Therapeutic cell engineering: designing programmable synthetic genetic circuits in mammalian cells

Cell therapy approaches that employ engineered mammalian cells for on-demand production of therapeutic agents in the patient’s body are moving beyond proof-of-concept in translational medicine. The therapeutic cells can be customized to sense user-defined signals, process them, and respond in a programmable and predictable way. In this paper, we introduce the available tools and strategies employed to design therapeutic cells. Then, various approaches to control cell behaviors, including open-loop and closed-loop systems, are discussed. We also highlight therapeutic applications of engineered cells for early diagnosis and treatment of various diseases in the clinic and in experimental disease models. Finally, we consider emerging technologies such as digital devices and their potential for incorporation into future cell-based therapies.     

Synthetic RNA circuits

Natural and engineered RNA 'parts' can perform a variety of functions, including hybridizing to targets, binding ligands and undergoing programmed conformational changes, and catalyzing reactions. These RNA parts can in turn be assembled into synthetic genetic circuits that regulate gene expression by acting either in cis or in trans on mRNAs. As more parts are discovered and engineered, it should be increasingly possible to create synthetic RNA circuits that are able to carry out complex logical operations in cells, either superimposed on or autonomous to extant gene regulation.     

Synthetic in vitro circuits

Inspired by advances in the ability to construct programmable circuits in living organisms, in vitro circuits are emerging as a viable platform for designing, understanding, and exploiting dynamic biochemical circuitry. In vitro systems allow researchers to directly access and manipulate biomolecular parts without the unwieldy complexity and intertwined dependencies that often exist in vivo. Experimental and computational foundations in DNA, DNA/RNA, and DNA/RNA/protein based circuitry have given rise to systems with more than 100 programmed molecular constituents. Functionally, they have diverse capabilities including: complex mathematical calculations, associative memory tasks, and sensing of small molecules. Progress in this field is showing that cell-free synthetic biology is a versatile testing ground for understanding native biological circuits and engineering novel functionality.     

Programmable genetic circuits for pathway engineering

1. Download : Download high-res image (109KB)
2. Download : Download full-size image

     

Synthetic mammalian signaling circuits for robust cell population control

1. Download : Download high-res image (184KB)
2. Download : Download full-size image

     

Synthetic circuits,devices and modules

The aim of synthetic biology is to design artificial biological systems for novel applications. From an engineering perspective, construction of biological systems of defined functionality in a hierarchical way is fundamental to this emerging field. Here, we highlight some current advances on design of several basic building blocks in synthetic biology including the artificial gene control elements, synthetic circuits and their assemblies into devices and modules. Such engineered basic building blocks largely expand the synthetic toolbox and contribute to our understanding of the underlying design principles of living cells.     

Synthetic analog and digital circuits for cellular computation and memory

1. Download : Download high-res image (49KB)
2. Download : Download full-size image

     

Synthetic therapeutic gene circuits in mammalian cells

In the emerging field of synthetic biology, scientists are focusing on designing and creating functional devices, systems, and organisms with novel functions by engineering and assembling standardised biological building blocks. The progress of synthetic biology has significantly advanced the design of functional gene networks that can reprogram metabolic activities in mammalian cells and provide new therapeutic opportunities for future gene- and cell-based therapies. In this review, we describe the most recent advances in synthetic mammalian gene networks designed for biomedical applications, including how these synthetic therapeutic gene circuits can be assembled to control signalling networks and applied to treat metabolic disorders, cancer, and immune diseases. We conclude by discussing the various challenges and future prospects of using synthetic mammalian gene networks for disease therapy.     

Neuronal circuits on a chip for biological network monitoring

Cultured neuronal networks (CNNs) are a robust model to closely investigate neuronal circuits’ formation and monitor their structural properties evolution. Typically, neurons are cultured in plastic plates or, more recently, in microfluidic platforms with potentially a wide variety of neuroscience applications. As a biological protocol, cell culture integration with a microfluidic system provides benefits such as accurate control of cell seeding area, culture medium renewal, or lower exposure to contamination. The objective of this report is to present a novel neuronal network on a chip device, including a chamber, fabricated from PDMS, vinyl and glass connected to a microfluidic platform to perfuse the continuous flow of culture medium. Network growth is compared in chips and traditional Petri dishes to validate the microfluidic chip performance. The network assessment is performed by computing relevant topological measures like the number of connected neurons, the clustering coefficient, and the shortest path between any pair of neurons throughout the culture's life. The results demonstrate that neuronal circuits on a chip have a more stable network structure and lifespan than developing in conventional settings, and therefore this setup is an advantageous alternative to current culture methods. This technology could lead to challenging applications such as batch drug testing of in vitro cell culture models. From the engineering perspective, a device's advantage is the chance to develop custom designs more efficiently than other microfluidic systems.     

Synthetic nonlinear computation for genetic circuit design

1. Download : Download high-res image (99KB)
2. Download : Download full-size image

     

Dynamical roles of biological regulatory circuits

Regulatory circuits are found at the basis of all non-trivial dynamical properties of biological networks. More specifically, positive circuits are involved in the generation of multiple differentiated states, whereas negative circuits can generate cyclic or homeostatic behaviours. These notions are briefly reviewed, from initial biological formulations to mathematical formalisations, further encompassing their application to the design of synthetic regulatory systems. Finally, current challenges for the analysis of increasingly complex regulatory networks are indicated, as well as prospects for our understanding of development and evolution.     

Engineering and applications of genetic circuits

In the field of synthetic biology, recent genetic engineering efforts have enabled the construction of novel genetic circuits with diverse functionalities and unique activation mechanisms. Because of these advances, artificial genetic networks are becoming increasingly complex, and are demonstrating more robust behaviors with reduced crosstalk between defined modules. These properties have allowed for the identification of a growing set of design principles that govern genetic networks, and led to an increased number of applications for genetic circuits in the fields of metabolic engineering and biomedical engineering. Such progress indicates that synthetic biology is rapidly evolving into an integrated engineering practice that uses rational and combinatorial design of synthetic gene networks to solve complex problems in biology, medicine, and human health.     

Toward scalable parts families for predictable design of biological circuits

Our current ability to engineer biological circuits is hindered by design cycles that are costly in terms of time and money, with constructs failing to operate as desired, or evolving away from the desired function once deployed. Synthetic biologists seek to understand biological design principles and use them to create technologies that increase the efficiency of the genetic engineering design cycle. Central to the approach is the creation of biological parts--encapsulated functions that can be composited together to create new pathways with predictable behaviors. We define five desirable characteristics of biological parts--independence, reliability, tunability, orthogonality and composability, and review studies of small natural and synthetic biological circuits that provide insights into each of these characteristics. We propose that the creation of appropriate sets of families of parts with these properties is a prerequisite for efficient, predictable engineering of new function in cells and will enable a large increase in the sophistication of genetic engineering applications.