A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues
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PROVIDER: PRJNA692990 | ENA |
REPOSITORIES: ENA
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Project description:Mechanobiologic signals play critical roles in regulating cellular responses under both physiologic and pathologic conditions. Using a combination of synthetic biology and tissue engineering, we developed a mechanically-responsive bioartificial tissue that responds to mechanical loading to produce a pre-programmed therapeutic biologic drug. By deconstructing the signaling networks induced by activation the mechanically-sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we synthesized synthetic TRPV4-responsive genetic circuits in chondrocytes. These cells were then engineered into living tissues that respond to mechanical compression to drive the production of the anti-inflammatory drug interleukin-1 receptor antagonist. Mechanical loading of these tissues in the presence of the cytokine interleukin-1 protected constructs from inflammatory degradation. This “mechanogenetic” approach enables long-term autonomous delivery of therapeutic compounds that is driven by physiologically-relevant mechanical loading with cell-scale mechanical force resolution. The development of synthetic mechanogenetic gene circuits provides a novel approach for the autonomous regulation of cell-based drug delivery systems.
2021-01-19 | GSE165027 | GEO
Project description:Engineered transactivation domains (TADs) combined with programmable DNA binding platforms have revolutionized synthetic transcriptional control. Despite recent progress in programmable CRISPR/Cas-based transactivation (CRISPRa) technologies, the TADs used in these systems often contain poorly tolerated elements and/or are prohibitively large for many applications. Here we defined and optimized minimal TADs built from human mechanosensitive transcription factors (MTFs). We used these components to construct potent and compact multipartite transactivation modules (MSN, NMS, and eN3×9) and to build the CRISPR-dCas9 recruited enhanced activation module (CRISPR-DREAM) platform. We found that CRISPR-DREAM was specific, robust across mammalian cell types, and efficiently stimulated transcription from diverse regulatory loci. We also showed that MSN and NMS were portable across Type I, II, and V CRISPR systems, TALEs, and ZF proteins. Further, as proofs of concepts, we used dCas9-NMS to efficiently reprogram human fibroblasts into iPSCs and demonstrated that MTF TADs are efficacious and well tolerated in therapeutically important primary human cell types. Finally, we leveraged the compact and potent features of these engineered TADs to build new dual and all-in-one CRISPRa AAV systems. Altogether, these compact human TADs, fusion modules, and new delivery architectures should be valuable for synthetic transcriptional control in biomedical applications.
2023-07-31 | GSE238178 | GEO
Project description:Obesity is becoming increasingly widespread in developed countries, and is often associated with heart diseases and diabetes. Elevated levels of plasma free fatty acids are a biochemical hallmark of obesity. Unlike plants and bacteria, mammals cannot utilize fatty acids to generate glucose because of the lack of glyoxylate shunt enzymes. Instead, fatty acids are used for energy storage, and their utilization is regulated at multiple levels ranging from hormonal to metabolic ensuring that glucose is preferentially oxidized before fatty acids. Here we designed a synthetic gene-metabolic circuit to alter the intracellular signaling and metabolic pathways that control fatty acid metabolism. By introducing the Escherichia coli glyoxylate shunt enzymes, isocitrate lyase (AceA) and malate synthase (AceB), into the mitochondria of human hepatocytes, we demonstrated that fatty acid utilization is preferentially increased while glucose uptake is significantly reduced. This bacterial pathway diverts signal metabolites (citrate, acetyl-CoA, and malonyl-CoA) that inhibits fatty acid uptake and provides an additional channel for fatty acid utilization. Remarkably, the hepatocytes readily adapted to the synthetic circuit by a series of global transcriptional and post-translational changes in metabolic and signal transduction pathways that further advanced our design objective. This systems approach illustrates the logic of the synergistic interaction between metabolism and signal transduction. The ready acceptance of this non-native pathway by hepatocytes suggests the plasticity of liver metabolism and opens a possibility for the synthetic approach in regulating human metabolism. Keywords: Fatty acid response, synthetic circuit, carbohydrate metabolism, stably transfected HepG2 cell line, glyoxylate shunt
2009-06-05 | GSE5903 | GEO
Project description:Synthetic negative feedback circuits using engineered small RNAs
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Project description:Vineetha Mandlik, Mayuri Gurav & Shailza Singh. Regulatory dynamics of network architecture and function in tristable genetic circuit of Leishmania: a mathematical biology approach. Journal of Biomolecular Structure and Dynamics 33, 12 (2015).
The emerging field of synthetic biology has led to the design of tailor-made synthetic circuits for several therapeutic applications. Biological networks can be reprogramed by designing synthetic circuits that modulate the expression of target proteins. IPCS (inositol phosphorylceramide synthase) has been an attractive target in the sphingolipid metabolism of the parasite Leishmania. In this study, we have constructed a tristable circuit for the IPCS protein. The circuit has been validated and its long-term behavior has been assessed. The robustness and evolvability of the circuit has been estimated using evolutionary algorithms. The tristable synthetic circuit has been specifically designed to improve the rate of production of phosphatidylcholine: ceramide cholinephosphotransferase 4 (SLS4 protein). Site-specific delivery of the circuit into the parasite-infected macrophages could serve as a possible therapeutic intervention of the infectious disease 'Leishmaniasis'.
2024-09-02 | BIOMD0000000584 | BioModels
Project description:We report the characterization of a synthetic genetic circuit using RNA-Seq data. Data is collected for all input inducer combinations and cells harboring the circuit are grown in two conditions (14 ml culture tubes and 250 ml Erlenmeyer flasks).
