Therapeutic Applications of Engineered Cell Death, Arrest, and Persistence.

Engineered cells provide versatile tools for precise, tunable drug delivery, especially when synthetic stimulus-responsive gene circuits are incorporated. In many complex disease conditions, endogenous pathologic signals such as inflammation can vary dynamically over different time scales. For example, in autoimmune conditions such as rheumatoid arthritis or juvenile idiopathic arthritis, local (joint) and systemic inflammatory signals fluctuate daily, peaking in the early morning, but can also persist over long periods of time, triggering flare-ups that can last weeks to months. However, treatment with disease-modifying anti-rheumatic drugs is typically provided at continuous high doses, regardless of disease activity and without consideration for levels of inflammatory signals. In previous studies, we have developed cell-based drug delivery systems that can automatically address the different scales of flares using either chronogenetic circuits (i.e., clock gene-responsive elements) that can be tuned for optimal drug delivery to dampen circadian variations in inflammatory levels or inflammation-responsive circuits (i.e., NF-κB-sensitive elements) that can respond to sustained arthritis flares on demand with proportional synthesis of drug. The goal of this study was to develop a novel dual-responsive synthetic gene circuit that responds to both circadian and inflammatory inputs using OR-gate logic for both daily timed therapeutic output and enhanced therapeutic output during chronic inflammatory conditions. We developed a synthetic gene circuit driven by tandem inflammatory NF-κB and circadian E'-box response elements. When engineered into induced pluripotent stem cells that were chondrogenically differentiated, the gene circuit demonstrated basal-level circadian output with enhanced stimulus-responsive output during an inflammatory challenge shown by bioluminescence monitoring. Similarly, this system exhibited enhanced therapeutic levels of biologic drug interleukin-1 receptor antagonist (IL-1Ra) during an inflammatory challenge in differentiated cartilage pellets. This dual-responsive therapeutic gene circuit mitigated both the inflammatory response as measured by bioluminescence reporter output and tissue-level degradation during conditions mimicking an arthritic flare. The dual-responsive synthetic gene circuit developed herein responds to input cues from two key homeostatic transcriptional networks, enabling dynamic and tunable output. This proof-of-concept approach has the potential to match drug delivery to disease activity for optimal outcomes that addresses the complex environment of inflammatory arthritis.

BackgroundEngineered cells provide versatile tools for precise, tunable drug delivery, especially when synthetic stimulus-responsive gene circuits are incorporated. In many complex disease conditions, endogenous pathologic signals such as inflammation can vary dynamically over different time scales. For example, in autoimmune conditions such as rheumatoid arthritis or juvenile idiopathic arthritis, local (joint) and systemic inflammatory signals fluctuate daily, peaking in the early morning, but can also persist over long periods of time, triggering flare-ups that can last weeks to months. However, treatment with disease-modifying anti-rheumatic drugs is typically provided at continuous high doses, regardless of disease activity and without consideration for levels of inflammatory signals. In previous studies, we have developed cell-based drug delivery systems that can automatically address the different scales of flares using either chronogenetic circuits (i.e., clock gene-responsive elements) that can be tuned for optimal drug delivery to dampen circadian variations in inflammatory levels or inflammation-responsive circuits (i.e., NF-κB-sensitive elements) that can respond to sustained arthritis flares on demand with proportional synthesis of drug. The goal of this study was to develop a novel dual-responsive synthetic gene circuit that responds to both circadian and inflammatory inputs using OR-gate logic for both daily timed therapeutic output and enhanced therapeutic output during chronic inflammatory conditions.ResultsWe developed a synthetic gene circuit driven by tandem inflammatory NF-κB and circadian E’-box response elements. When engineered into induced pluripotent stem cells that were chondrogenically differentiated, the gene circuit demonstrated basal-level circadian output with enhanced stimulus-responsive output during an inflammatory challenge shown by bioluminescence monitoring. Similarly, this system exhibited enhanced therapeutic levels of biologic drug interleukin-1 receptor antagonist (IL-1Ra) during an inflammatory challenge in differentiated cartilage pellets. This dual-responsive therapeutic gene circuit mitigated both the inflammatory response as measured by bioluminescence reporter output and tissue-level degradation during conditions mimicking an arthritic flare.ConclusionsThe dual-responsive synthetic gene circuit developed herein responds to input cues from two key homeostatic transcriptional networks, enabling dynamic and tunable output. This proof-of-concept approach has the potential to match drug delivery to disease activity for optimal outcomes that addresses the complex environment of inflammatory arthritis.

