A programmable targeted protein-degradation platform for versatile applications in mammalian cells and mice.

Myriad physiological and pathogenic processes are governed by protein levels and modifications. Controlled protein activity perturbation is essential to studying protein function in cells and animals. Based on Trim-Away technology, we screened for truncation variants of E3 ubiquitinase Trim21 with elevated efficiency (ΔTrim21) and developed multiple ΔTrim21-based targeted protein-degradation systems (ΔTrim-TPD) that can be transfected into host cells. Three ΔTrim-TPD variants are developed to enable chemical and light-triggered programmable activation of TPD in cells and animals. Specifically, we used ΔTrim-TPD for (1) red-light-triggered inhibition of HSV-1 virus proliferation by degrading the packaging protein gD, (2) for chemical-triggered control of the activity of Cas9/dCas9 protein for gene editing, and (3) for blue-light-triggered degradation of two tumor-associated proteins for spatiotemporal inhibition of melanoma tumor growth in mice. Our study demonstrates that multiple ΔTrim21-based controllable TPD systems provide powerful tools for basic biology research and highlight their potential biomedical applications.

An Optogenetic-Controlled Cell Reprogramming System for Driving Cell Fate and Light-Responsive Chimeric Mice.

Pluripotent stem cells (PSCs) hold great promise for cell-based therapies, disease modeling, and drug discovery. Classic somatic cell reprogramming to generate induced pluripotent stem cells (iPSCs) is often achieved based on overexpression of transcription factors (TFs). However, this process is limited by side effect of overexpressed TFs and unpredicted targeting of TFs. Pinpoint control over endogenous TFs expression can provide the ability to reprogram cell fate and tissue function. Here, a light-inducible cell reprogramming (LIRE) system is developed based on a photoreceptor protein cryptochrome system and clustered regularly interspaced short palindromic repeats/nuclease-deficient CRISPR-associated protein 9 for induced PSCs reprogramming. This system enables remote, non-invasive optogenetical regulation of endogenous Sox2 and Oct4 loci to reprogram mouse embryonic fibroblasts into iPSCs (iPSCLIRE ) under light-emitting diode-based illumination. iPSCLIRE cells can be efficiently differentiated into different cells by upregulating a corresponding TF. iPSCLIRE cells are used for blastocyst injection and optogenetic chimeric mice are successfully generated, which enables non-invasive control of user-defined endogenous genes in vivo, providing a valuable tool for facile and traceless controlled gene expression studies and genetic screens in mice. This LIRE system offers a remote, traceless, and non-invasive approach for cellular reprogramming and modeling of complex human diseases in basic biological research and regenerative medicine applications.

Engineering antiviral immune-like systems for autonomous virus detection and inhibition in mice.

The ongoing COVID-19 pandemic has demonstrated that viral diseases represent an enormous public health and economic threat to mankind and that individuals with compromised immune systems are at greater risk of complications and death from viral diseases. The development of broad-spectrum antivirals is an important part of pandemic preparedness. Here, we have engineer a series of designer cells which we term autonomous, intelligent, virus-inducible immune-like (ALICE) cells as sense-and-destroy antiviral system. After developing a destabilized STING-based sensor to detect viruses from seven different genera, we have used a synthetic signal transduction system to link viral detection to the expression of multiple antiviral effector molecules, including antiviral cytokines, a CRISPR-Cas9 module for viral degradation and the secretion of a neutralizing antibody. We perform a proof-of-concept study using multiple iterations of our ALICE system in vitro, followed by in vivo functionality testing in mice. We show that dual output ALICESaCas9+Ab system delivered by an AAV-vector inhibited viral infection in herpetic simplex keratitis (HSK) mouse model. Our work demonstrates that viral detection and antiviral countermeasures can be paired for intelligent sense-and-destroy applications as a flexible and innovative method against virus infection.

