Patrick Guye

Rapid neurogenesis through transcriptional activation in human stem cells

Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human‐induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems‐level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types.

Rapid, modular and reliable construction of complex mammalian gene circuits

We developed a framework for quick and reliable construction of complex gene circuits for genetically engineering mammalian cells. Our hierarchical framework is based on a novel nucleotide addressing system for defining the position of each part in an overall circuit. With this framework, we demonstrate construction of synthetic gene circuits of up to 64 kb in size comprising 11 transcription units and 33 basic parts. We show robust gene expression control of multiple transcription units by small molecule inducers in human cells with transient transfection and stable chromosomal integration of these circuits. This framework enables development of complex gene circuits for engineering mammalian cells with unprecedented speed, reliability and scalability and should have broad applicability in a variety of areas including mammalian cell fermentation, cell fate reprogramming and cell-based assays.

Genetically engineering self-organization of human pluripotent stem cells into a liver bud-like tissue using Gata6.

Human induced pluripotent stem cells (hiPSCs) have potential for personalized and regenerative medicine. While most of the methods using these cells have focused on deriving homogenous populations of specialized cells, there has been modest success in producing hiPSC-derived organotypic tissues or organoids. Here we present a novel approach for generating and then co-differentiating hiPSC-derived progenitors. With a genetically engineered pulse of GATA-binding protein 6 (GATA6) expression, we initiate rapid emergence of all three germ layers as a complex function of GATA6 expression levels and tissue context. Within 2 weeks we obtain a complex tissue that recapitulates early developmental processes and exhibits a liver bud-like phenotype, including haematopoietic and stromal cells as well as a neuronal niche. Collectively, our approach demonstrates derivation of complex tissues from hiPSCs using a single autologous hiPSCs as source and generates a range of stromal cells that co-develop with parenchymal cells to form tissues.

A platform for rapid prototyping of synthetic gene networks in mammalian cells

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 genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present 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. We demonstrate the potential of this framework 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. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This 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.

Design and Connection of Robust Genetic Circuits

Phenotypic robustness is a highly sought after goal for synthetic biology. There are many well-studied examples of robust systems in biology, and for the advancement of synthetic biology, particularly in performance-critical applications, fundamental understanding of how robustness is both achieved and maintained is very important. A synthetic circuit may fail to behave as expected for a multitude of reasons, and since many of these failures are difficult to predict a priori, a better understanding of a circuits behavior as well as its possible failures are needed. In this chapter, we outline work that has been done in developing design principles for robust synthetic circuits, as well as sharing our experiences designing and constructing gene circuits.

Customized signaling with reconfigurable protein scaffolds

VOLUME 26 NUMBER 5 MAY 2008 NATURE BIOTECHNOLOGY dengue viruses. These candidate vaccines seem promising, but studies are still underway to determine whether they can confer a balanced immune response against all four dengue viruses and avoid immunological interference. For West Nile virus, the lead candidate vaccines—a DNA vaccine and a chimeric yellow fever 17D virus—also appear promising, but there is still no consensus on what constitutes a long-term protective immune response. Chang et al.5 build on earlier work by Kofler et al.9,10, who showed that the tick-borne encephalitis flavivirus still forms immunogenic virus particles even when much of the capsid gene sequence has been deleted. Using Kunjin, a subtype of West Nile virus found in Australia, Chang et al.5 have developed a ‘split-genome’ vaccine that generates two RNA species, one encoding the entire Kunjin virus genome except the capsid gene and the other encoding only the capsid gene. As both RNAs are encoded on the same DNA plasmid under the control of two cytomegalovirus promoters configured in a back-toback orientation, transfected cells transcribe and translate all the viral genes. The capsid protein acts as a helper to assemble virus particles containing the viral genomic RNA lacking the capsid gene. These so-called single-round infectious particles (SRIPs) then infect adjacent cells (Fig. 1), in contrast to DNA vaccines that produce viral antigens only in the cells initially infected. Because the viral genome transmitted to neighboring cells does not encode capsid protein, no further viral replication can occur. Chang et al.5 compare the immunogenicity of SRIPs in mice to a live virus, a traditional DNA vaccine (encoding the viral genome, with the exception of functional capsid) and a DNA vaccine that produces virus-like particles composed of the pre-membrane and envelope proteins. SRIPs confer a superior antibodymediated immune response in mice and horses, as well as protective immunity in mice, at lower doses of DNA compared with the traditional DNA vaccine. CD8+ T-cell responses elicited by SRIPs in mice were also significantly greater than those produced by the virus-like particle vaccine, although smaller than those following immunization with live virus. Neutralizing antibodies are considered critical for achieving protective immunity, but it is clear that a vaccine must elicit both antibodyand cell-mediated immunity to ensure long-term protection. Although these results represent an important proof of concept of a technology that should in theory be applicable to any flavivirus, a couple of important points should be considered. First, comparison of DNA-based vaccine strategies is very difficult given the many variables involved (e.g., viral strain, viral gene(s) selected, different parental virus strains and codon optimization). Second, it remains to be seen whether the present findings translate to primates. Several candidate DNA vaccines have performed impressively in lower animals only to disappoint in clinical trials. Prospects for using a SRIP-based approach in veterinary vaccines, such as those against Japanese encephalitis and West Nile virus infections of horses, seem more promising in the short term, especially as killed vaccines do not induce long-term protective immunity and booster doses are required to maintain immunity. The major issues surrounding new candidate vaccines always concern efficacy and safety. With regard to efficacy, we know that current licensed flavivirus vaccines have neutralizing antibody as the correlate of protection and that only low levels of neutralizing antibodies are required for protective immunity. We do not know whether this will be true for vaccines against dengue and West Nile viruses—and even if it is, as most investigators believe, it is unclear what level of neutralizing antibodies will be required. This question is particularly complicated for a dengue vaccine, as the disease is caused by four genetically and serologically related viruses. For a tetravalent vaccine, higher levels of neutralizing antibodies might be needed to control four viruses simultaneously. Candidate vaccines, such as those involving SRIPs, may help achieve this goal, possibly through a prime-boost regimen, although it remains to be shown that SRIP-based vaccines are effective over the long term. In the 21st century, safety has become the paramount attribute of a vaccine, even more so then efficacy, as society will not accept any adverse events associated with a vaccine. In this study, Chang et al.5 have boosted efficacy using viral particles that have clear safety advantages over live attenuated vaccines.