The induction of senescence, an irreversible growth arrest, in cancer cells is regarded as a mean to halt tumor progression. The phytoalexin resveratrol (RV) is known to possess a variety of cancer-preventive, -therapeutic, and -chemosensitizing properties. We report here that chronic treatment with RV in a subapoptotic concentration induces senescence-like growth arrest in tumor cells. In contrast to the widely accepted antioxidant property of RV, we demonstrate that one causative stimulus for senescence induction by chronic RV is an increased level of reactive oxygen species (ROS). The ROS formed upon RV exposure include hydrogen peroxide and superoxide and originate largely from mitochondria. Consistently, co-incubation with the antioxidant N-acetyl cysteine interfered with RV-mediated reactivation of the senescence program. Molecular mediators on the way from increased ROS levels to the observed growth arrest include p38 MAPK, p53, and p21. Moreover, we provide evidence that RV-initiated replication stress, apparent by activation of the ataxia telangiectasia-mutated kinase pathway, is associated with increased ROS levels and senescence induction. This is the first report linking cell cycle effects with a pro-oxidant and pro-senescent effect of RV in cancer cells.

Adipose-derived stem cells (ASCs) are an important stem cell type separated from adipose tissue, with the properties of multilineage differentiation, easy availability, high proliferation potential, and self-renewal. Exosomes are novel frontiers of intercellular communication regulating the biological behaviors of cells, such as angiogenesis, immune modulation, proliferation, and migration. ASC-derived exosomes (ASC-exos) are important components released by ASCs paracrine, possessing multiple biological activities. Tissue regeneration requires coordinated “vital networks” of multiple growth factors, proteases, progenitors, and immune cells producing inflammatory cytokines. Recently, as cell-to-cell messengers, ASC-exos have received much attention for the fact that they are important paracrine mediators contributing to their suitability for tissue regeneration. ASC-exos, with distinct properties by encapsulating various types of bioactive cargoes, are endowed with great application potential in tissue regeneration, mechanically via the migration and proliferation of repair cells, facilitation of the neovascularization, and other specific functions in different tissues. Here, this article elucidated the research progress of ASC-exos about tissue regeneration in plastic and cosmetic surgery, including skin anti-aging therapy, dermatitis improvement, wound healing, scar removal, flap transplantation, bone tissue repair and regeneration, obesity prevention, fat grafting, breast cancer, and breast reconstruction. Deciphering the biological properties of ASC-exos will provide further insights for exploring novel therapeutic strategies of tissue regeneration in plastic and cosmetic surgery.

Fucoidan based hydrogel biomaterials for tissue engineering.

Keratose hydrogel for tissue regeneration and drug delivery

MicroRNAs (miRNAs) have been shown to modulate gene expression noise, but less is known about how miRNAs with different properties may regulate noise differently. Here, we investigate the role of competing RNAs and the composition of miRNA response elements (MREs) in modulating noise. We find that weak competing RNAs could introduce lower noise than strong competing RNAs. In comparison with a single MRE, both repetitive and composite MREs can reduce the noise at low expression, but repetitive MREs can elevate the noise remarkably at high expression. We further observed the behavior of a synthetic cell-type classifier with miRNAs as inputs and find that miRNAs and MREs that could introduce higher noise tend to enhance cell state transition. These results provide a systematic and quantitative understanding of the function of miRNAs in controlling gene expression noise and the utilization of miRNAs to modulate the behavior of synthetic gene circuits.

In situ forming hydrogels based on chitosan for drug delivery and tissue regeneration

Chapter 12 - Bionanofibers in drug delivery

The field of synthetic gene circuits is concerned with engineering novel gene expression dynamics into organisms. This field, a subset of synthetic biology, was started almost two decades ago with the creation of two synthetic circuits: a bistable toggle switch and an oscillator. From the very outset, modeling has played a role in the development of synthetic circuits. However, modeling has been used to gain qualitative insight into dynamics, and actual quantitative modeling has been lagging behind. Parameters for quantitative models for biological systems often cannot be adequately estimated from measured data, because far too many sets of parameters can produce the same set of limited measured outputs. Additionally, models for synthetic gene circuits are often not correct the first time, and iterating on cycles of modeling and parameter estimation is difficult. Finally, there is a gap between development of modeling and system identification tools and their application to experiments on actual synthetic gene circuits. This thesis attempts to work towards addressing these issues with quantitative modeling for synthetic gene circuits. First, we derive theoretical conditions for identifiability of stochastic linear systems from heterogenous types of measurement data. These identifiability conditions can provide insight into what type of model to use and what measurements to collect in order to ensure that the resulting model can be identified. Second, we develop a software package for fast and flexible modeling and parameter estimation for synthetic gene circuits. The user can input models into our software using a simple text format and perform simulations of all types at optimized speeds. By inputting measured experimental data along with the model, the software can be used to perform Bayesian parameter estimation in an automated manner. To bridge the gap between computation and application, we apply this software to parameter estimation of DNA recombinase dynamics using real experimental data collected in an in vitro cell extract. Finally, we use modeling to guide the design of an improved single gene synthetic oscillator. While the original synthetic genetic oscillator contained three genes, we show that a simple circuit with a single gene can produce robust and synchronized oscillations across a population. Our results constitute an additional step towards the incorporation of quantitative modeling and parameter inference as part of the design-build-test cycle for synthetic gene circuits.

