bims-drudre Biomed News
on Targeted drug delivery and programmed release mechanisms
Issue of 2022‒12‒04
eighteen papers selected by
Ceren Kimna
Technical University of Munich
  1. Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.
  2. Matrix viscoelasticity controls spatiotemporal tissue organization.
  3. Selective targeting of visceral adiposity by polycation nanomedicine.
  4. Trapping of protein cargo molecules inside DNA origami nanocages.
  5. Engineered Phage with Aggregation-Induced Emission Photosensitizer in Cocktail Therapy against Sepsis.
  6. Complex Living Materials made by Light-based Printing of Genetically Programmed Bacteria.
  7. Overcoming the Blood-Brain Barrier for Gene Therapy via Systemic Administration of GSH-Responsive Silica Nanocapsules.
  8. In Response to Precision Medicine: Current Subcellular Targeting Strategies for Cancer Therapy.
  9. A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance.
  10. Single Low-Dose Nanovaccine for Long-Term Protection against Anthrax Toxins.
  11. Immunosuppressive biomaterial-based therapeutic vaccine to treat multiple sclerosis via re-establishing immune tolerance.
  12. Lipid Giant Vesicles Engulf Living Bacteria Triggered by Minor Enhancement in Membrane Fluidity.
  13. DNA-Encoded Libraries: Towards Harnessing their Full Power with Darwinian Evolution.
  14. A self-powered ingestible wireless biosensing system for real-time in situ monitoring of gastrointestinal tract metabolites.
  15. Scaffold-free Tracheal Engineering via a Modular Strategy Based on Cartilage and Epithelium Sheets.
  16. Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.
  17. Modular stimuli-responsive hydrogel sealants for early gastrointestinal leak detection and containment.
  18. A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex.
  1. Nat Mater. 2022 Nov 28.
    Species-agnostic polymeric formulations for inhalable messenger RNA delivery to the lung.
    Laura Rotolo, Daryll Vanover, Nicholas C Bruno, Hannah E Peck, Chiara Zurla, Jackelyn Murray, Richard K Noel, Laura O'Farrell, Mariluz Araínga, Nichole Orr-Burks, Jae Yeon Joo, Lorena C S Chaves, Younghun Jung, Jared Beyersdorf, Sanjeev Gumber, Ricardo Guerrero-Ferreira, Santiago Cornejo, Merrilee Thoresen, Alicia K Olivier, Katie M Kuo, James C Gumbart, Amelia R Woolums, Francois Villinger, Eric R Lafontaine, Robert J Hogan, M G Finn, Philip J Santangelo.
      Messenger RNA has now been used to vaccinate millions of people. However, the diversity of pulmonary pathologies, including infections, genetic disorders, asthma and others, reveals the lung as an important organ to directly target for future RNA therapeutics and preventatives. Here we report the screening of 166 polymeric nanoparticle formulations for functional delivery to the lungs, obtained from a combinatorial synthesis approach combined with a low-dead-volume nose-only inhalation system for mice. We identify P76, a poly-β-amino-thio-ester polymer, that exhibits increased expression over formulations lacking the thiol component, delivery to different animal species with varying RNA cargos and low toxicity. P76 allows for dose sparing when delivering an mRNA-expressed Cas13a-mediated treatment in a SARS-CoV-2 challenge model, resulting in similar efficacy to a 20-fold higher dose of a neutralizing antibody. Overall, the combinatorial synthesis approach allowed for the discovery of promising polymeric formulations for future RNA pharmaceutical development for the lungs.
    DOI:  https://doi.org/10.1038/s41563-022-01404-0
  2. Nat Mater. 2022 Dec 01.
    Matrix viscoelasticity controls spatiotemporal tissue organization.
    Alberto Elosegui-Artola, Anupam Gupta, Alexander J Najibi, Bo Ri Seo, Ryan Garry, Christina M Tringides, Irene de Lázaro, Max Darnell, Wei Gu, Qiao Zhou, David A Weitz, L Mahadevan, David J Mooney.
