scholarly journals A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Joep Beumer ◽  
Maarten H. Geurts ◽  
Mart M. Lamers ◽  
Jens Puschhof ◽  
Jingshu Zhang ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Genetically Engineered ◽  
Host Factors ◽  
Therapeutic Interventions ◽  
Target Cells ◽  
Genetic Screens ◽  
Transformed Cell Lines ◽  
Viral Target ◽  
Host Genes

AbstractRapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.

scholarly journals A CRISPR/Cas9 genetically engineered organoid biobank reveals essential host factors for coronaviruses

2021 ◽  
Author(s):  
Joep Beumer ◽  
Maarten H Geurts ◽  
Mart M Lamers ◽  
Jens Puschhof ◽  
Jingshu Zhang ◽  
...  
Keyword(s):  
Rapid Identification ◽  
Genetically Engineered ◽  
Host Factors ◽  
Therapeutic Interventions ◽  
Target Cells ◽  
Genetic Screens ◽  
Transformed Cell Lines ◽  
Viral Target ◽  
Host Genes

Rapid identification of host genes essential for virus replication may expedite the generation of therapeutic interventions. Genetic screens are often performed in transformed cell lines that poorly represent viral target cells in vivo, leading to discoveries that may not be translated to the clinic. Intestinal organoids (IOs) are increasingly used to model human disease and are amenable to genetic engineering. To discern which host factors are reliable anti-coronavirus therapeutic targets, we generate mutant clonal IOs for 19 host genes previously implicated in coronavirus biology. We verify ACE2 and DPP4 as entry receptors for SARS-CoV/SARS-CoV-2 and MERS-CoV respectively. SARS-CoV-2 replication in IOs does not require the endosomal Cathepsin B/L proteases, but specifically depends on the cell surface protease TMPRSS2. Other TMPRSS family members were not essential. The newly emerging coronavirus variant B.1.1.7, as well as SARS-CoV and MERS-CoV similarly depended on TMPRSS2. These findings underscore the relevance of non-transformed human models for coronavirus research, identify TMPRSS2 as an attractive pan-coronavirus therapeutic target, and demonstrate that an organoid knockout biobank is a valuable tool to investigate the biology of current and future emerging coronaviruses.

scholarly journals CRISPR-NSID: anin vivoCRISPR/Cas9 negative selection screen reveals EZH2 as a druggable dependency factor in a genetic desmoid tumor model

2019 ◽  
Author(s):  
Thomas Naert ◽  
Tom Van Nieuwenhuysen ◽  
Suzan Demuynck ◽  
Sven de Grande ◽  
Joanna Przybyl ◽  
...  
Keyword(s):  
Negative Selection ◽  
Unmet Need ◽  
Tumor Model ◽  
Rapid Identification ◽  
Genetically Engineered ◽  
Model Systems ◽  
Desmoid Tumors ◽  
Cancer Models

AbstractIdentification of true dependencies in cancer is pivotal to the elucidation of novel therapeutic strategies to increase prospects for cancer patients. Unfortunately,in vivoidentification of genetic dependencies has long relied on expensive and time-consuming breeding of genetically engineered animal models. Recently,in vitroCRISPR/Cas9 screens provided a new method for rapid and genome-wide identification of genetic dependencies. Nevertheless, genetic dependencies would ideally be identified usingin vivocancer models initiated by clinically relevant oncogenic driver or tumor suppressor insults. Here, we report a new methodology calledCRISPR/Cas9-mediatedNegativeSelectionIdentification of geneticDependencies (CRISPR-NSID) that allowsin vivoelucidation of cancer cell vulnerabilities in genetic cancer models. The methodology hinges on the fact that for a genetic dependency there is an incapability for recovering tumors carrying biallelic frameshift mutations in this gene. We demonstrate how integrating experimentally determined, orin silicopredicted, probabilities of frameshift editing for any given gRNA can be employed to ascertain negative selection pressure on inactivation of a genetic dependency during tumorigenesis. As a proof-of-principle, we use CRISPR-NSID to identifyezh2andcreb3l1as genetic dependencies in desmoid tumors (desmoid-type fibromatosis) occurring in aXenopus tropicaliscancer model driven byapcmutations. Bridging CRISPR-NSID to a clinically unmet need, we further demonstrate the promise for EZH2 inhibition as a new therapeutic strategy for desmoid tumors. This study establishes a new methodology for rapid identification of genetic dependencies in monoclonal disorders with wide adaptability to other model systems and organisms.

