scholarly journals CRISPR/Cas9 as an antiviral against Orthopoxviruses using an AAV vector

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Cathryn M. Siegrist ◽  
Sean M. Kinahan ◽  
Taylor Settecerri ◽  
Adrienne C. Greene ◽  
Joshua L. Santarpia
Keyword(s):  
Targeted Delivery ◽  
Model Organism ◽  
Host Cells ◽  
Hek293 Cells ◽  
Viral Titer ◽  
Human Embryonic Kidney ◽  
Adeno Associated Virus ◽  
Guide Rnas ◽  
Per Gene

Abstract A vaccine for smallpox is no longer administered to the general public, and there is no proven, safe treatment specific to poxvirus infections, leaving people susceptible to infections by smallpox and other zoonotic Orthopoxviruses such as monkeypox. Using vaccinia virus (VACV) as a model organism for other Orthopoxviruses, CRISPR–Cas9 technology was used to target three essential genes that are conserved across the genus, including A17L, E3L, and I2L. Three individual single guide RNAs (sgRNAs) were designed per gene to facilitate redundancy in rendering the genes inactive, thereby reducing the reproduction of the virus. The efficacy of the CRISPR targets was tested by transfecting human embryonic kidney (HEK293) cells with plasmids encoding both SaCas9 and an individual sgRNA. This resulted in a reduction of VACV titer by up to 93.19% per target. Following the verification of CRISPR targets, safe and targeted delivery of the VACV CRISPR antivirals was tested using adeno-associated virus (AAV) as a packaging vector for both SaCas9 and sgRNA. Similarly, AAV delivery of the CRISPR antivirals resulted in a reduction of viral titer by up to 92.97% for an individual target. Overall, we have identified highly specific CRISPR targets that significantly reduce VACV titer as well as an appropriate vector for delivering these CRISPR antiviral components to host cells in vitro.

scholarly journals Comparative Genomics, Infectivity and Cytopathogenicity of American Isolates of Zika Virus that Developed Persistent Infections in Human Embryonic Kidney (HEK293) Cells

2019 ◽  
Vol 20 (12) ◽  
pp. 3035 ◽  
Author(s):  
Hebing Liu ◽  
Hsiao-Mei Liao ◽  
Bingjie Li ◽  
Shien Tsai ◽  
Guo-Chiuan Hung ◽  
...  
Keyword(s):  
Cell Line ◽  
Zika Virus ◽  
Hek293 Cell ◽  
Clonal Selection ◽  
Gene Mutations ◽  
Host Cells ◽  
Hek293 Cells ◽  
Specific Gene ◽  
Human Embryonic Kidney ◽  
Hek293 Cell Line

Zika virus (ZIKV) transmission can cause serious fetal neurological abnormalities. ZIKV persistence in various human cells and tissues can serve as infectious reservoirs and post serious threats to public health. The human embryonic kidney (HEK293) cell line with known neuronal developmental properties was readily infected by ZIKV in a strain-dependent fashion. Significant cytopathic effect in HEK293 cells infected by the prototype MR 766 strain of ZIKV resulted in complete loss of cells, while small numbers of HEK293 cells infected by contemporary ZIKV isolates (PRV or FLR strain) continued to survive and regrow to confluency in the culture around two months after initial infection. Most, if not all, of the cells in the two resulting persistently ZIKV-infected HEK293 cell lines tested positive for ZIKV antigen. Compared to HEK293 control cells, the persistently ZIKV-infected HEK293 cells had slower growth rates with some cells undergoing apoptosis in culture. The “persistent ZIKVs” produced constitutively by both PRV and FLR strains ZIKV-infected HEK293 cells had significantly attenuated cell infectivity and/or cytopathogenicity. Comparative genome sequence analyses between the persistent ZIKVs and the original inoculum ZIKVs showed no clonal selection with specific gene mutations in the prolonged process of establishing persistently PRV strain ZIKV-infected HEK293 cells; while selection of ZIKV subclones with mutations in the envelope, protein pr and multiple NS genes was evident in developing persistently FLR strain ZIKV-infected HEK293 cell line. Our study provides molecular insights into the complex interplays of ZIKV and human host cells in establishing ZIKV persistence.

scholarly journals Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

2005 ◽  
Vol 79 (1) ◽  
pp. 28-38 ◽  
Author(s):  
John M. Casper ◽  
Jennifer M. Timpe ◽  
John David Dignam ◽  
James P. Trempe
Keyword(s):  
Gene Expression ◽  
Random Sequence ◽  
Gene Promoter ◽  
Helper Virus ◽  
Host Cells ◽  
Mobility Shift ◽  
Adeno Associated Virus ◽  
Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

Synthesis of Chiral N-Nitro-oxazolidin-2-ones and O-(β-Nitraminoalkyl) Carbamates in Liquefied 1,1,1,2-Tetrafluoroethane Medium

