scholarly journals Extracellular vesicles engineered with valency-controlled DNA nanostructures deliver CRISPR/Cas9 system for gene therapy

2020 ◽  
Vol 48 (16) ◽  
pp. 8870-8882 ◽  
Author(s):  
Jialang Zhuang ◽  
Jizhou Tan ◽  
Chenglin Wu ◽  
Jie Zhang ◽  
Ting Liu ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Primary Liver Cancer ◽  
The Body ◽  
Dna Aptamer ◽  
Great Promise ◽  
Tumor Growth Inhibition ◽  
Specific Cell ◽  
Dna Nanostructures

Abstract Extracellular vesicles (EVs) hold great promise for transporting CRISPR–Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.

The Role of Polyphenols in the Modulation of Plasma Non-Enzymatic Antioxidant Capacity (NEAC)

2012 ◽  
Vol 82 (3) ◽  
pp. 228-232 ◽  
Author(s):  
Mauro Serafini ◽  
Giuseppa Morabito
Keyword(s):  
Antioxidant Capacity ◽  
Dietary Intervention ◽  
Ex Vivo ◽  
The Body ◽  
Dietary Polyphenols ◽  
Enzymatic Antioxidant ◽  
Endogenous Antioxidant

Dietary polyphenols have been shown to scavenge free radicals, modulating cellular redox transcription factors in different in vitro and ex vivo models. Dietary intervention studies have shown that consumption of plant foods modulates plasma Non-Enzymatic Antioxidant Capacity (NEAC), a biomarker of the endogenous antioxidant network, in human subjects. However, the identification of the molecules responsible for this effect are yet to be obtained and evidences of an antioxidant in vivo action of polyphenols are conflicting. There is a clear discrepancy between polyphenols (PP) concentration in body fluids and the extent of increase of plasma NEAC. The low degree of absorption and the extensive metabolism of PP within the body have raised questions about their contribution to the endogenous antioxidant network. This work will discuss the role of polyphenols from galenic preparation, food extracts, and selected dietary sources as modulators of plasma NEAC in humans.

scholarly journals Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Microbiome ◽  
2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Leeann Klassen ◽  
Greta Reintjes ◽  
Jeffrey P. Tingley ◽  
Darryl R. Jones ◽  
Jan-Hendrik Hehemann ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Ex Vivo ◽  
Rapid Assessment ◽  
Vital Role ◽  
Strain Level ◽  
Metabolic Phenotype ◽  
Specific Cell ◽  
Bacterial Cells ◽  
Carbohydrate Utilization

AbstractGut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

scholarly journals 695 Oral delivery of a microbial extracellular vesicle induces potent anti-tumor immunity in mice

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A737-A737
Author(s):  
Loise Francisco-Anderson ◽  
Loise Francisco-Anderson ◽  
Mary Abdou ◽  
Michael Goldberg ◽  
Erin Troy ◽  
...  
Keyword(s):  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Extracellular Vesicles ◽  
Tumor Immunity ◽  
Extracellular Vesicle ◽  
The Body ◽  
Checkpoint Inhibition ◽  
Small Intestinal ◽  
The Impact