scholarly journals Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo

2017 ◽  
Vol 312 (1) ◽  
pp. L110-L121 ◽  
Author(s):  
Duo Zhang ◽  
Heedoo Lee ◽  
Ziwen Zhu ◽  
Jasleen K. Minhas ◽  
Yang Jin
Keyword(s):  
High Efficiency ◽  
Therapeutic Interventions ◽  
Target Cells ◽  
Transfection Method ◽  
Exosomal Mirnas ◽  
Mother Cells ◽  
Antisense Oligos ◽  
Exosomal Mirna

Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells.

scholarly journals Implementation of CRISPR/Cas9 Genome Editing to Generate Murine Lung Cancer Models That Depict the Mutational Landscape of Human Disease

2021 ◽  
Vol 9 ◽  
Author(s):  
Oliver Hartmann ◽  
Michaela Reissland ◽  
Carina R. Maier ◽  
Thomas Fischer ◽  
Cristian Prieto-Garcia ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Translational Research ◽  
Genome Editing ◽  
Mouse Models ◽  
Genetic Alterations ◽  
Surrogate Models ◽  
Genetically Engineered ◽  
Lung Epithelial Cells ◽  
Therapeutic Interventions

Lung cancer is the most common cancer worldwide and the leading cause of cancer-related deaths in both men and women. Despite the development of novel therapeutic interventions, the 5-year survival rate for non-small cell lung cancer (NSCLC) patients remains low, demonstrating the necessity for novel treatments. One strategy to improve translational research is the development of surrogate models reflecting somatic mutations identified in lung cancer patients as these impact treatment responses. With the advent of CRISPR-mediated genome editing, gene deletion as well as site-directed integration of point mutations enabled us to model human malignancies in more detail than ever before. Here, we report that by using CRISPR/Cas9-mediated targeting of Trp53 and KRas, we recapitulated the classic murine NSCLC model Trp53fl/fl:lsl-KRasG12D/wt. Developing tumors were indistinguishable from Trp53fl/fl:lsl-KRasG12D/wt-derived tumors with regard to morphology, marker expression, and transcriptional profiles. We demonstrate the applicability of CRISPR for tumor modeling in vivo and ameliorating the need to use conventional genetically engineered mouse models. Furthermore, tumor onset was not only achieved in constitutive Cas9 expression but also in wild-type animals via infection of lung epithelial cells with two discrete AAVs encoding different parts of the CRISPR machinery. While conventional mouse models require extensive husbandry to integrate new genetic features allowing for gene targeting, basic molecular methods suffice to inflict the desired genetic alterations in vivo. Utilizing the CRISPR toolbox, in vivo cancer research and modeling is rapidly evolving and enables researchers to swiftly develop new, clinically relevant surrogate models for translational research.

scholarly journals Intracellular Vesicle Entrapment of Nanobubble Ultrasound Contrast Agents Targeted to PSMA Promotes Prolonged Enhancement and Stability In Vivo and In Vitro

2021 ◽  
Author(s):  
Reshani Perera ◽  
Eric Abenojar ◽  
Pinunta Nittayacharn ◽  
Xinning Wang ◽  
Gopal Ramamurthy ◽  
...  
Keyword(s):  
Contrast Agents ◽  
Ultrasound Contrast Agents ◽  
Therapeutic Interventions ◽  
Active Targeting ◽  
Target Cells ◽  
Acoustic Activity ◽  
Ultrasound Contrast ◽  
Imaging Plane

