, I exemplify the power of this synthetic biology strategy to track HSV1-tk reporter gene expression present in CAR-engineered T-cells in humans using 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG) positron emission tomography (Figure 1) (7).

Relevant Publications

K.V. Keu*, T.H. Witney*, S. Yaghoubi*, J. Rosenberg, A. Kurien, R. Magnusson, J. Williams, F. Habte, J.R. Wagner, S. Forman, C. Brown, M. Allen-Auerbach, J. Czernin, W. Tang, M.C. Jensen, B. Badie, S.S. Gambhir (2017). Reporter Gene Imaging of Targeted T-Cell Immunotherapy in Recurrent Glioma. Sci Transl Med 9, eeag2196.

References

1.         M. Vanneman, G. Dranoff. Nat Rev Cancer 12, 237-251 (2012).

2.         F. S. Hodi et al. N Engl J Med 363, 711-723 (2010).

3.         S. Kelderman, T. N. Schumacher, J. B. Haanen. Mol Oncol 8, 1132-1139 (2014).

4.         M. F. Kircher, S. S. Gambhir, J. Grimm. Nat Rev Clin Oncol 8, 677-688 (2011).

5.         D. M. Kurtz, S. S. Gambhir. Adv Cancer Res 124, 257-296 (2014).

6.         C. Botti, D. R. et al. Eur J Nucl Med 24, 497-504 (1997).

7.         K.V. Keu et al. Sci Transl Med 9, eeag2196 (2017).

Acknowledgement

I wish to acknowledge salary and research support from The Wellcome Trust and The Royal Society and am grateful for the donation of slides by Dr Tammy Kalber.

Figure 1. Reporter Gene Imaging of Targeted T-Cell Immunotherapy in Recurrent Glioma

Strategy for imaging engineered cytotoxic T lymphocytes (left) and for monitoring response to this novel cellular immunotherapy (right) using a highly specific in vivo PET imaging agent, 18F-FHBG.