BackgroundThe small intestinal axis (SINTAX) is a network of anatomic and functional connections between the small intestine and the rest of the body. It acts as an immunosurveillance system, integrating signals from the environment that affect physiological processes throughout the body. The impact of events in the gut in the control of tumor immunity is beginning to be appreciated. We have previously shown that an orally delivered single strain of commensal bacteria induces anti-tumor immunity preclinically via pattern recognition receptor-mediated activation of innate and adaptive immunity. Some bacteria produce extracellular vesicles (EVs) that share molecular content with the parent bacterium in a particle that is roughly 1/1000th the volume in a non-replicating form. We report here an orally-delivered and gut-restricted bacterial EV which potently attenuates tumor growth to a greater extent than whole bacteria or checkpoint inhibition.MethodsEDP1908 is a preparation of extracellular vesicles produced by a gram-stain negative strain of bacterium of the Oscillospiraceae family isolated from a human donor. EDP1908 was selected for its immunostimulatory profile in a screen of EVs from a range of distinct microbial strains. Its mechanism of action was determined by ex vivo analysis of the tumor microenvironment (TME) and by in vitro functional studies with murine and human cells.ResultsOral treatment of tumor-bearing mice with EDP1908 shows superior control of tumor growth compared to checkpoint inhibition (anti-PD-1) or an intact microbe. EDP1908 significantly increased the percentage of IFNγ and TNF producing CD8+ CTLs, NK cells, NKT cells and CD4+ cells in the tumor microenvironment (TME). EDP1908 also increased tumor-infiltrating dendritic cells (DC1 and DC2). Analysis of cytokines in the TME showed significant increases in IP-10 and IFNg production in mice treated with EDP1908, creating an environment conducive to the recruitment and activation of anti-tumor lymphocytes.ConclusionsThis is the first report of striking anti-tumor effects of an orally delivered microbial extracellular vesicle. These data point to oral EVs as a new class of immunotherapeutic drugs. They are particularly effective at harnessing the biology of the small intestinal axis, acting locally on host cells in the gut to control distal immune responses within the TME. EDP1908 is in preclinical development for the treatment of cancer.Ethics ApprovalPreclinical murine studies were conducted under the approval of the Avastus Preclinical Services’ Ethics Board. Human in vitro samples were attained by approval of the IntegReview Ethics Board; informed consent was obtained from all subjects.

scholarly journals Uptake and Distribution of Administered Bone Marrow Mesenchymal Stem Cell Extracellular Vesicles in Retina

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 730
Author(s):  
Biji Mathew ◽  
Leianne A. Torres ◽  
Lorea Gamboa Gamboa Acha ◽  
Sophie Tran ◽  
Alice Liu ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Extracellular Vesicles ◽  
Time Course ◽  
Ganglion Cells ◽  
Ex Vivo ◽  
Dependent Manner ◽  
Paracrine Effects

Cell replacement therapy using mesenchymal (MSC) and other stem cells has been evaluated for diabetic retinopathy and glaucoma. This approach has significant limitations, including few cells integrated, aberrant growth, and surgical complications. Mesenchymal Stem Cell Exosomes/Extracellular Vesicles (MSC EVs), which include exosomes and microvesicles, are an emerging alternative, promoting immunomodulation, repair, and regeneration by mediating MSC’s paracrine effects. For the clinical translation of EV therapy, it is important to determine the cellular destination and time course of EV uptake in the retina following administration. Here, we tested the cellular fate of EVs using in vivo rat retinas, ex vivo retinal explant, and primary retinal cells. Intravitreally administered fluorescent EVs were rapidly cleared from the vitreous. Retinal ganglion cells (RGCs) had maximal EV fluorescence at 14 days post administration, and microglia at 7 days. Both in vivo and in the explant model, most EVs were no deeper than the inner nuclear layer. Retinal astrocytes, microglia, and mixed neurons in vitro endocytosed EVs in a dose-dependent manner. Thus, our results indicate that intravitreal EVs are suited for the treatment of retinal diseases affecting the inner retina. Modification of the EV surface should be considered for maintaining EVs in the vitreous for prolonged delivery.

scholarly journals A Comprehensive Proteomic SWATH-MS Workflow for Profiling Blood Extracellular Vesicles: A New Avenue for Glioma Tumour Surveillance

2020 ◽  
Vol 21 (13) ◽  
pp. 4754 ◽  
Author(s):  
Susannah Hallal ◽  
Ali Azimi ◽  
Heng Wei ◽  
Nicholas Ho ◽  
Maggie Yuk Ting Lee ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Biomarker Discovery ◽  
Ex Vivo ◽  
Tumour Progression ◽  
Principal Component ◽  
P Value ◽  
Diffuse Glioma ◽  
Blood Proteins ◽  
Treatment Protocols

