Smart medication through combination of synthetic biology and cell microencapsulation

Recent advances in the field of synthetic biology have led to the design of a new generation of complex, man-made biological networks that operate inside living cells in a desired manner. Key elements of these systems are often controllable genetic switches that are capable of processing therapeutic signals by sensing and responding to the environment. For biomedical applications, however, it is necessary to seal these engineered cells in order to protect them from the host immune system and enable straightforward removal after completion of the therapy. A promising and successful approach is the microencapsulation of defined cells into a semi-permeable and biocompatible microcapsule. Shielding from the external environment still allows exchange to occur on a molecular basis. Thus, the powerful combination of synthetic biology and microencapsulation has been opening the door to novel and innovative cell-based biomedical applications, such as smart implantable drug delivery systems. This review highlights recent developments in the overlap of these two areas, thereby presenting promising developments and perspectives for future treatment strategies.     

Engineered riboswitches: Expanding researchers' toolbox with synthetic RNA regulators

Riboswitches are natural RNA-based genetic switches that sense small-molecule metabolites and regulate in response the expression of the corresponding metabolic genes. Within the last years, several engineered riboswitches have been developed that act on various stages of gene expression. These switches can be engineered to respond to any ligand of choice and are therefore of great interest for synthetic biology. In this review, we present an overview of engineered riboswitches and discuss their application in conditional gene expression systems. We will provide structural and mechanistic insights and point out problems and recent trends in the development of engineered riboswitches.     

Synthetic biology in multicellular organisms: Opportunities in nematodes

Synthetic biology has mainly focused on introducing new or altered functionality in single cell systems: primarily bacteria, yeast, or mammalian cells. Here, we describe the extension of synthetic biology to nematodes, in particular the well-studied model organism Caenorhabditis elegans, as a convenient platform for developing applications in a multicellular setting. We review transgenesis techniques for nematodes, as well as the application of synthetic biology principles to construct nematode gene switches and genetic devices to control motility. Finally, we discuss potential applications of engineered nematodes.     

Cancer gene-therapy: clinical trials

The objective of gene therapy for the treatment of cancer is to kill tumour cells but preserve normal tissue; therefore, the ideal gene therapy agent would be targeted for specific transduction of tumour cells and have specificity in its cytotoxic action. A variety of strategies to achieve these aims have demonstrated promising results in the laboratory, including enzyme-pro-drug activating systems, correction of genetic mutations contributing to the malignant phenotype and stimulation of a T-cell-mediated anti-tumour immune response. The key to the success of all these strategies is an effective vector that can direct appropriate expression of the therapeutic gene. Viruses have many properties that can be adapted to achieve this therapeutic endpoint; furthermore, they can be engineered to replicate selectively in cancer cells and lyse them. The challenge now is to translate these features into effective therapies that can supplement or supplant existing treatments.     

Prospects for stem cell-based therapy

Resident pools of somatic stem cells in many organs are responsible for tissue maintenance and repair. The goal of regenerative medicine is to exploit these cells either by transplanting them from an exogenous source or by activating endogenous stem cells pharmacologically. For diseases caused by mutations in a single gene, the therapeutic goal is tissue replacement using stem cells engineered to correct the genetic defect. However, a number of technical hurdles must be overcome before therapies based on pluripotent human stem cells can enter the clinic.     

Receptor-based assays.

Receptor-based assays have benefitted from the newest advances in biotechnology and electronics in three main ways: genetically engineered cells expressing single receptor subtypes have been developed for many natural and synthetic ligands; assays have been designed which take advantage of a variety of signals triggered in cells by binding, or inhibition of binding, of ligands to surface-bound receptors; and radiolabelled ligand assays have been considerably improved and simplified by novel electronic devices.     

工程生物活药在肿瘤免疫治疗中的应用

人体细胞、细菌、病毒等生命体可以改造为工程生物活药,可在患者体内维持生物活性、自我复制并表达基因。相比于传统药物,工程生物活药在体内维持疗效时间长,具备外源基因表达能力,可实现多功能性和稳态调控,且具有独特的靶向、响应等能力。近年来,工程生物活药在肿瘤免疫治疗中的应用受到广泛关注,CAR-T等细胞治疗、溶瘤病毒疗法已在临床中获得良好的疗效,工程菌也在临床和临床前研究中发展迅猛。细胞、细菌、病毒三类活药的特性和治疗机制不同,因此具有不同的设计目的与思路。随着合成生物学技术的发展,工程生物活药将更安全、更高效,也将为肿瘤治疗带来新的机遇。针对工程生物活药在肿瘤免疫治疗中应用的最新进展开展了综述,阐述了不同生物活药的合成生物学设计和免疫治疗机制。     