Previous work has shown that active targeting of nanobubble (NB) ultrasound contrast agents to the prostate-specific membrane antigen (PSMA) significantly prolongs ultrasound signal enhancement in PSMA-expressing prostate cancer. However, the specific mechanism behind this effect is not well understood. Furthermore, prior studies were carried out using clinical ultrasound scanners in a single imaging plane. Because tumor heterogeneity can have a drastic effect on bubble kinetics and resulting contrast enhancement, a single region of interest in one imaging plane over time may not fully represent the contrast dynamics of the entire tumor. Accordingly, in the current work, we used high-frequency dynamic parametric contrast-enhanced ultrasound (DCE-US) imaging to gain a detailed understanding of NB kinetics in prostate tumors in mice. Specifically, we examined the differences in enhancement between the tumor periphery and tumor core in the same imaging plane. We also quantified intact nanobubble retention in the entire tumor volume. To better understand the mechanism behind prolonged tumor enhancement, intracellular retention and the acoustic activity of PSMA-NB were evaluated in cell culture. DCE-US US data suggest that both tumor wash-in and retention of PSMA-NB are delayed due to biomarker interaction and binding. The longer retention of PSMA-NB signal in tumor core supported target-driven bubble extravasation. In vitro studies demonstrated a higher level of internalization and prolonged-acoustic activity of internalized PSMA-NB. GC/MS analysis confirmed gas persistence in the cells after PSMA-NB internalization. The active-targeting of NB results in cellular internalization via receptor-mediated endocytosis, and the location with intracellular vesicles (late-stage endosomes/lysosomes) significantly prolongs gas retention within the cells. These features can enable background-free diagnostic imaging of the target cells/tissues, as well as highly focused ultrasound-modulated therapeutic interventions.

scholarly journals 202 In vivo and in vitro characterization of AIM ACT, a novel nanoparticle-based technology, expanded MART-1 specific T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A213-A213
Author(s):  
Ruipeng Wang ◽  
Lauren Suarez ◽  
Emily Lu ◽  
Pratima Kunwar ◽  
Daniel Dembrow ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Manufacturing Process ◽  
Genetically Engineered ◽  
Target Cells ◽  
Clinical Scale

BackgroundNexImmune is developing highly differentiated immunotherapies to target, activate and expand tumor antigen-specific T cells using the proprietary Artificial Immune Modulation (AIM™) nanotechnology platform. The AIM nanoparticle (AIM-np) technology functions as synthetic dendritic cells capable of directing a specific T cell-mediated immune response. By mimicking natural T cell biology, NexImmune’s non-genetically engineered cellular therapy product candidates (AIM ACT) are designed to combine the attributes of cellular precision, potency, and persistence with reduced potential for undesired toxicities.MethodsHere we present an example of AIM ACT expanded MART-1 specific T cells and their phenotypic and functional characterization in vitro and in vivo. Leukopaks from healthy donors were used to produce AIM ACT T cell products with our proprietary AIM ACT enrichment and expansion (E+E) manufacturing process and antigen peptide-loaded AIM-nanoparticles.ResultsThe final MART1 T cell products include up to 62.8% (20.8% in average) MART-1-specific CD8+ T cells as determined by MART1 peptide (ELAGIGILTV)-loaded multimer staining. MART1-specific T cells were tested in flow cytometry-based and live cell imaging-based cytotoxicity assays using HLA-A2 positive MART1 peptide-loaded target cells. The AIM ACT-generated T cells showed potent cytotoxicity to MART1 peptide-loaded target cells in vitro, while unloaded control cells were not killed. In over 30 independent AIM ACT E+E clinical scale runs, the expanded T cells consisted of a combined average of 91.7% T stem cell like, central and effector memory T cells, as determined by CD62L, CD45RA and CD95 staining. These phenotypes have been associated with long term in vivo persistence and anti-tumor efficacy. In a human melanoma PDX model, we confirmed that transfusion of AIM ACT T cells resulted in long term survival in vivo and significant reduction of tumor growth with complete tumor clearance in 6 out of 15 animals.ConclusionsThe results demonstrate that AIM ACT MART1 T cells have long term persistence and anti-tumor activity in solid tumors such as melanoma, and that the AIM ACT E+E approach is a reproducible clinical scale manufacturing process for non-genetically engineered antigen-specific T cells. The AIM ACT platform is currently being used for generating T cell products for our current clinical trials, NEXI-001 (NCT04284228) and NEXI-002 (NCT04505813), and our pre-clinical development for HPV-associated malignancies. The findings support initiating Phase I trials of adoptive T cell therapy in solid tumors.Ethics ApprovalThe study was approved by the Institutional Animal Care and Use Committee (IACUC).