Synthesis ◽  
2020 ◽  
Vol 52 (22) ◽  
pp. 3485-3491
Author(s):  
Sergei G. Zlotin ◽  
Svetlana S. Arabadzhi ◽  
Mikhail N. Zharkov ◽  
Ilya V. Kuchurov
Keyword(s):  
Amino Acid ◽  
Nitro Compounds ◽  
Hek293 Cells ◽  
Human Embryonic Kidney ◽  
Enantiomerically Pure ◽  
Dinitrogen Pentoxide ◽  
Convenient Synthesis ◽  
Stereogenic Centers

AbstractA convenient synthesis of chiral N-nitro-oxazolidin-2-ones by nitration of α-amino acid derived 1,3-oxazolidin-2-ones containing one or two stereogenic centers with dinitrogen pentoxide in liquefied 1,1,1,2-tetrafluoroethane medium has been developed. The obtained N-nitroheterocycles were converted into enantiomerically pure O-(β-nitraminoalkyl) carbamates by treatment with ammonia or amines in the same solvent. The synthesized N-nitro compounds are slightly toxic in vitro to Human Embryonic Kidney 293 (HEK293) cells.

scholarly journals Identification of an Insulator in AAVS1, a Preferred Region for Integration of Adeno-Associated Virus DNA

2003 ◽  
Vol 77 (16) ◽  
pp. 9000-9007 ◽  
Author(s):  
Toshihiko Ogata ◽  
Takuyo Kozuka ◽  
Tadahito Kanda
Keyword(s):  
Dnase I ◽  
Hek293 Cells ◽  
Open Chromatin ◽  
Chromatin Conformation ◽  
Adeno Associated Virus ◽  
Barr Virus ◽  
Cell Clones ◽  
Enhancer Blocking ◽  
Epstein Barr

ABSTRACT In latent adeno-associated virus (AAV) infection, the viral genome is integrated preferentially into the human chromosome 19 q arm at a specific region designated AAVS1, which has an open chromatin conformation as indicated by the presence of a DNase I-hypersensitive site (DHS-S1). We examined whether an insulator, which defines the domain of gene expression by directionally blocking the action of enhancers and by preventing the spread of heterochomatin, is present near the DHS-S1 in the middle of a 2.6-kbp AAVS1-related DNA fragment used in this study. The fragment, cloned into an Epstein-Barr virus (EBV)-based eukaryotic episomal plasmid, was introduced into HEK293 cells. The DHS-S1 on the plasmid replicating in the nuclei was hypersensitive to DNase I digestion, and thus, the EBV plasmid system was used in an enhancer-blocking assay with the 2.6-kbp DNA and two shortened DNAs, of 1.6 kbp and 336 bp, containing DHS-S1. The three DNA fragments, when inserted in the proper direction between the cytomegalovirus immediate-early enhancer and minimal promoter, repressed the expression of a reporter gene. Thus, the enhancer-blocking activity was located within the 336-bp DNA containing the entire region (300 bp) of DHS-S1. To investigate the prevention of repression caused by heterochromatin, a transgene-expressing cassette flanked by the two 336-bp DNAs placed in the enhancer-blocking direction was introduced into HEK293 and HeLa cells. All the cell clones examined with the cassette integrated into cell DNA continued to express the transgene, which indicates that the pair of 336-bp DNA apparently prevented the spread of heterochromatin. The results show that an insulator lies between nucleotides 17 and 354 near the DHS-S1 in AAVS1. In a gel shift test, the 336-bp DNA did not bind an in vitro-prepared CCCTC-binding factor that binds to the chicken β-globin insulator, suggesting that the AAVS1 insulator requires an as yet unidentified binding protein. The newly identified AAVS1 insulator is likely to contribute to the maintenance of an open chromatin conformation that affects the life cycle of AAV.

scholarly journals Hepatocellular-Targeted mRNA Delivery Using Functionalized Selenium Nanoparticles In Vitro

Pharmaceutics ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 298
Author(s):  
Dhireshan Singh ◽  
Moganavelli Singh
Keyword(s):  
Targeted Delivery ◽  
Hepg2 Cells ◽  
Hek293 Cells ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Vis Spectroscopy ◽  
Gene Assay ◽  
Nuclease Digestion ◽  
Diphenyl Tetrazolium Bromide

