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The effects of immunotherapeutics on interactions between immune and cancer cells are modulated by multiple components in the tumour microenvironment (TME), including endothelium and tumour stroma, which provide both a physical barrier and immunosuppressive stimuli. Herein, we report a recirculating chip to enable continuous immune cell recirculation through a microfluidic cell array to include these crucial players. This system consists of a three-layered cell array (μFCA) spatially emulating the TME, with tailored fluidic circuits establishing T cell recirculation. This platform enables the study of dynamics among the TME, immune cells in a circulatory system and cancer cell responses thereof. Through this system, we found that tumour endothelium hindered T cell infiltration into the reconstructed breast cancer tumour compartment. This negative effect was alleviated when treated with anti-human PD-L1 (programmed cell death ligand 1) antibody. Another key stromal component – cancer associated fibroblasts – attenuated T cell infiltration, compared against normal fibroblasts, and led to reduced apoptotic activity in cancer cells. These results confirm the capability of our tumour-on-a-chip system in identifying some key axes to target in overcoming barriers to immunotherapy by recapitulating immune cell interactions with the reconstructed TME. Our results also attest to the feasibility of scaling up this system for high-throughput cancer immunotherapeutic screening. [ABSTRACT FROM AUTHOR]

Abstract: A new quantum dot (QD)-aptamer (apt) beacon that acts by folding-induced dissociation of a DNA intercalating dye, BOBO-3(B), is demonstrated with label-free thrombin detection. The beacon, denoted as QD-apt:B, is constructed by (1) coupling of a single-stranded thrombin aptamer to Qdot 565 via EDC/Sulfo-NHS chemistry and (2) staining the duplex regions of the aptamer on QD with excess BOBO-3 before thrombin binding. When mixing a thrombin sample with QD-apt:B, BOBO-3 is competed away from the beacon due to target-induced aptamer folding, which then causes a decrease in QD fluorescence resonance energy transfer (FRET)-mediated BOBO-3 emission and achieves thrombin quantitation. In this work, the effects of Mg2+, coupling time, and aptamer type on the beacon''s performances are investigated and discussed thoroughly with various methods, including transmission electron microscopy (TEM), dynamic light scattering (DLS), and two-color differential gel electrophoresis. Using the best aptamer beacon (HTQ37), we attain highly specific and wide-range detection (from nM to μM) of thrombin in buffer, and the beacon can sense nM-range thrombin in 15% diluted serum. Compared to the reported QD aptamer assays, our method is advantageous from the aspect of using a simple sensory unit design without losing the detection sensitivity. Therefore, we consider the QD-apt:B beacon a potential alternative to immuno-reagents and an effective tool to study nucleic acid folding on QD as well. [Copyright &y& Elsevier]

Chronic hepatitis B infection remains incurable due to the stable presence of various forms of hepatitis B virus (HBV) genome, especially the HBV covalently closed circular DNA (cccDNA). The emergence of clustered regularly interspaced short palindromic repeat (CRISPR) technology provides a new opportunity to potentially cure the HBV infection. However, the efficiency and specificity remain unsatisfactory, especially for nonviral CRISPR/Cas9 delivery. To tackle these, a liver‐specific CRISPR/Cas9 magnetic nanosystem FMNPpAG333/sgXPP is constructed based on fluorinated polyethylenimine‐coated magnetic nanoparticles and liver‐specific ApoE.HCR.hAAT promoter‐driven Cas9‐T2A‐EGFP plasmid with dual sgRNAs. The elaborate system enables magnetic field‐induced spatially specific distribution and hepatocyte‐specific promoter‐driven liver‐specific gene editing. Moreover, this CRISPR/Cas9 magnetic nanosystem is designed to disrupt the two conserved sites in X opening reading frame and Pol opening reading frame of the HBV genome, thereby significantly inactivating the HBV genome without showing off‐target effects. Treatment with FMNPpAG333/sgXPP for 7 days reduces serum HBsAg levels by 76% with a total editing efficiency of ≈20% in the two conserved sites. Collectively, this study demonstrates spatiotemporal liver genome editing as well as the feasibility of applying a nonviral CRISPR/Cas9 vector for HBV treatment, which may open up a new application for CRISPR therapeutics. [ABSTRACT FROM AUTHOR]

For patients carrying BRCA1 mutations, at least one‐third develop triple negative breast cancer (TNBC). Not only is TNBC difficult to treat due to the lack of molecular target receptors, but BRCA1 mutations (BRCA1m) also result in chemotherapeutic resistance, making disease recurrence more likely. Although BRCA1m are highly heterogeneous and therefore difficult to target, BRCA1 gene's synthetic lethal pair, PARP1, is conserved in BRCA1m cancer cells. Therefore, we hypothesize that targeting PARP1 might be a fruitful direction to sensitize BRCA1m cancer cells to chemotherapy. We used CRISPR/Cas9 technology to generate PARP1 deficiency in two TNBC cell lines, MDA‐MB‐231 (BRCA1 wild‐type) and MDA‐MB‐436 (BRCA1m). We explored whether this PARP1 disruption (PARP1m) could significantly lower the chemotherapeutic dose necessary to achieve therapeutic efficacy in both a 2D and 3D tumor‐on‐a‐chip model. With both BRCA1m and PARP1m, the TNBC cells were more sensitive to three representative chemotherapeutic breast cancer drugs, doxorubicin, gemcitabine and docetaxel, compared with the PARP1 wild‐type counterpart in the 2D culture environment. However, PARP1m did not result in this synergy in the 3D tumor‐on‐a‐chip model, suggesting that drug dosing in the tumor microenvironment may influence the synergy. Taken together, our results highlight a discrepancy in the efficacy of the combination of PARP1 inhibition and chemotherapy for TNBC treatment, which should be clarified to justify further clinical testing. [ABSTRACT FROM AUTHOR]

Abstract: CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics. [ABSTRACT FROM AUTHOR]

Keywords: CRISPR/Cas9; gene editing; hepatitis B virus (HBV); liver-specific promoters; magnetic nanosystems EN CRISPR/Cas9 gene editing hepatitis B virus (HBV) liver-specific promoters magnetic nanosystems 1 1 1 02/13/23 20230209 NES 230209 B Spatiotemporal Liver Genome Editing b In article number 2210860, Mingqiang Li, Yu Tao, Yeh-Hsing Lao, and co-workers report spatiotemporally-controlled gene editing to inactivate hepatitis B infection based on magnetic nanoparticles and liver-specific promoter-driven CRISPR/Cas9 plasmid. CRISPR/Cas9, gene editing, hepatitis B virus (HBV), liver-specific promoters, magnetic nanosystems Magnetic-Activated Nanosystem with Liver-Specific CRISPR Nonviral Vector to Achieve Spatiotemporal Liver Genome Editing as Hepatitis B Therapeutics (Adv. Funct. [Extracted from the article]