      Biomolecular and physical cues of the extracellular matrix environment regulate collective cell dynamics and tissue patterning. Nonetheless, how the viscoelastic properties of the matrix regulate collective cell spatial and temporal organization is not fully understood. Here we show that the passive viscoelastic properties of the matrix encapsulating a spheroidal tissue of breast epithelial cells guide tissue proliferation in space and in time. Matrix viscoelasticity prompts symmetry breaking of the spheroid, leading to the formation of invading finger-like protrusions, YAP nuclear translocation and epithelial-to-mesenchymal transition both in vitro and in vivo in a Arp2/3-complex-dependent manner. Computational modelling of these observations allows us to establish a phase diagram relating morphological stability with matrix viscoelasticity, tissue viscosity, cell motility and cell division rate, which is experimentally validated by biochemical assays and in vitro experiments with an intestinal organoid. Altogether, this work highlights the role of stress relaxation mechanisms in tissue growth dynamics, a fundamental process in morphogenesis and oncogenesis.
    DOI:  https://doi.org/10.1038/s41563-022-01400-4
  3. Nat Nanotechnol. 2022 Dec 01.
    Selective targeting of visceral adiposity by polycation nanomedicine.
    Qianfen Wan, Baoding Huang, Tianyu Li, Yang Xiao, Ying He, Wen Du, Branden Z Wang, Gregory F Dakin, Michael Rosenbaum, Marcus D Goncalves, Shuibing Chen, Kam W Leong, Li Qiang.
      Obesity is a pandemic health problem with poor solutions, especially for targeted treatment. Here we develop a polycation-based nanomedicine polyamidoamine generation 3 (P-G3) that-when delivered intraperitoneally-selectively targets visceral fat due to its high charge density. Moreover, P-G3 treatment of obese mice inhibits visceral adiposity, increases energy expenditure, prevents obesity and alleviates the associated metabolic dysfunctions. In vitro adipogenesis models and single-cell RNA sequencing revealed that P-G3 uncouples adipocyte lipid synthesis and storage from adipocyte development to create adipocytes that possess normal functions but are deficient in hypertrophic growth, at least through synergistically modulating nutrient-sensing signalling pathways. The visceral fat distribution of P-G3 is enhanced by modifying P-G3 with cholesterol to form lipophilic nanoparticles, which is effective in treating obesity. Our study highlights a strategy to target visceral adiposity and suggests that cationic nanomaterials could be exploited for treating metabolic diseases.
    DOI:  https://doi.org/10.1038/s41565-022-01249-3
  4. Nanoscale. 2022 Nov 29.
    Trapping of protein cargo molecules inside DNA origami nanocages.
    Merle Scherf, Florian Scheffler, Christopher Maffeo, Ulrich Kemper, Jingjing Ye, Aleksei Aksimentiev, Ralf Seidel, Uta Reibetanz.
      The development of the DNA origami technique has directly inspired the idea of using three-dimensional DNA cages for the encapsulation and targeted delivery of drug or cargo molecules. The cages would be filled with molecules that would be released at a site of interest upon cage opening triggered by an external stimulus. Though different cage variants have been developed, efficient loading of DNA cages with freely-diffusing cargo molecules that are not attached to the DNA nanostructure and their efficient retention within the cages has not been presented. Here we address these challenges using DNA origami nanotubes formed by a double-layer of DNA helices that can be sealed with tight DNA lids at their ends. In a first step we attach DNA-conjugated cargo proteins to complementary target strands inside the DNA tubes. After tube sealing, the cargo molecules are released inside the cavity using toehold-mediated strand displacement by externally added invader strands. We show that DNA invaders are rapidly entering the cages through their DNA walls. Retention of ∼70 kDa protein cargo molecules inside the cages was, however, poor. Guided by coarse-grained simulations of the DNA cage dynamics, a tighter sealing of the DNA tubes was developed which greatly reduced the undesired escape of cargo proteins. These improved DNA nanocages allow for efficient encapsulation of medium-sized cargo molecules while remaining accessible to small molecules that can be used to trigger reactions, including a controlled release of the cargo via nanocage opening.
    DOI:  https://doi.org/10.1039/d2nr05356j
  5. Adv Mater. 2022 Nov 28. e2208578
    Engineered Phage with Aggregation-Induced Emission Photosensitizer in Cocktail Therapy against Sepsis.
    Ming-Yu Wu, Luojia Chen, Qingrong Chen, Rui Hu, Xiaoyu Xu, Yifei Wang, Jie Li, Shun Feng, Changjiang Dong, Xiao-Lian Zhang, Zhiqiang Li, Lianrong Wang, Shi Chen, Meijia Gu.
