An engineered mammalian band-pass network.

Gene expression circuitries, which enable cells to detect precise levels within a morphogen concentration gradient, have a pivotal impact on biological processes such as embryonic pattern formation, paracrine and autocrine signalling, and cellular migration. We present the rational synthesis of a synthetic genetic circuit exhibiting band-pass detection characteristics. The components, involving multiply linked mammalian trans-activator and -repressor control systems, were selected and fine-tuned to enable the detection of 'low-threshold' morphogen (tetracycline) concentrations, in which target gene expression was triggered, and a 'high-threshold' concentration, in which expression was muted. In silico predictions and supporting experimental findings indicated that the key criterion for functional band-pass detection was the matching of componentry that enabled sufficient separation of the low and high threshold points. Using the circuitry together with a fluorescence-encoded target gene, mammalian cells were genetically engineered to be capable of forming a band-like pattern of differentiation in response to a tetracycline chemical gradient. Synthetic gene networks designed to emulate naturally occurring gene behaviours provide not only insight into biological processes, but may also foster progress in future tissue engineering, gene therapy and biosensing applications.

A synthetic low-frequency mammalian oscillator.

Circadian clocks have long been known to be essential for the maintenance of physiological and behavioral processes in a variety of organisms ranging from plants to humans. Dysfunctions that subvert gene expression of oscillatory circadian-clock components may result in severe pathologies, including tumors and metabolic disorders. While the underlying molecular mechanisms and dynamics of complex gene behavior are not fully understood, synthetic approaches have provided substantial insight into the operation of complex control circuits, including that of oscillatory networks. Using iterative cycles of mathematical model-guided design and experimental analyses, we have developed a novel low-frequency mammalian oscillator. It incorporates intronically encoded siRNA-based silencing of the tetracycline-dependent transactivator to enable the autonomous and robust expression of a fluorescent transgene with periods of 26 h, a circadian clock-like oscillatory behavior. Using fluorescence-based time-lapse microscopy of engineered CHO-K1 cells, we profiled expression dynamics of a destabilized yellow fluorescent protein variant in single cells and real time. The novel oscillator design may enable further insights into the system dynamics of natural periodic processes as well as into siRNA-mediated transcription silencing. It may foster advances in design, analysis and application of complex synthetic systems in future gene therapy initiatives.

Intronically encoded siRNAs improve dynamic range of mammalian gene regulation systems and toggle switch.

Applications of conditional gene expression, whether for therapeutic or basic research purposes, are increasingly requiring mammalian gene control systems that exhibit far tighter control properties. While numerous approaches have been used to improve the widely used Tet-regulatory system, many applications, particularly with respect to the engineering of synthetic gene networks, will require a broader range of tightly performing gene control systems. Here, a generically applicable approach is described that utilizes intronically encoded siRNA on the relevant transregulator construct, and siRNA sequence-specific tags on the reporter construct, to minimize basal gene activity in the off-state of a range of common gene control systems. To demonstrate tight control of residual expression the approach was successfully used to conditionally express the toxic proteins RipDD and Linamarase. The intronic siRNA concept was also extended to create a new generation of compact, single-vector, autoinducible siRNA vectors. Finally, using improved regulation systems a mammalian epigenetic toggle switch was engineered that exhibited superior in vitro and in vivo induction characteristics in mice compared to the equivalent non-intronic system.

Economic benefits of subcutaneous trastuzumab administration: A single institutional study from Karolinska University Hospital in Sweden.