Far-red light-activated human islet-like designer cells enable sustained fine-tuned secretion of insulin for glucose control.

Diabetes affects almost half a billion people, and all individuals with type 1 diabetes (T1D) and a large portion of individuals with type 2 diabetes rely on self-administration of the peptide hormone insulin to achieve glucose control. However, this treatment modality has cumbersome storage and equipment requirements and is susceptible to fatal user error. Here, reasoning that a cell-based therapy could be coupled to an external induction circuit for blood glucose control, as a proof of concept we developed far-red light (FRL)-activated human islet-like designer (FAID) cells and demonstrated how FAID cell implants achieved safe and sustained glucose control in diabetic model mice. Specifically, by introducing a FRL-triggered optogenetic device into human mesenchymal stem cells (hMSCs), which we encapsulated in poly-(l-lysine)-alginate and implanted subcutaneously under the dorsum of T1D model mice, we achieved FRL illumination-inducible secretion of insulin that yielded improvements in glucose tolerance and sustained blood glucose control over traditional insulin glargine treatment. Moreover, the FAID cell implants attenuated both oxidative stress and development of multiple diabetes-related complications in kidneys. This optogenetics-controlled "living cell factory" platform could be harnessed to develop multiple synthetic designer therapeutic cells to achieve long-term yet precisely controllable drug delivery.

Engineering genetic devices for in vivo control of therapeutic T cell activity triggered by the dietary molecule resveratrol.

Chimeric antigen receptor (CAR)-engineered T cell therapies have been recognized as powerful strategies in cancer immunotherapy; however, the clinical application of CAR-T is currently constrained by severe adverse effects in patients, caused by excessive cytotoxic activity and poor T cell control. Herein, we harnessed a dietary molecule resveratrol (RES)-responsive transactivator and a transrepressor to develop a repressible transgene expression (RESrep) device and an inducible transgene expression (RESind) device, respectively. After optimization, these tools enabled the control of CAR expression and CAR-mediated antitumor function in engineered human cells. We demonstrated that a resveratrol-repressible CAR expression (RESrep-CAR) device can effectively inhibit T cell activation upon resveratrol administration in primary T cells and a xenograft tumor mouse model. Additionally, we exhibit how a resveratrol-inducible CAR expression (RESind-CAR) device can achieve fine-tuned and reversible control over T cell activation via a resveratrol-titratable mechanism. Furthermore, our results revealed that the presence of RES can activate RESind-CAR T cells with strong anticancer cytotoxicity against cells in vitro and in vivo. Our study demonstrates the utility of RESrep and RESind devices as effective tools for transgene expression and illustrates the potential of RESrep-CAR and RESind-CAR devices to enhance patient safety in precision cancer immunotherapies.

Molecular method for rapid detection of the red tide dinoflagellate Karenia mikimotoi in the coastal region of Xiangshan Bay, China.

The species Karenia mikimotoi is a common nearshore red tide alga that can secrete hemolytic exotoxin and ichthyotoxin, which can induce the death of fish and shellfish, causing severe economic losses. In this study, loop-mediated isothermal amplification (LAMP) was employed in combination with the lateral flow dipstick (LFD) visual detection method to establish the LAMP-LFD rapid detection method for K. mikimotoi. The internal transcribed spacer ITS1-5.8S-ITS2 of K. mikimotoi was used as the target sequence and was amplified with specific primers designed in this study. The results indicated that the amplification optimal reaction conditions for LAMP in this paper were for 20 min at 65 °C. Moreover, LAMP had excellent specificity, showing negative results for other common red tide causing algal species. In field samples, we successfully reduced the total time, with only 23 min needed from LAMP amplification to LFD result display, which was shorter than that of conventional PCR. Consequently, LAMP-LFD should be useful for rapid field detection of low-density K. mikimotoi and for the early prevention of red tide induced by such algae.

A Novel Chemoenzymatic Approach to Produce Cilengitide Using the Thioesterase Domain from Microcystis aeruginosa Microcystin Synthetase C.