2017-09-29 | GSE88835 | GEO
Project description:Differential responses to stimuli can affect how cells succumb to disease. In yeast, DNA damage can create heterogeneous responses. To delineate how a response contributes to a cell's future behavior, we constructed a transcription-based memory circuit that detects DNA repair to isolate subpopulations with heritable damage responses. Strongly responsive cells show multigenerational effects, including growth defects and iron-associated gene expression. Less-responsive cells exhibit increased mutation frequencies but resume wild-type behavior. These two subpopulations remain distinct for multiple generations, indicating a transmissible memory of damage. Collectively, this work demonstrates the efficacy of using synthetic biology to define how environmental exposure contributes to distinct cell fates. Cells containing the HUG1 memory device (Burrill, 2011) were exposed to 0.3% EMS for 24 h and recovered for 36 h, at which point cells were sorted based on YFP expression, i.e. memory and non-memory cells. These two sorted populations were then grown for another 12 h, at which point transcriptional profiling was performed.
2012-03-02 | E-GEOD-36210 | biostudies-arrayexpress
Project description:Mesenchymal stem cells (MSC) secrete naturally extracellular vesicles (EV) with a regenerative profile, and an increasing number of studies have focused on the employment of EV for therapeutic drug delivery. However, EV are usually produced by the cells at low quantities and packed with unnecessary cytoplasmic components that are unfavorable for further drug loading. In this study, we developed a simple vesicle generation process from the cell membranes directly, after which they are called synthetic eukaryotic vesicles (SyEV or reNV). We hypothesized that MSC-derived SyEV can be efficiently loaded with an anti-inflammatory drug and the loaded vesicles can strongly suppress bacterial outer membrane vesicle (OMV)-induced sepsis. SyEV were generated from MSC membranes through serial extrusion, ionic stress and subsequent revesiculation, leading to high yield and purity with very few cytosolic contaminants. When these SyEV were given to macrophages and mice exposed to OMV, the release of pro-inflammatory cytokines was significantly inhibited comparable to natural EV. Then, we encapsulated SyEV with a peptide targeting Myd88 and showed the enhanced therapeutic potential of the loaded vesicles in OMV-induced macrophages. Further, in vivo study indicated that the peptide-encapsulated SyEV could dramatically suppress cytokine release into the systemic circulation via a synergistic way. Taken together, these data suggest that SyEV-based therapeutics is a highly interesting platform to deliver an advanced therapeutic drug for the treatment of inflammatory diseases without severe side effects.
2023-03-11 | PXD033512 | Pride
Project description:Differential responses to stimuli can affect how cells succumb to disease. In yeast, DNA damage can create heterogeneous responses. To delineate how a response contributes to a cell's future behavior, we constructed a transcription-based memory circuit that detects DNA repair to isolate subpopulations with heritable damage responses. Strongly responsive cells show multigenerational effects, including growth defects and iron-associated gene expression. Less-responsive cells exhibit increased mutation frequencies but resume wild-type behavior. These two subpopulations remain distinct for multiple generations, indicating a transmissible memory of damage. Collectively, this work demonstrates the efficacy of using synthetic biology to define how environmental exposure contributes to distinct cell fates.
2012-03-02 | GSE36210 | GEO
Project description:Obesity is becoming increasingly widespread in developed countries, and is often associated with heart diseases and diabetes. Elevated levels of plasma free fatty acids are a biochemical hallmark of obesity. Unlike plants and bacteria, mammals cannot utilize fatty acids to generate glucose because of the lack of glyoxylate shunt enzymes. Instead, fatty acids are used for energy storage, and their utilization is regulated at multiple levels ranging from hormonal to metabolic ensuring that glucose is preferentially oxidized before fatty acids. Here we designed a synthetic gene-metabolic circuit to alter the intracellular signaling and metabolic pathways that control fatty acid metabolism. By introducing the Escherichia coli glyoxylate shunt enzymes, isocitrate lyase (AceA) and malate synthase (AceB), into the mitochondria of human hepatocytes, we demonstrated that fatty acid utilization is preferentially increased while glucose uptake is significantly reduced. This bacterial pathway diverts signal metabolites (citrate, acetyl-CoA, and malonyl-CoA) that inhibits fatty acid uptake and provides an additional channel for fatty acid utilization. Remarkably, the hepatocytes readily adapted to the synthetic circuit by a series of global transcriptional and post-translational changes in metabolic and signal transduction pathways that further advanced our design objective. This systems approach illustrates the logic of the synergistic interaction between metabolism and signal transduction. The ready acceptance of this non-native pathway by hepatocytes suggests the plasticity of liver metabolism and opens a possibility for the synthetic approach in regulating human metabolism. Experiment Overall Design: Wild type (WT) human hepatocyte HepG2 and HepG2 cells stably transfected with pBudaceAB (designated ACE), a vector containing isocitrate lyase and malate synthase from E. coli, were analyzed after 24hour exposure to palmitate. Cells were seeded at 70% confluency in 10cm plates with DMEM growth media augmented with 300uM palmitate. After 24hours, total RNA was harvested and hybrized to Affymetrix HG_U133A 2.0 GeneChips. Data was processed using GCOS 1.2 software. Experiment Overall Design: 2 WT biological replicates and 2 ACE biological replicates were analyzed. Experiment Overall Design:
2009-06-24 | E-GEOD-5903 | biostudies-arrayexpress