Improving outcomes for diffuse glioma patients requires methods that can accurately and sensitively monitor tumour activity and treatment response. Extracellular vesicles (EV) are membranous nanoparticles that can traverse the blood–brain-barrier, carrying oncogenic molecules into the circulation. Measuring clinically relevant glioma biomarkers cargoed in circulating EVs could revolutionise how glioma patients are managed. Despite their suitability for biomarker discovery, the co-isolation of highly abundant complex blood proteins has hindered comprehensive proteomic studies of circulating-EVs. Plasma-EVs isolated from pre-operative glioma grade II–IV patients (n = 41) and controls (n = 11) were sequenced by Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) and data extraction was performed by aligning against a custom 8662-protein library. Overall, 4054 proteins were measured in plasma-EVs. Differentially expressed proteins and putative circulating-EV markers were identified (adj. p-value < 0.05), including those reported in previous in-vitro and ex-vivo glioma-EV studies. Principal component analysis showed that plasma-EV protein profiles clustered according to glioma histological-subtype and grade, and plasma-EVs resampled from patients with recurrent tumour progression grouped with more aggressive glioma samples. The extensive plasma-EV proteome profiles achieved here highlight the potential for SWATH-MS to define circulating-EV biomarkers for objective blood-based measurements of glioma activity that could serve as ideal surrogate endpoints to assess tumour progression and allow more dynamic, patient-centred treatment protocols.

scholarly journals microRNA-155 Is Decreased During Atherosclerosis Regression and Is Increased in Urinary Extracellular Vesicles During Atherosclerosis Progression

2020 ◽  
Vol 11 ◽  
Author(s):  
Stephen Fitzsimons ◽  
Silvia Oggero ◽  
Robyn Bruen ◽  
Cathal McCarthy ◽  
Moritz J. Strowitzki ◽  
...  
Keyword(s):  
Extracellular Vesicles ◽  
Ex Vivo ◽  
Macrophage Polarization ◽  
Chronic Inflammatory Disease ◽  
Cholesterol Diet ◽  
Human Coronary Artery ◽  
Inflammatory Signaling ◽  
Anti Inflammatory

BackgroundAtherosclerosis is a chronic inflammatory disease driven by macrophage accumulation in medium and large sized arteries. Macrophage polarization and inflammation are governed by microRNAs (miR) that regulate the expression of inflammatory proteins and cholesterol trafficking. Previous transcriptomic analysis led us to hypothesize that miR-155-5p (miR-155) is regulated by conjugated linoleic acid (CLA), a pro-resolving mediator which induces regression of atherosclerosis in vivo. In parallel, as extracellular vesicles (EVs) and their miR content have potential as biomarkers, we investigated alterations in urinary-derived EVs (uEVs) during the progression of human coronary artery disease (CAD).MethodsmiR-155 expression was quantified in aortae from ApoE−/− mice fed a 1% cholesterol diet supplemented with CLA blend (80:20, cis-9,trans-11:trans-10,cis-12 respectively) which had been previously been shown to induce atherosclerosis regression. In parallel, human polarized THP-1 macrophages were used to investigate the effects of CLA blend on miR-155 expression. A miR-155 mimic was used to investigate its inflammatory effects on macrophages and on ex vivo human carotid endarterectomy (CEA) plaque specimens (n = 5). Surface marker expression and miR content were analyzed in urinary extracellular vesicles (uEVs) obtained from patients diagnosed with unstable (n = 12) and stable (n = 12) CAD.ResultsHere, we report that the 1% cholesterol diet increased miR-155 expression while CLA blend supplementation decreased miR-155 expression in the aorta during atherosclerosis regression in vivo. CLA blend also decreased miR-155 expression in vitro in human THP-1 polarized macrophages. Furthermore, in THP-1 macrophages, miR-155 mimic decreased the anti-inflammatory signaling proteins, BCL-6 and phosphorylated-STAT-3. In addition, miR-155 mimic downregulated BCL-6 in CEA plaque specimens. uEVs from patients with unstable CAD had increased expression of miR-155 in comparison to patients with stable CAD. While the overall concentration of uEVs was decreased in patients with unstable CAD, levels of CD45+ uEVs were increased. Additionally, patients with unstable CAD had increased CD11b+ uEVs and decreased CD16+ uEVs.ConclusionmiR-155 suppresses anti-inflammatory signaling in macrophages, is decreased during regression of atherosclerosis in vivo and is increased in uEVs from patients with unstable CAD suggesting miR-155 has potential as a prognostic indicator and a therapeutic target.