      Sepsis, a widely recognized disease, is characterized by multiple pathogen infections. Therefore, it is imperative to develop methods that can efficiently identify and neutralize pathogen species. Phage cocktail therapy utilizes the host specificity of phages to adapt to infect resistant bacteria. However, its low sterilization stability efficiency and lack of imaging units seriously restrict its application. Here, we propose a novel strategy combining the aggregation-induced emission photosensitizer (AIE-PS) TBTCP-PMB with phages through a nucleophilic substitution reaction between benzyl bromide and sulfhydryl groups to remove pathogenic bacteria for sepsis treatment. This strategy retains the phage's host specificity while possessing AIE-PS characteristics with a fluorescence imaging function and reactive-oxygen-species (ROS) for detecting and sterilizing bacteria. This synergetic strategy combining phage cocktail therapy and photodynamic therapy (PDT) showed a strong "1+1>2" bactericidal efficacy and superior performance in sepsis mouse models with good biocompatibility. Furthermore, the strategy can quickly diagnose blood infections of clinical blood samples. This simple and accurate strategy provides a promising therapeutic platform for rapid pathogen detection and point-of-care diagnosis. Moreover, it presents a new method for expanding the library of antibacterial drugs to develop new strain identification and improve infectious disease treatment, thereby demonstrating strong translational potential. This article is protected by copyright. All rights reserved.
    Keywords:  Sepsis; aggregation-induced emission; phage cocktail therapy; photodynamic therapy; photosensitizers
    DOI:  https://doi.org/10.1002/adma.202208578
  6. Adv Mater. 2022 Nov 29. e2207483
    Complex Living Materials made by Light-based Printing of Genetically Programmed Bacteria.
    Marco R Binelli, Anton Kan, Luis E A Rozas, Giovanni Pisaturo, Namita Prakash, André R Studart.
      Living materials with embedded microorganisms can genetically encode attractive sensing, self-repairing and responsive functionalities for applications in medicine, robotics and infrastructure. While the synthetic toolbox for genetically engineering bacteria continues to expand, technologies to shape bacteria-laden living materials into complex three-dimensional geometries are still rather limited. Here, we show that bacteria-laden hydrogels can be shaped into living materials with unusual architectures and functionalities using readily available light-based printing techniques. Bioluminescent and melanin-producing bacteria are used to create complex materials with autonomous chemical-sensing capabilities by harnessing the metabolic activity of wild-type and engineered microorganisms. The shaping freedom offered by printing technologies and the rich biochemical diversity available in bacteria provides ample design space for the creation and exploration of complex living materials with programmable functionalities for a broad range of applications. This article is protected by copyright. All rights reserved.
    Keywords:  DLP printing; engineered living materials; printed living materials; volumetric printing
    DOI:  https://doi.org/10.1002/adma.202207483
  7. Adv Mater. 2022 Nov 29. e2208018
    Overcoming the Blood-Brain Barrier for Gene Therapy via Systemic Administration of GSH-Responsive Silica Nanocapsules.
    Yuyuan Wang, Xiuxiu Wang, Ruosen Xie, Jacobus C Burger, Yao Tong, Shaoqin Gong.
      CRISPR genome editing has demonstrated great potential to treat the root causes of many genetic diseases, including central nervous system (CNS) disorders. However, the promise of brain-targeted therapeutic genome editing relies on the efficient delivery of biologics bypassing the blood-brain barrier (BBB), which represents a substantial challenge in the development of CRISPR therapeutics. In this study, we created a library of GSH-responsive silica nanocapsules (SNCs) and screened them for brain targeting via systemic delivery of nucleic acids and CRISPR genome editors. In vivo studies demonstrated that systemically delivered SNCs conjugated with glucose and RVG peptide under glycemic control can efficiently bypass the intact BBB, enabling brain-wide delivery of various biologics (mRNA, Cas9 mRNA/sgRNA, and Cas9/sgRNA ribonucleoprotein) targeting both exogenous genes (i.e., Ai14 stop cassette) and disease-relevant endogenous genes (i.e., App and Th genes) in Ai14 reporter mice and wild-type mice, respectively. In particular, we observed up to 28% neuron editing via systemic delivery of Cre mRNA in Ai14 mice, up to 6.1% amyloid precursor protein (App) gene editing (resulting in 19.1% reduction in the expression level of intact APP), and up to 3.9% tyrosine hydroxylase (Th) gene editing (resulting in 30.3% reduction in the expression level of TH) in wild-type mice. This versatile SNC nanoplatform may offer a novel strategy for the treatment of CNS disorders including Alzheimer's, Parkinson's, and Huntington's disease. This article is protected by copyright. All rights reserved.