Gene therapy for diabetes mellitus

There are diverse strategies for gene therapy of diabetes mellitus. Prevention of beta-cell autoimmunity is a specific gene therapy for prevention of type 1 (insulin-dependent) diabetes in a preclinical stage, whereas improvement in insulin sensitivity of peripheral tissues is a specific gene therapy for type 2 (non-insulin-dependent) diabetes. Suppression of beta-cell apoptosis, recovery from insulin deficiency, and relief of diabetic complications are common therapeutic approaches to both types of diabetes. Several approaches to insulin replacement by gene therapy are currently employed: 1) stimulation of beta-cell growth, 2) induction of beta-cell differentiation and regeneration, 3) genetic engineering of non-beta cells to produce insulin, and 4) transplantation of engineered islets or beta cells. In type 1 diabetes, the therapeutic effect of beta-cell proliferation and regeneration is limited as long as the autoimmune destruction of beta cells continues. Therefore, the utilization of engineered non-beta cells free from autoimmunity and islet transplantation with immunological barriers are considered potential therapies for type 1 diabetes. Proliferation of the patients' own beta cells and differentiation of the patients' own non-beta cells to beta cells are desirable strategies for gene therapy of type 2 diabetes because immunological problems can be circumvented. At present, however, these strategies are technically difficult, and transplantation of engineered beta cells or islets with immunological barriers is also a potential gene therapy for type 2 diabetes.     

Programming microbial population dynamics by engineered cell-cell communication

A major aim of synthetic biology is to program novel cellular behavior using engineered gene circuits. Early endeavors focused on building simple circuits that fulfill simple functions, such as logic gates, bistable toggle switches, and oscillators. These gene circuits have primarily focused on single-cell behaviors since they operate intracellularly. Thus, they are often susceptible to cell-cell variations due to stochastic gene expression. Cell-cell communication offers an efficient strategy to coordinate cellular behavior at the population level. To this end, we review recent advances in engineering cell-cell communication to achieve reliable population dynamics, spanning from communication within single species to multispecies, from one-way sender-receiver communication to two-way communication in synthetic microbial ecosystems. These engineered systems serve as well-defined model systems to better understand design principles of their naturally occurring counterparts and to facilitate novel biotechnology applications.     

Current status and perspectives of regulatory T cell-based therapy

The CD4⁺FOXP3⁺ regulatory T (Treg) cells are essential for maintaining immune homeostasis in healthy individuals. Results from clinical trials of Treg cell-based therapies in patients with graft versus host disease (GVHD), type 1 diabetes (T1D), liver transplantation, and kidney transplantation have demonstrated that adoptive transfer of Treg cells is emerging as a promising strategy to promote immune tolerance. Here we provide an overview of recent progresses and current challenges of Treg cell-based therapies. We summarize the completed and ongoing clinical trials with human Treg cells. Notably, a few of the chimeric antigen receptor (CAR)-Treg cell therapies are currently undergoing clinical trials. Meanwhile, we describe the new strategies for engineering Treg cells used in preclinical studies. Finally, we envision that the use of novel synthetic receptors, metabolic regulators, combined therapies, and in vivo generated antigen-specific or engineered Treg cells through the delivery of modified mRNA and CRISPR-based gene editing will further promote the advances of next-generation Treg cell therapies.     

Stochastic simulations of a synthetic bacteria-yeast ecosystem

ABSTRACT: BACKGROUND: The eld of synthetic biology has greatly evolved and numerous functions can now be implemented by articially engineered cells carrying the appropriate genetic information. However, in order for the cells to robustly perform complex or multiple tasks, co-operation between them may be necessary. Therefore, various synthetic biological systems whose functionality requires cell-cell communication are being designed. These systems, microbial consortia, are composed of engineered cells and exhibit a wide range of behaviors. These include yeast cells whose growth is dependent on one another, or bacteria that kill or rescue each other, synchronize, behave as predator-prey ecosystems or invade cancer cells. RESULTS: In this paper, we study a synthetic ecosystem comprising of bacteria and yeast that communicate with and benet from each other using small diffusible molecules. We explore the behavior of this heterogeneous microbial consortium, composed of Saccharomyces cerevisiae and Escherichia coli cells, using stochastic modeling. The stochastic model captures the relevant intra-cellular and inter-cellular interactions taking place in and between the eukaryotic and prokaryotic cells. Integration of well-characterized molecular regulatory elements into these two microbes allows for communication through quorum sensing. A gene controlling growth in yeast is induced by bacteria via chemical signals and vice versa. Interesting dynamics that are common in natural ecosystems, such as obligatory and facultative mutualism, extinction, commensalism and predator-prey like dynamics are observed. We investigate and report on the conditions under which the two species can successfully communicate and rescue each other. CONCLUSIONS: This study explores the various behaviors exhibited by the cohabitation of engineered yeast and bacterial cells. The way that the model is built allows for studying the dynamics of any system consisting of two species communicating with one another via chemical signals. Therefore, key information acquired by our model may potentially drive the experimental design of various synthetic heterogeneous ecosystems.     