This article presents an overview focusing on the structural control of electrospun membranes on a multilevel scale ranging from the morphology of single nanofibers to the packing and alignment of nanofibers and the patterns and shapes of fibrous scaffolds. The typical structures of electrospun membranes and the specific electrospinning strategies used to produce these structures are reviewed. In addition, potential applications of these controlled structures in tissue engineering and drug delivery are highlighted. Finally, this review concludes with a perspective on the challenges and future directions for the design and fabrication of electrospun scaffolds using controlled structures along with an investigation of the relationship between the structures of electrospun membranes and the cell and drug delivery behaviors.

Three-dimensional (3D) printing, which is a valuable technique for the fabrication of tissue-engineered constructs and biomedical devices with complex architectures, has brought about considerable progress in regenerative medicine, drug delivery, and diagnosis of diseases. However, because of the static and inanimate properties of conventional 3D-printed structures, it is difficult to use them in therapies for active and precise medicine, such as improved tissue regeneration, targeted or controlled drug delivery, and advanced pathophysiological monitoring. The integration of stimuli-responsive biomaterials into 3D printing provides a potential strategy for designing and building smart constructs that exhibit programmed functions and controllable changes in properties in response to exogenous and autogenous stimuli. These features make 3D-printed smart constructs the next generation of tissue-engineered products. In this review, we introduce the prevalent 3D printing techniques (with an emphasis on the differences between 3D printing and bioprinting, and biomaterials and bioink), the working principle of each technique, and the advantages of using 3D printing for the fabrication of smart constructs. Stimuli-responsive biomaterials that are widely used for 3D printing of smart constructs are categorized, followed by a summary of their applications in tissue regeneration, drug delivery, and biosensors. Finally, the challenges and future perspectives of 3D-printed smart constructs are discussed.

ABSTRACTA new family of A-B di-block copolymers based on the amino acid sequences of Nephila clavipes major ampulate dragline spider silk, which have a strong potential for applications in tissue regeneration and drug delivery, was synthesized and characterized. The morphology was assessed by SEM: HBA3 formed fibrillar structure and 2 μm diameter hollow micelles, while HBA4 and HBA6 formed hollow micelles in water solution. The secondary structures of water-cast spider silk-like block copolymer films were assessed by FTIR. The crystallinity was determined by Fourier self-deconvolution of the amide I spectra and confirmed by wide angle X-ray diffraction. Results indicate that the self-assembled morphology and the crystallinity can be varied by changing the length of A-block, and a minimum of 3 A-blocks are required to form β sheet crystalline regions in water-cast spider silk block copolymers. A theoretical model was used to predict the specific reversing heat capacity, Cp(T), which is crucial to the design of smart biomaterials. Excellent agreement was found between the theoretical value and the Cp(T) determined by temperature modulated differential scanning calorimetry. This method can serve as a standard by which to assess the thermal properties for other biologically inspired block copolymers, and then be further applied to control the biological interactions for use in drug delivery and smart biomaterials applications.

Synthetic gene circuits are powerful tools for precisely programming gene expression and introducing novel cellular functions. However, their development and application in plants has lagged behind other systems, due mainly to the limited availability of modular genetic parts. We recently developed a CRISPR interference (CRISPRi)-based synthetic gene circuit system for programming gene expression in plants. Using a robust and high-throughput protoplast-based dual luciferase assay, we demonstrated the development, testing and functionality of these circuits in various plant species. Here we detail the key design principles and considerations for building and testing programmable and reversible CRISPRi-based gene circuits in plants. We also provide detailed procedures for isolating protoplasts from multiple plant species, including Arabidopsis thaliana, Brassica napus, Triticum aestivum and Physcomitrium patens. Furthermore, we provide step-by-step instructions for the 96-well plate-based protoplast transfection assay for testing genetic parts and synthetic circuits, using a dual luciferase assay. The detailed descriptions of these developed systems will enhance the efficiency and reproducibility of the construction, testing, and implementation of synthetic gene circuits in a variety of plant species. This protocol enables the design and testing of CRISPRi-based gene circuits in plants within ~4 weeks.

Electrospun nanofibers of collagen and chitosan for tissue engineering and drug delivery applications: A review.