    Keywords:  CRISPR gene editing; blood-brain barrier; brain-wide delivery; mRNA; silica nanocapsule
    DOI:  https://doi.org/10.1002/adma.202208018
  8. Adv Mater. 2022 Nov 29. e2209529
    In Response to Precision Medicine: Current Subcellular Targeting Strategies for Cancer Therapy.
    Zheng Li, Jianhua Zou, Xiaoyuan Chen.
      Emerging as a potent anti-cancer treatment, subcellular targeted cancer therapy has drawn increasing attention, bringing great opportunities for clinical application. In this review, we summarize two targeting strategies for four main subcellular organelles (mitochondria, lysosome, endoplasmic reticulum, and nucleus), including molecule and nanomaterial (inorganic nanoparticles, micelles, organic polymers, and others)-based targeted delivery or therapeutic strategies. Phototherapy, chemotherapy, radiotherapy, immunotherapy, and "all-in-one" combination therapy are among the strategies covered in detail. Such materials have been constructed based on the specific properties and relevant mechanisms of organelles, enabling the elimination of tumors by inducing dysfunction in the corresponding organelles or destroying specific structures. The challenges faced by organelle-targeting cancer therapies are also summarized. Looking forward, we envision a paradigm for organelle-targeting therapy with enhanced therapeutic efficacy compared to current clinical approaches. This article is protected by copyright. All rights reserved.
    Keywords:  cancer therapy; molecules; nanomaterials; precision medicine; subcellular organelle targeting
    DOI:  https://doi.org/10.1002/adma.202209529
  9. Nat Commun. 2022 Nov 30. 13(1): 7361
    A nuclease-mimetic platinum nanozyme induces concurrent DNA platination and oxidative cleavage to overcome cancer drug resistance.
    Fangyuan Li, Heng Sun, Jiafeng Ren, Bo Zhang, Xi Hu, Chunyan Fang, Jiyoung Lee, Hongzhou Gu, Daishun Ling.
      Platinum (Pt) resistance in cancer almost inevitably occurs during clinical Pt-based chemotherapy. The spontaneous nucleotide-excision repair of cancer cells is a representative process that leads to Pt resistance, which involves the local DNA bending to facilitate the recruitment of nucleotide-excision repair proteins and subsequent elimination of Pt-DNA adducts. By exploiting the structural vulnerability of this process, we herein report a nuclease-mimetic Pt nanozyme that can target cancer cell nuclei and induce concurrent DNA platination and oxidative cleavage to overcome Pt drug resistance. We show that the Pt nanozyme, unlike cisplatin and conventional Pt nanoparticles, specifically induces the nanozyme-catalyzed cleavage of the formed Pt-DNA adducts by generating in situ reactive oxygen species, which impairs the damage recognition factors-induced DNA bending prerequisite for nucleotide-excision repair. The recruitment of downstream effectors of nucleotide-excision repair to DNA lesion sites, including xeroderma pigmentosum groups A and F, is disrupted by the Pt nanozyme in cisplatin-resistant cancer cells, allowing excessive accumulation of the Pt-DNA adducts for highly efficient cancer therapy. Our study highlights the potential benefits of applying enzymatic activities to the use of the Pt nanomedicines, providing a paradigm shift in DNA damaging chemotherapy.
    DOI:  https://doi.org/10.1038/s41467-022-35022-w
  10. Nano Lett. 2022 Nov 30.
    Single Low-Dose Nanovaccine for Long-Term Protection against Anthrax Toxins.
    Maya Holay, Nishta Krishnan, Jiarong Zhou, Yaou Duan, Zhongyuan Guo, Weiwei Gao, Ronnie H Fang, Liangfang Zhang.