Modern organic chemistry faces many difficulties in the reliable production of cyclopeptides, such as poor yields and insufficient regio- and stereoselectivity. Thioesterase (TE) shows impressive stereospecificity, region- and chemoselectivity during the cyclization of peptide substrates. The biocatalytic properties of TE provide high value for industrial applications. Herein, a novel chemoenzymatic method to synthesize cilengitide is described based on the cyclic activity of the TE domain from microcystin synthetase C (McyC) of Microcystis aeruginosa. In addition, a single active site mutation in the McyC TE was engineered to generate a more effective macrocyclization catalyst. Compared to the chemical approach to synthesize cilengitide, this novel enzyme-catalysed methodology exhibits a higher synthetic efficiency with an approximately 3.4-fold higher yield (49.2%).

A real-time recombinase polymerase amplification assay for the rapid detection of Vibrio harveyi.

Vibrio harveyi is a pathogen that infects fish and shellfish worldwide, causing severe economic losses for the aquaculture industry. As the early diagnosis of V. harveyi infection is crucial to disease surveillance and prevention in cultured marine animals, a fast and accurate method to detect V. harveyi is required. Here, we performed recombinase polymerase amplification (RPA) using novel primers specifically designed to recognize the V. harveyi toxR gene, which encodes a transmembrane protein, and then hybridized this gene with a carboxy fluorescein (FAM)-labeled probe. The optimal conditions for the real-time RPA assay were a probe concentration of 90 nM and a 20 min incubation at 37 °C. The sensitivity of our real-time RPA assay was 50 copies of the standard plasmid, while that of real-time PCR was 500 copies. In V. harveyi-spiked Pseudosciaena crocea samples, the sensitivity of our real-time RPA was 60 CFUs per reaction, while that of PCR was 600 CFUs per reaction. SPSS probit regression analysis indicated that the limit of detection (LOD) of our RPA assay, with 95% probability, was 18 copies. The LOD was reached within 20 min and was highly reproducible across eight independent assays. Our novel RPA method successfully differentiated V. harveyi from all other tested Vibrio species, including some that were closely related. Our real-time RPA assay, in combination with a rapid DNA extraction protocol, is a fast and accurate tool for the detection of V. harveyi and for monitoring disease outbreaks. This tool will be valuable for the aquaculture industry.

Effect of methylated tea catechins from Chinese oolong tea on the proliferation and differentiation of 3T3-L1 preadipocyte.

As the important component of tea catechins in oolong tea, (-)-epigallocatechin 3-O-(3-O-methyl) gallate (EGCG3″Me) has exhibited various beneficial effects, however, little attention about its obesity prevention effect is available. In this study, the inhibitory effects of tea catechin monomers, including their methylated forms on the proliferation and differentiation of 3T3-L1 preadipocyte were studied. The major methylated tea catechins in oolong tea were identified as EGCG3″Me and ECG3″Me. The accumulation of triglyceride was significantly reduced in a concentration-dependent manner in groups treated with EGCG3″Me at concentrations of 20, 40 and 80µg/mL, and the accumulation of lipid was decreased to 89.42±2.66%, 64.36±3.13% and 39.37±2.79%, respectively. Both EGCG3″Me and EGCG treatments showed a significant inhibitory effect on adipogenesis, while EGCG3″Me showed a relatively higher effect than EGCG, which was contrary to the results of cytotoxic activity. For ECG and ECG3″Me, ECG3″Me also showed a relatively higher antiobesity effect and lower cytotoxic activity. The results of activity screening showed that methylated tea catechins, including EGCG3″Me and ECG3″Me inhibited the proliferation and differentiation of 3T3-L1 preadipocyte. The difference of inhibitory effects for tested compounds may be due to their structural difference (the hydroxyl group at C-3 in D ring substituted by methoxy group).