scholarly journals Genetically engineered cell membrane–coated nanoparticles for targeted delivery of dexamethasone to inflamed lungs

2021 ◽  
Vol 7 (25) ◽  
pp. eabf7820
Author(s):  
Joon Ho Park ◽  
Yao Jiang ◽  
Jiarong Zhou ◽  
Hua Gong ◽  
Animesh Mohapatra ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Targeted Delivery ◽  
Biological Membrane ◽  
Genetically Engineered ◽  
Target Cells ◽  
Coated Nanoparticles ◽  
Ligand Interactions ◽  
Late Antigen

As numerous diseases are associated with increased local inflammation, directing drugs to the inflamed sites can be a powerful therapeutic strategy. One of the common characteristics of inflamed endothelial cells is the up-regulation of vascular cell adhesion molecule–1 (VCAM-1). Here, the specific affinity between very late antigen–4 (VLA-4) and VCAM-1 is exploited to produce a biomimetic nanoparticle formulation capable of targeting inflammation. The plasma membrane from cells genetically modified to constitutively express VLA-4 is coated onto polymeric nanoparticle cores, and the resulting cell membrane–coated nanoparticles exhibit enhanced affinity to target cells that overexpress VCAM-1 in vitro. A model anti-inflammatory drug, dexamethasone, is encapsulated into the nanoformulation, enabling improved delivery of the payload to inflamed lungs and significant therapeutic efficacy in vivo. Overall, this work leverages the unique advantages of biological membrane coatings to engineer additional targeting specificities using naturally occurring target-ligand interactions.

Recent Patents on Anti-Cancer Potential of Helenalin

2020 ◽  
Vol 15 (2) ◽  
pp. 132-142
Author(s):  
Priyanka Kriplani ◽  
Kumar Guarve
Keyword(s):  
Synergistic Effects ◽  
Therapeutic Interventions ◽  
Active Constituent ◽  
Scientific Impact ◽  
Isolation Techniques ◽  
Anti Cancer ◽  
Prevention Of Cancer ◽  
Synthetic Derivatives

Background: Arnica montana, containing helenalin as its principal active constituent, is the most widely used plant to treat various ailments. Recent studies indicate that Arnica and helenalin provide significant health benefits, including anti-inflammatory, neuroprotective, antioxidant, cholesterol-lowering, immunomodulatory, and most important, anti-cancer properties. Objective: The objective of the present study is to overview the recent patents of Arnica and its principal constituent helenalin, including new methods of isolation, and their use in the prevention of cancer and other ailments. Methods: Current prose and patents emphasizing the anti-cancer potential of helenalin and Arnica, incorporated as anti-inflammary agents in anti-cancer preparations, have been identified and reviewed with particular emphasis on their scientific impact and novelty. Results: Helenalin has shown its anti-cancer potential to treat multiple types of tumors, both in vitro and in vivo. It has also portrayed synergistic effects when given in combination with other anti- cancer drugs or natural compounds. New purification/isolation techniques are also developing with novel helenalin formulations and its synthetic derivatives have been developed to increase its solubility and bioavailability. Conclusion: The promising anti-cancer potential of helenalin in various preclinical studies may open new avenues for therapeutic interventions in different tumors. Thus clinical trials validating its tumor suppressing and chemopreventive activities, particularly in conjunction with standard therapies, are immediately required.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

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