      Anthrax infections caused by Bacillus anthracis are an ongoing bioterrorism and livestock threat worldwide. Current approaches for management, including extended passive antibody transfusion, antibiotics, and prophylactic vaccination, are often cumbersome and associated with low patient compliance. Here, we report on the development of an adjuvanted nanotoxoid vaccine based on macrophage membrane-coated nanoparticles bound with anthrax toxins. This design leverages the natural binding interaction of protective antigen, a key anthrax toxin, with macrophages. In a murine model, a single low-dose vaccination with the nanotoxoids generates long-lasting immunity that protects against subsequent challenge with anthrax toxins. Overall, this work provides a new approach to address the ongoing threat of anthrax outbreaks and bioterrorism by taking advantage of an emerging biomimetic nanotechnology.
    Keywords:  anthrax; antivirulence vaccine; biomimetic nanoparticle; cell membrane coating; nanotoxoid
    DOI:  https://doi.org/10.1021/acs.nanolett.2c03881
  11. Nat Commun. 2022 Dec 02. 13(1): 7449
    Immunosuppressive biomaterial-based therapeutic vaccine to treat multiple sclerosis via re-establishing immune tolerance.
    Thanh Loc Nguyen, Youngjin Choi, Jihye Im, Hyunsu Shin, Ngoc Man Phan, Min Kyung Kim, Seung Woo Choi, Jaeyun Kim.
      Current therapies for autoimmune diseases, such as multiple sclerosis (MS), induce broad suppression of the immune system, potentially promoting opportunistic infections. Here, we report an immunosuppressive biomaterial-based therapeutic vaccine carrying self-antigen and tolerance-inducing inorganic nanoparticles to treat experimental autoimmune encephalomyelitis (EAE), a mouse model mimicking human MS. Immunization with self-antigen-loaded mesoporous nanoparticles generates Foxp3+ regulatory T-cells in spleen and systemic immune tolerance in EAE mice, reducing central nervous system-infiltrating antigen-presenting cells (APCs) and autoreactive CD4+ T-cells. Introducing reactive oxygen species (ROS)-scavenging cerium oxide nanoparticles (CeNP) to self-antigen-loaded nanovaccine additionally suppresses activation of APCs and enhances antigen-specific immune tolerance, inducing recovery in mice from complete paralysis at the late, chronic stage of EAE, which shows similarity to chronic human MS. This study clearly shows that the ROS-scavenging capability of catalytic inorganic nanoparticles could be utilized to enhance tolerogenic features in APCs, leading to antigen-specific immune tolerance, which could be exploited in treating MS.
    DOI:  https://doi.org/10.1038/s41467-022-35263-9
  12. Nano Lett. 2022 Nov 28.
    Lipid Giant Vesicles Engulf Living Bacteria Triggered by Minor Enhancement in Membrane Fluidity.
    Shaoying Dai, Xiaoyu Tang, Na Zhang, Haofei Li, Chengzhi He, Yuchun Han, Yilin Wang.
      Antibacterial amphiphiles normally kill bacteria by destroying the bacterial membrane. Whether and how antibacterial amphiphiles alter normal cell membrane and lead to subsequent effects on pathogen invasion into cells have been scarcely promulgated. Herein, by taking four antibacterial gemini amphiphiles with different spacer groups to modulate cell-mimic phospholipid giant unilamellar vesicles (GUVs), bacteria adhesion on the modified GUVs surface and bacteria engulfment process by the GUVs are clearly captured by confocal laser scanning microscopy. Further characterization shows that the enhanced cationic surface charge of GUVs by the amphiphiles determines the bacteria adhesion amount, while the involvement of amphiphile in GUVs results in looser molecular arrangement and concomitant higher fluidity in the bilayer membranes, facilitating the bacteria intruding into GUVs. This study sheds new light on the effect of amphiphiles on membrane bilayer and the concurrent effect on pathogen invasion into cell mimics and broadens the nonprotein-mediated endocytosis pathway for live bacteria.
    Keywords:  bacteria; engulfment; gemini amphiphile; giant unilamellar vesicle; membrane fluidity
    DOI:  https://doi.org/10.1021/acs.nanolett.2c03475
  13. Angew Chem Int Ed Engl. 2022 Dec 02.
    DNA-Encoded Libraries: Towards Harnessing their Full Power with Darwinian Evolution.