Selenium’s (Se) chemopreventative and therapeutic properties have attracted attention in nanomedicine. Se nanoparticles (SeNPs) retain these properties of Se while possessing lower toxicity and higher bioavailability, potentiating their use in gene delivery. This study aimed to formulate SeNPs for efficient binding and targeted delivery of FLuc-mRNA to hepatocellular carcinoma cells (HepG2) in vitro. The colorectal adenocarcinoma (Caco-2) and normal human embryonic kidney (HEK293) cells that do not have the asialoorosomucoid receptor (ASGPR) were utilized for comparison. SeNPs were functionalized with chitosan (CS), polyethylene glycol (PEG), and lactobionic acid (LA) for ASGPR targeting on HepG2 cells. Nanoparticles (NPs) and their mRNA-nanocomplexes were characterized by Fourier transform infra-red (FTIR) and UV-vis spectroscopy, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). Gel and fluorescence-based assays assessed the NP’s ability to bind and protect FLuc-mRNA. Cytotoxicity was determined using the -(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, while transgene expression was evaluated using the luciferase reporter gene assay. All NPs appeared spherical with sizes ranging 57.2–130.0 nm and zeta potentials 14.9–31.4 mV. NPs bound, compacted, and protected the mRNA from nuclease digestion and showed negligible cytotoxicity in vitro. Targeted gene expression was highest in the HepG2 cells using the LA targeted NPs. These NPs portend to be efficient nanocarriers of nucleic acids and warrant further investigation.

scholarly journals The Surface Antigen 1(SAG1) of Toxoplasma Gondii Interacts With RACK1 Inducing Autophagy and Maintaining The Viability of Host Cells

2021 ◽  
Author(s):  
Ni An ◽  
Bing Li ◽  
Yu-ming Liu ◽  
Zhi-xuan Zhang ◽  
Rongjian Su ◽  
...  
Keyword(s):  
Surface Antigen ◽  
Underlying Mechanism ◽  
Host Cells ◽  
Hek293 Cells ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
Multifunctional Protein ◽  
Fluorescent Microscope ◽  
Tnf Α ◽  
Enhanced Expression

Abstract BackgroundIt has been well known that the surface antigen 1(SAG 1) of T.gondii plays an important role in the invasion of Tachyzoite into host cells. However whether it also play a role in the intracellular parasitism of T.gondii remains unclear. The main purpose of this study was to determine the effect of SAG1 on host cells and investigate the underlying mechanism. MethodsSAG1 was overexpressed in human embryonic kidney cell 293(HEK) by transfection. Autophagy was determined by fluorescent microscope and flow cytometry (FCM) in HEK293 cells co-transfected with Flag-SAG1 and EGFP-LC3. The interaction of SAG1 and RACK1 was measured by co-immunoprecipitation(Co-IP), GST pulldown and fluorescent microscope. The expression of cytokines including IL-1β, IL-6, and IL-12 was determined by qRT-PCR. The expression of LC3, Ki67 and RACK1 was detected by Western blot. Cellular senescence was measured by β-galactosidase staining.ResultsWe found that overexpression of SAG1 in human embryonic kidney cells (HEK293) induces non-canonical autophagy and inhibition of autophagy using hydroxychloroquine (HCQ) significantly decreases the cell viability of HEK293 cells. Mechanically, we identified RACK1, an intracellular multifunctional protein as a binding partner of SAG1. Depletion of RACK1 inhibited SAG1 induces non-autophagy and decreases the enhanced expression of cytokines including IL-1β, IL-6, IL-12 and TNF-α in SAG1 overexpressing cells. ConclusionThese data showed that SAG1 could induce non-canonical autophagy and facilitate the expression of IL-1β, IL-6, IL-12 and TNF-α by interacting with RACK1, maintaining the viability of host cells. Our results suggests a new contribution of SAG1 in the intracellular parasitism.

scholarly journals Evaluation of Graphene Oxide Induced Cellular Toxicity and Transcriptome Analysis in Human Embryonic Kidney Cells

Nanomaterials ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 969 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Muhammad Arsalan Iqbal ◽  
Muhammad Qasim ◽  
Chan Hyeok Park ◽  
Hyunjin Yoo ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Molecular Mechanisms ◽  
Cellular Responses ◽  
Hek293 Cells ◽  
Single Atom ◽  
Ros Generation ◽  
Human Embryonic Kidney ◽  
Human Embryonic Kidney Cells ◽  
In Vitro Investigation