scholarly journals Optimized cell culture conditions promote ex-vivo manipulation and expansion of primitive hematopoietic stem cells for therapeutic gene editing.

2022 ◽  
Author(s):  
Rajeev Rai ◽  
Winston Vetharoy ◽  
Asma Naseem ◽  
Zohar Steinberg ◽  
Adrian James Thrasher ◽  
...  
Keyword(s):  
Gene Editing ◽  
Culture Media ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Fine Tuning ◽  
Great Promise ◽  
Hematopoietic Stem ◽  
Monogenic Diseases

During the last few years, gene editing has emerged as a powerful tool for the therapeutic correction of monogenic diseases. CRISPR/Cas9 applied to hematopoietic stem and progenitor cells (HSPCs) has shown great promise in proof-of-principle preclinical studies to treat haematological disorders, and clinical trials using these tools are now underway. Nonetheless, there remain important challenges that need to be addressed, such as the efficiency of targeting primitive, long-term repopulating HSPCs and expand them in vitro for clinical purposes. Here we have tested the effect exerted by different culture media compositions on the ability of HSPCs to proliferate and undergo homology directed repair-mediated knock-in of a reporter gene, while preserving their stemness features during ex-vivo culture. We tested different combinations of compounds and demonstrated that by supplementing the culture media with inhibitors of histone deacetylases, and/or by fine-tuning its cytokine composition it is possible to achieve high levels of gene targeting in long-term repopulating HSPCs both in vitro and in vivo, with a beneficial balance between preservation of stemness and cell expansion, thus allowing to obtain a significant amount of edited, primitive HSPCs compared to established, state-of-the-art culture conditions. Overall, the implantation of this optimized ex vivo HSPC culture protocol will improve the efficacy, feasibility and applicability of gene editing and will likely provide one step further to unlock the full therapeutic potential of such powerful technology.

scholarly journals Platelet generation from circulating megakaryocytes is triggered in the lung vasculature

2021 ◽  
Author(s):  
Xiaojuan Zhao ◽  
Dominic Alibhai ◽  
Tony G. Walsh ◽  
Nathalie Tarassova ◽  
Semra Z. Birol ◽  
...  
Keyword(s):  
Blood Cells ◽  
Ex Vivo ◽  
Critical Role ◽  
The Body ◽  
Mouse Lung ◽  
Generation Process ◽  
Large Size ◽  
Large Numbers ◽  
Microfluidic Chamber

Platelets, small hemostatic blood cells, are derived from megakaryocytes, although the generation process is not clear. Only small numbers of platelets have been produced in systems outside the body, where bone marrow and lung are proposed as sites of platelet generation. Here we show that perfusion of megakaryocytes ex vivo through the mouse lung vasculature generates very large numbers of platelets, up to 3,000 per megakaryocyte. Despite their large size, megakaryocytes were able repeatedly to passage through the lung vasculature, leading to enucleation and fragmentation to generate platelets intravascularly. Using the ex vivo lung and a novel in vitro microfluidic chamber we determined the contributions of oxygenation, ventilation and endothelial cell health to platelet generation, and showed a critical role for the actin regulator TPM4.