    Millicent Dockerill, Nicolas Winssinger.
      DNA-encoded library (DEL) technologies are transforming the drug discovery process, enabling the identification of ligands at unprecedented speed and scale. DEL makes use of libraries that are orders of magnitude larger than traditional high throughput screens. While a DNA tag alludes to a genotype-phenotype connection that is exploitable for evolutionary cycles, most of the work in the field is performed with libraries where the tag serves as an amplifiable bar code but does not allow 'translation' into the synthetic product it is linked to. In this review, we cover technologies that enable the 'translation' of the genetic tag into synthetic molecules, both biochemically and chemically, and explore how it can be used to harness Darwinian evolutionary pressure.
    Keywords:  DNA encoded libraries, Combinatorial Chemistry, Self-assembly, Supramolecular Chemistry, Darwinian evolution
    DOI:  https://doi.org/10.1002/anie.202215542
  14. Nat Commun. 2022 Dec 01. 13(1): 7405
    A self-powered ingestible wireless biosensing system for real-time in situ monitoring of gastrointestinal tract metabolites.
    Ernesto De la Paz, Nikhil Harsha Maganti, Alexander Trifonov, Itthipon Jeerapan, Kuldeep Mahato, Lu Yin, Thitaporn Sonsa-Ard, Nicolas Ma, Won Jung, Ryan Burns, Amir Zarrinpar, Joseph Wang, Patrick P Mercier.
      Information related to the diverse and dynamic metabolite composition of the small intestine is crucial for the diagnosis and treatment of various diseases. However, our current understanding of the physiochemical dynamics of metabolic processes within the small intestine is limited due to the lack of in situ access to the intestinal environment. Here, we report a demonstration of a battery-free ingestible biosensing system for monitoring metabolites in the small intestine. As a proof of concept, we monitor the intestinal glucose dynamics on a porcine model. Battery-free operation is achieved through a self-powered glucose biofuel cell/biosensor integrated into a circuit that performs energy harvesting, biosensing, and wireless telemetry via a power-to-frequency conversion scheme using magnetic human body communication. Such long-term biochemical analysis could potentially provide critical information regarding the complex and dynamic small intestine metabolic profiles.
    DOI:  https://doi.org/10.1038/s41467-022-35074-y
  15. Adv Healthc Mater. 2022 Dec 02. e2202022
    Scaffold-free Tracheal Engineering via a Modular Strategy Based on Cartilage and Epithelium Sheets.
    Minglei Yang, Jiafei Chen, Yi Chen, Weikang Lin, Hai Tang, Ziwen Fan, Long Wang, Yunlang She, Feng Jin, Lei Zhang, Weiyan Sun, Chang Chen.
      Tracheal defects lead to devastating problems, and practical clinical substitutes that have complex functional structures and can avoid adverse influences from exogenous bioscaffolds are lacking. Herein, a modular strategy for scaffold-free tracheal engineering was developed. A cartilage sheet (Cart-S) prepared by high-density culture was laminated and reshaped to construct a cartilage tube as the main load-bearing structure in which the chondrocytes exhibited a stable phenotype and secreted considerable cartilage-specific matrix, presenting a native-like grid arrangement. To further build a tracheal epithelial barrier, a temperature-sensitive technique was used to construct the monolayer epithelium sheet (Epi-S), in which the airway epithelial cells presented integrated tight junctions, good transepithelial electrical resistance, and favorable ciliary differentiation capability. Epi-S could be integrally transferred to inner wall of cartilage tube, forming a scaffold-free complex tracheal substitute (SC-trachea). Interestingly, when Epi-S was attached to the cartilage surface, epithelium-specific gene expression was significantly enhanced. SC-trachea established abundant blood supply via heterotopic vascularization and then was pedicle transplanted for tracheal reconstruction, achieving 83.3% survival outcomes in rabbit models. Notably, the scaffold-free engineered trachea simultaneously satisfied sufficient mechanical properties and barrier function due to its matrix-rich cartilage structure and well-differentiated ciliated epithelium, demonstrating great clinical potential for long-segmental tracheal reconstruction. This article is protected by copyright. All rights reserved.