活体生物药的应用及在遗传性代谢缺陷病治疗中的展望

活体生物药(live biotherapeutic products,LBPs)是指来自于人体肠道内或自然界中能够治疗人类疾病的活性菌。但天然筛选的活菌存在治疗效果不明显、差异性较大等缺点,难以满足个性化诊疗的需要。近年来,随着合成生物学的发展,研究者利用生命科学及工程科学手段,设计并构建了若干可响应外界复杂环境信号的工程菌株,加快了活体生物药的研发和应用过程。遗传性代谢缺陷病(inherited metabolic disease)是因体内某些酶的遗传缺陷致使体内相应的代谢物不能正常代谢而引发一系列临床症状的一类疾病,因此利用合成生物学技术,针对特定缺陷的酶设计重组活体生物药,未来有希望用于遗传性代谢缺陷病的治疗。本综述以活体生物药为切入点,并结合国内外文献综述,来探讨活体生物药在疾病治疗中的应用,以及对遗传性代谢缺陷病治疗的潜力。     

Orthogonal control of endogenous gene expression in mammalian cells using synthetic ligands

Gene switches have wide utility in synthetic biology, gene therapy, and developmental biology, and multiple orthogonal gene switches are needed to construct advanced circuitry or to control complex phenotypes. Endogenous vascular endothelial growth factor (VEGF‐A) is crucial to angiogenesis, and it has been shown that multiple alternately spliced VEGF‐A isoforms are necessary for proper blood vessel formation. Such a necessity limits the utility of direct transgene delivery, which can provide only one splice variant. To overcome this limitation, we constructed a gene switch that can regulate the (VEGF‐A) locus in mammalian cells by combining an engineered estrogen receptor (ER) ligand‐binding domain (LBD), a p65 activation domain, and an artificial zinc‐finger DNA binding domain (DBD). Our gene switch is specifically and reversibly controlled by 4,4′‐dyhydroxybenzil (DHB), a small molecule, non‐steroid synthetic ligand, which acts orthogonally in a mammalian system. After optimization of the gene switch architecture, an endogenous VEGF‐A induction ratio of >100‐fold can be achieved in HEK293 cells at 1 µM DHB, which is the highest endogenous induction reported to date. In addition, induction has been shown to be reversible, repeatable, and sustainable. Another advantage is that the ligand response is tunable by varying the clonal composition of a stably integrated cell line. The integration of our findings with the technology to change ligand specificity and DNA binding specificity will provide the framework for generating a wide array of orthogonal gene switches that can control multiple genes with multiple orthogonal ligands. Biotechnol. Bioeng. 2013; 110: 1419–1429. © 2012 Wiley Periodicals, Inc.     

Synthetic spatial patterning in bacteria: advances based on novel diffusible signals

Engineering multicellular patterning may help in the understanding of some fundamental laws of pattern formation and thus may contribute to the field of developmental biology. Furthermore, advanced spatial control over gene expression may revolutionize fields such as medicine, through organoid or tissue engineering. To date, foundational advances in spatial synthetic biology have often been made in prokaryotes, using artificial gene circuits. In this review, engineered patterns are classified into four levels of increasing complexity, ranging from spatial systems with no diffusible signals to systems with complex multi-diffusor interactions. This classification highlights how the field was held back by a lack of diffusible components. Consequently, we provide a summary of both previously characterized and some new potential candidate small-molecule signals that can regulate gene expression in Escherichia coli. These diffusive signals will help synthetic biologists to successfully engineer increasingly intricate, robust and tuneable spatial structures.     

合成生物学的医学应用

合成生物学旨在基于工程学原理,通过人工合成生物调控元件、模块和基因调控网络等对细胞进行设计和改造,以实现细胞和生命体的定向演化.在医学研究中,合成生物学主要采用人工设计合成治疗性的基因回路,制备工程化细胞植入体内,纠正机体已发生缺陷的生物调控元件,以达到治疗疾病的目的.本文对合成生物学的兴起、发展及其在医学中的应用和研...