INTRODUCTION: Adjuvant trastuzumab is a standard of care in the treatment of Human Epidermal growth factor Receptor 2 (HER2) positive early breast cancer (eBC). Initially trastuzumab could only be administered intravenously (IV), however since 2013, a subcutaneous (SC) formulation with comparable efficacy and safety profile is available and preferred by patients. Trastuzumab SC does not require pharmacy preparation and has shorter administration time. The objective of this study was to estimate the economic efficiency of the SC formulation of trastuzumab by assessing the economic benefits of actual SC-driven process changes at one single Swedish healthcare institution. METHODS: This study analyzes changes in trastuzumab administration practice after the SC formulation was introduced at the Karolinska University Hospital. Process changes were identified and introduced in order to capitalize on the inherent work efficiency benefits of the SC formulation. Actual hospital data for 2015 were used to quantitatively estimate the annual economic impact of the changes. It encompassed administrative (i.e. non-medical) data of 178 newly diagnosed HER2-positive eBC patients and a total of 2,769 SC administrations. Realized economic benefits were expressed in hours saved by nurses, direct monetary cost savings and potential infusion fee revenue that could be earned through infrastructural revenue gains. RESULTS: In 2015, the replacement of IV infusion to SC administration generated total time savings of more than 1,100 hours, and led to direct monetary cost savings of 603,000 EUR. It unlocked a capacity gain of 1-2 additional administrations daily within the existing facility infrastructure. Given the current remuneration structure per administration, this revenue gain translated into an incremental revenue potential of up to 3 million EUR. CONCLUSION: Data from this study showed that the shift from trastuzumab IV to SC formulation resulted in significant economic effects in terms of departmental resources related to time, direct monetary cost savings, and infrastructural revenue gains.

Therapeutic protein transduction of mammalian cells and mice by nucleic acid-free lentiviral nanoparticles.

The straightforward production and dose-controlled administration of protein therapeutics remain major challenges for the biopharmaceutical manufacturing and gene therapy communities. Transgenes linked to HIV-1-derived vpr and pol-based protease cleavage (PC) sequences were co-produced as chimeric fusion proteins in a lentivirus production setting, encapsidated and processed to fusion peptide-free native protein in pseudotyped lentivirions for intracellular delivery and therapeutic action in target cells. Devoid of viral genome sequences, protein-transducing nanoparticles (PTNs) enabled transient and dose-dependent delivery of therapeutic proteins at functional quantities into a variety of mammalian cells in the absence of host chromosome modifications. PTNs delivering Manihot esculenta linamarase into rodent or human, tumor cell lines and spheroids mediated hydrolysis of the innocuous natural prodrug linamarin to cyanide and resulted in efficient cell killing. Following linamarin injection into nude mice, linamarase-transducing nanoparticles impacted solid tumor development through the bystander effect of cyanide.

Mammalian synthetic biology: engineering of sophisticated gene networks.

With the recent development of a wide range of inducible mammalian transgene control systems it has now become possible to create functional synthetic gene networks by linking and connecting systems into various configurations. The past 5 years has thus seen the design and construction of the first synthetic mammalian gene regulatory networks. These networks have built upon pioneering advances in prokaryotic synthetic networks and possess an impressive range of functionalities that will some day enable the engineering of sophisticated inter- and intra-cellular functions to become a reality. At a relatively simple level, the modular linking of transcriptional components has enabled the creation of genetic networks that are strongly analogous to the architectural design and functionality of electronic circuits. Thus, by combining components in different serial or parallel configurations it is possible to produce networks that follow strict logic in integrating multiple independent signals (logic gates and transcriptional cascades) or which temporally modify input signals (time-delay circuits). Progressing in terms of sophistication, synthetic transcriptional networks have also been constructed which emulate naturally occurring genetic properties, such as bistability or dynamic instability. Toggle switches which possess "memory" so as to remember transient administered inputs, hysteric switches which are resistant to stochastic fluctuations in inputs, and oscillatory networks which produce regularly timed expression outputs, are all examples of networks that have been constructed using such properties. Initial steps have also been made in designing the above networks to respond not only to exogenous signals, but also endogenous signals that may be associated with aberrant cellular function or physiology thereby providing a means for tightly controlled gene therapy applications. Moving beyond pure transcriptional control, synthetic networks have also been created which utilize phenomena, such as post-transcriptional silencing, translational control, or inter-cellular signaling to produce novel network-based control both within and between cells. It is envisaged in the not-too-distant future that these networks will provide the basis for highly sophisticated genetic manipulations in biopharmaceutical manufacturing, gene therapy and tissue engineering applications.

Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform.

Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.