    Keywords:  Cartilage regeneration; Epithelium regeneration; Scaffold-free; Tissue engineering; Tracheal reconstruction
    DOI:  https://doi.org/10.1002/adhm.202202022
  16. Nat Commun. 2022 Nov 30. 13(1): 7391
    Self-cleaving guide RNAs enable pharmacological selection of precise gene editing events in vivo.
    Amita Tiyaboonchai, Anne Vonada, Jeffrey Posey, Carl Pelz, Leslie Wakefield, Markus Grompe.
      Expression of guide RNAs in the CRISPR/Cas9 system typically requires the use of RNA polymerase III promoters, which are not cell-type specific. Flanking the gRNA with self-cleaving ribozyme motifs to create a self-cleaving gRNA overcomes this limitation. Here, we use self-cleaving gRNAs to create drug-selectable gene editing events in specific hepatocyte loci. A recombinant Adeno Associated Virus vector targeting the Albumin locus with a promoterless self-cleaving gRNA to create drug resistance is linked in cis with the therapeutic transgene. Gene expression of both are dependent on homologous recombination into the target locus. In vivo drug selection for the precisely edited hepatocytes allows >30-fold expansion of gene-edited cells and results in therapeutic levels of a human Factor 9 transgene. Importantly, self-cleaving gRNA expression is also achieved after targeting weak hepatocyte genes. We conclude that self-cleaving gRNAs are a powerful system to enable cell-type specific in vivo drug resistance for therapeutic gene editing applications.
    DOI:  https://doi.org/10.1038/s41467-022-35097-5
  17. Nat Commun. 2022 Nov 27. 13(1): 7311
    Modular stimuli-responsive hydrogel sealants for early gastrointestinal leak detection and containment.
    Alexandre H C Anthis, Maria Paulene Abundo, Anna L Neuer, Elena Tsolaki, Jachym Rosendorf, Thomas Rduch, Fabian H L Starsich, Bernhard Weisse, Vaclav Liska, Andrea A Schlegel, Mikhail G Shapiro, Inge K Herrmann.
      Millions of patients every year undergo gastrointestinal surgery. While often lifesaving, sutured and stapled reconnections leak in around 10% of cases. Currently, surgeons rely on the monitoring of surrogate markers and clinical symptoms, which often lack sensitivity and specificity, hence only offering late-stage detection of fully developed leaks. Here, we present a holistic solution in the form of a modular, intelligent suture support sealant patch capable of containing and detecting leaks early. The pH and/or enzyme-responsive triggerable sensing elements can be read out by point-of-need ultrasound imaging. We demonstrate reliable detection of the breaching of sutures, in as little as 3 hours in intestinal leak scenarios and 15 minutes in gastric leak conditions. This technology paves the way for next-generation suture support materials that seal and offer disambiguation in cases of anastomotic leaks based on point-of-need monitoring, without reliance on complex electronics or bulky (bio)electronic implantables.
    DOI:  https://doi.org/10.1038/s41467-022-34272-y
  18. Nat Commun. 2022 Dec 03. 13(1): 7460
    A quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex.
    Marius Rutkauskas, Inga Songailiene, Patrick Irmisch, Felix E Kemmerich, Tomas Sinkunas, Virginijus Siksnys, Ralf Seidel.
      CRISPR-Cas effector complexes recognise nucleic acid targets by base pairing with their crRNA which enables easy re-programming of the target specificity in rapidly emerging genome engineering applications. However, undesired recognition of off-targets, that are only partially complementary to the crRNA, occurs frequently and represents a severe limitation of the technique. Off-targeting lacks comprehensive quantitative understanding and prediction. Here, we present a detailed analysis of the target recognition dynamics by the Cascade surveillance complex on a set of mismatched DNA targets using single-molecule supercoiling experiments. We demonstrate that the observed dynamics can be quantitatively modelled as a random walk over the length of the crRNA-DNA hybrid using a minimal set of parameters. The model accurately describes the recognition of targets with single and double mutations providing an important basis for quantitative off-target predictions. Importantly the model intrinsically accounts for observed bias regarding the position and the proximity between mutations and reveals that the seed length for the initiation of target recognition is controlled by DNA supercoiling rather than the Cascade structure.
    DOI:  https://doi.org/10.1038/s41467-022-35116-5
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