Lipocortin-1 distribution in bronchoalveolar lavage from healthy human lung: effect of prednisolone

1995 ◽  
Vol 79 (1) ◽  
pp. 121-128 ◽  
Author(s):  
S. F. Smith ◽  
T. D. Tetley ◽  
A. K. Datta ◽  
T. Smith ◽  
A. Guz ◽  
...  
Keyword(s):  
Cell Surface ◽  
Bronchoalveolar Lavage ◽  
Ex Vivo ◽  
Specific Cell ◽  
Surface Binding ◽  
Healthy Human ◽  
Before And After ◽  
The Difference ◽  
Specific Cell Surface

Lipocortin-1 (LC-1; annexin-1) may mediate some anti-inflammatory actions of the glucocorticoids, probably after binding to specific cell surface binding sites. We have quantified LC-1 levels in bronchoalveolar lavage (BAL) fluid and cells collected from seven healthy volunteers before and after 7 days of treatment with an oral glucocorticoid, prednisolone (30 mg/day). Extracellular BAL LC-1 was higher and cellular LC-1 was lower after prednisolone than before [extracellular: before, median 98 ng/mg albumin (range 48–350 ng/mg albumin); after, 236 ng/mg albumin (19–414 ng/mg albumin); P < 0.05. Cellular: before, 23.3 ng/10(6) cells (14.6–26.9 ng/10(6) cells); after, 18.0 ng/10(6) cells (122–268 ng/10(6) cells); P < 0.05]. The distribution of LC-1 within BAL cells ex vivo (cell surface = 25%, cytosol = 50%, membrane = 25%) was unaffected by prednisolone treatment. However, in adherent cells that had been cultured for 4 h, 70–80% of the LC-1 was on the cell surface. In summary, prednisolone appears to promote cellular release of LC-1. The difference in distribution of cellular LC-1 in BAL cells ex vivo and in vitro may reflect adherence and/or activation.

scholarly journals Targeted photodynamic therapy selectively kills activated fibroblasts in experimental arthritis

Rheumatology ◽  
2020 ◽  
Vol 59 (12) ◽  
pp. 3952-3960 ◽  
Author(s):  
Daphne N Dorst ◽  
Mark Rijpkema ◽  
Marti Boss ◽  
Birgitte Walgreen ◽  
Monique M A Helsen ◽  
...  
Keyword(s):  
Photodynamic Therapy ◽  
Therapeutic Potential ◽  
Ex Vivo ◽  
Synovial Fibroblasts ◽  
Knee Joints ◽  
Specific Cell ◽  
Collagen Induced Arthritis ◽  
Targeted Photodynamic Therapy

Abstract Objective In RA, synovial fibroblasts become activated. These cells express fibroblast activation protein (FAP) and contribute to the pathogenesis by producing cytokines, chemokines and proteases. Selective depletion in inflamed joints could therefore constitute a viable treatment option. To this end, we developed and tested a new therapeutic strategy based on the selective destruction of FAP-positive cells by targeted photodynamic therapy (tPDT) using the anti-FAP antibody 28H1 coupled to the photosensitizer IRDye700DX. Methods After conjugation of IRDye700DX to 28H1, the immunoreactive binding and specificity of the conjugate were determined. Subsequently, tPDT efficiency was established in vitro using a 3T3 cell line stably transfected with FAP. The biodistribution of [111In]In-DTPA-28H1 with and without IRDye700DX was assessed in healthy C57BL/6N mice and in C57BL/6N mice with antigen-induced arthritis. The potential of FAP-tPDT to induce targeted damage was determined ex vivo by treating knee joints from C57BL/6N mice with antigen-induced arthritis 24 h after injection of the conjugate. Finally, the effect of FAP-tPDT on arthritis development was determined in mice with collagen-induced arthritis. Results 28H1-700DX was able to efficiently induce FAP-specific cell death in vitro. Accumulation of the anti-FAP antibody in arthritic knee joints was not affected by conjugation with the photosensitizer. Arthritis development was moderately delayed in mice with collagen-induced arthritis after FAP-tPDT. Conclusion Here we demonstrate the feasibility of tPDT to selectively target and kill FAP-positive fibroblasts in vitro and modulate arthritis in vivo using a mouse model of RA. This approach may have therapeutic potential in (refractory) arthritis.

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