Developing new tools and platforms for mammalian synthetic biology: From the assembly and chromosomal integration of complex DNA circuits to the engineering of artificial intercellular communication systems

Abstract : Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to program cells is currently hampered both by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks, and also by the complexity of mammalian systems and our poor understanding of cellular processes context-dependencies. To address these problems, I proposed and validated a number of concepts and approaches during my PhD. First, I created a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. The potential of this framework was demonstrated by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Such a rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. Second, I developed a platform to identify and characterize new serine recombinase systems from Mycobacteriophage genomes in order to extend the toolbox of genome engineering tools available for mammalian cells programing. I validated the approach by identifying 26 new large serine recombinases from 400 Mycobacteriophage genomes, from which 4 were using new recombination sites. These recombinases could mediate site-specific recombination events in both E. coli and mammalian cells. Additionally, I demonstrated that a library of 6 orthogonal recombination site pairs could be engineered for each of these recombinases. To overcome the apparent limitations in our single-cell rational engineering capacity, I also engineered two new artificial intercellular communication systems for mammalian cells, in order to facilitate the spatial decoupling of different modules of a synthetic circuit. The first one consists in synthetic sender/receiver modules that can be either integrated within the same cell population to create an autocrine-like system or integrated into two distinct populations of cell to create a paracrine-like system. To create the sender module, I assembled and stable integrated a synthetic metabolic pathway using different plants enzymes to produce a small diffusible molecule: phloretin. This small molecule, orthogonal to endogenous signaling pathways, can be sensed by the receiver module I engineered, which relies on a de-novo synthetic inducible gene expression system combining bacterial and mammalian genetic parts. Based on previous development of Virus Like Particles (VLPs), I created a second intercellular communication system which enables the transfer of proteins from a sender cell population to a separate receiver cell population. I demonstrated that I could induce the budding of particles carrying recombinases (Cre and B3) from senders cell that could be delivered to the receiver cells and perform a targeted genomic rearrangement to activate transgene expression. Even though we are still years away from therapies using engineered cells carrying synthetic circuits to repair damaged or non-functional organs or to create de-novo tissues, I believe the contributions developed during the course of my PhD could potentially be used to push mammalian synthetic biology forward, whether it is by helping fasten the development of therapeutically relevant synthetic circuits or by providing new means to better understand the underlying mechanisms of cellular processes.
Keywords : Synthetic Biology
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Xavier Duportet. Developing new tools and platforms for mammalian synthetic biology: From the assembly and chromosomal integration of complex DNA circuits to the engineering of artificial intercellular communication systems. Biotechnology. Université Paris Diderot (Paris 7), 2014. English. ⟨tel-01108520⟩

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