Graphene, a two-dimensional carbon sheet with single-atom thickness, shows immense promise in several nanoscientific and nanotechnological applications, including in sensors, catalysis, and biomedicine. Although several studies have shown the cytotoxicity of graphene oxide in different cell types, there are no comprehensive studies on human embryonic kidney (HEK293) cells that include transcriptomic analysis and an in vitro investigation into the mechanisms of cytotoxicity following exposure to graphene oxide. Therefore, we exposed HEK293 cells to different concentrations of graphene oxide for 24 h and performed several cellular assays. Cell viability and proliferation assays revealed a significant dose-dependent cytotoxic effect on HEK293 cells. Cytotoxicity assays showed increased lactate dehydrogenase (LDH) leakage and reactive oxygen species (ROS) generation, and decreased levels of reduced glutathione (GSH) and increased level of oxidized glutathione indicative of oxidative stress. This detailed mechanistic approach showed that graphene oxide exposure elicits significant decreases in mitochondrial membrane potential and ATP synthesis, as well as in DNA damage and caspase 3 activity. Furthermore, our RNA-Seq analysis revealed that HEK293 cells exposed to graphene oxide significantly altered the expression of genes involved in multiple apoptosis-related biological pathways. Moreover, graphene oxide exposure perturbed the expression of key transcription factors, promoting these apoptosis-related pathways by regulating their downstream genes. Our analysis provides mechanistic insights into how exposure to graphene oxide induces changes in cellular responses and massive cell death in HEK293 cells. To our knowledge, this is the first study describing a combination of cellular responses and transcriptome in HEK293 cells exposed to graphene oxide nanoparticles, providing a foundation for understanding the molecular mechanisms of graphene oxide-induced cytotoxicity and for the development of new therapeutic strategies.

Investigation of the properties and activity of DfCas9 and DsCas9 nucleases in eucaryotic cells

2021 ◽  
Author(s):  
Yelizaveta V. Vlasova ◽  
Dmitry A. Madera ◽  
Pavel M. Gershovich
Keyword(s):  
Protein Detection ◽  
Nuclease Activity ◽  
Hek293 Cells ◽  
Rna Molecules ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cloning Method ◽  
Science And Education ◽  
Genetic Constructs

This study is focused on the two novel nucleases of the CRISPR/Cas9 family, which were found in bacterial genomes of DfCas9 (Defluviimonas sp) и DsCas9 (Demequina sediminicola). Discovery of these nucleases was part of the results of a joint study conducted by BIOCAD together with Skoltech Institute of Science and Technology and Saint-Petersburg Polytechnical University (SPPU) under a grant agreement with the Department of Science and Education of Russian Federation (Agreement number 14.606.21.0006 from September, 26th 2017). Under the agreement the nucleases DfCas9 and DsCas9 were characterized in vitro by Skoltech and SPPU. Based on the aforementioned results, in this study we characterized the genome-modifying nuclease activity of these enzymes in a mammalian cell line HEK293. Specifically, we created genetic constructs designed to express the nucleases DsCas9 and DfCas9 together with the necessary guide RNA molecules (sequences of the guide RNAs were described previously) [1]. We demonstrated expression of the nucleases on a protein level, as well as activity of DfCas9 at the VEGF2 locus in HEK293 cells. The theoretical study was conducted by analyzing international and national literature. The experimental part was performed with a restriction-ligation cloning method, transient transfections, Western blot protein detection method, and a T7 nuclease-based method of detection of heteroduplex double-stranded DNA.

DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM

2020 ◽  
Author(s):  
M.A. Tyumentseva ◽  
◽  
A.I. Tyumentsev ◽  
V.G. Akimkin ◽  
◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Health Monitoring ◽  
Biological Samples ◽  
Proviral Dna ◽  
Guide Rnas ◽  
Ribonucleoprotein Complexes ◽  
Low Concentrations ◽  
Immunodeficiency Virus ◽  
Hiv 1

For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.

Development and Characterization Colchicine-Loaded PEGylated Gelatin Nanoparticles for Targeted Delivery to Tumor

2013 ◽  
Vol 6 (2) ◽  
pp. 2058-2063
Author(s):  
G D Chandrethiya ◽  
P K Shelat ◽  
M N Zaveri
Keyword(s):  
Drug Delivery ◽  
Targeted Delivery ◽  
High Performance ◽  
Entrapment Efficiency ◽  
Stability Study ◽  
Spherical Particles ◽  
Precipitation Method ◽  
Antitumoral Activity ◽  
Gelatin Nanoparticles

PEGylated gelatin nanoparticles loaded with colchicine were prepared by ethanol precipitation method. Poly-(ethylene glycol)-5000-monomethylether (MPEG 5000), a hydrophilic polymer, was used to pegylate gelatin.  Gluteraldehyde was used as cross-linking agent. To obtain a high quality product, major formulation parameters were optimized.  Spherical particles with mean particles of 193 nm were measured by a Malvern particle size analyzer. Entrapment efficiency was found to be 71.7 ± 1.4% and determined with reverse phase high performance liquid charomatography (RP-HPLC). The in vitro drug release study was performed by dialysis bag method for a period of 168 hours. Lyophilizaton study showed sucrose at lower concentrations proved the best cryoprotectant for this formulation.  Stability study revealed that lyophilized nanoparticles were equally effective (p < 0.05) after one year of storage at 2-8°C with ambient humidity. In vitro antitumoral activity was accessed using the MCF-7 cell line by MTT assay.  The IC50 value was found to be 0.034 μg/ml for the prepared formulation. The results indicate that PEGylated gelatin nanoparticles could be utilized as a potential drug delivery for targeted drug delivery of tumors.  

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