A novel co-delivery system consisting of a Tomato bushy stunt virus and a defective interfering RNA for studying gene silencing

2003 ◽  
Vol 111 (1) ◽  
pp. 37-42 ◽  
Author(s):  
Hesheng Hou ◽  
Wenping Qiu
Keyword(s):  
Gene Silencing ◽  
Delivery System ◽  
Tomato Bushy Stunt Virus ◽  
Defective Interfering Rna ◽  
Interfering Rna

scholarly journals Cetuximab–siRNA Conjugate Linked Through Cationized Gelatin Knocks Down KRAS G12C Mutation in NSCLC Sensitizing the Cells Toward Gefitinib

2021 ◽  
Vol 20 ◽  
pp. 153303382110414
Author(s):  
K. Sreedurgalakshmi ◽  
R. Srikar ◽  
K. Harikrishnan ◽  
Lakshmi Srinivasan ◽  
Reena Rajkumari
Keyword(s):  
Gene Silencing ◽  
Delivery System ◽  
Sirna Delivery ◽  
Clinical Translation ◽  
Endosomal Escape ◽  
Nsclc Cell Line ◽  
Exon 2 ◽  
Molecule Inhibitor ◽  
Antibody Conjugates ◽  
Interfering Rna

Delivery of small-interfering RNA (siRNA) has been of great interest in the past decade for effective gene silencing. To overcome synthetic and regulatory challenges posed by nanoparticle-mediated siRNA delivery, antibody–siRNA conjugate (ARC) platform is emerging as a potential siRNA delivery system suitable for clinical translation. Herein, we have developed a delivery technology based on the ARC platform for stable delivery of siRNA called as Gelatin-Antibody Delivery System (GADS). In GADS, positively charged gelatin acts as a linker between antibody–siRNA and enables the endosomal escape of siRNA for gene silencing postcellular internalization. For proof of concept, we synthesized a scalable GADS conjugate comprising of Cetuximab (CTB), cationized gelatin (cGel) and NSCLC KRASG12C-specific siRNA. CTB was chemically conjugated to cGel through an amide link to form the CTB–cGel complex. Thereafter, siRNA was chemically conjugated to the cGel moiety of the complex through the thioether link to form CTB–cGel–siRNA conjugate. RP-HPLC analysis was used to monitor the reaction while gel retardation assay was used to determine siRNA loading capacity. SPR analysis showed the preservation of ligand binding affinity of antibody conjugates with KD of ∼0.3 nM. Furthermore, cellular internalization study using florescent microscopy revealed receptor-mediated endocytosis. The conjugate targeted EGFR receptor of KRAS mutant NSCLC to specifically knockdown G12C mutation. The oncogene knockdown sensitized the cells toward small molecule inhibitor—Gefitinib causing ∼70% loss in cell viability. Western blot analysis revealed significant downregulation for various RAS downstream proteins postoncogene knockdown. Comparison of the efficiency of GADS vis-à-vis positive siRNA control and CRISPR–Cas9-based knockout of KRAS Exon 2 in the NCI-H23 NSCLC cell line suggests GADS as a potential technology for clinical translation of gene therapy.

scholarly journals Integrity of Nonviral Fragments in Recombinant Tomato bushy stunt virus and Defective Interfering RNA Is Influenced by Silencing and the Type of Inserts

2005 ◽  
Vol 18 (8) ◽  
pp. 800-807 ◽  
Author(s):  
Xueyan Zhong ◽  
Hesheng Hou ◽  
Wenping Qiu
Keyword(s):  
Fluorescent Protein ◽  
Plant Viruses ◽  
Tomato Bushy Stunt Virus ◽  
Dependent Manner ◽  
Wild Type ◽  
Magnesium Chelatase ◽  
Recombinant Plant ◽  
Defective Interfering Rna ◽  
Green Fluorescent ◽  
Interfering Rna

Recombinant plant viruses have the propensity to remove foreign inserts during replication. This process is virusspecific and occurs in a host-dependent manner. In the present study, we investigated the integrity of foreign inserts in recombinant plant viruses using a model system consisting of Tomato bushy stunt virus (TBSV) and its defective interfering RNA (DI). These were tested in Nicotiana benthamiana plants that were either wild type or transgenic for the green fluorescent protein (GFP) gene. GFP-derived inserts were retained in the recombinant TBSV and DI population that were inoculated onto GFPtransgenic N. benthamiana plants in which silencing of the GFP transgene was initiated, but they were removed from the virus and DIs that were maintained on wild-type plants. A foreign insert derived from an endogenous N. benthamiana gene encoding the H subunit of the magnesium chelatase (NbChlH) was deleted, whereas the fragment of an RNA-dependent RNA polymerase gene (NbRdRP1m) was retained in the recombinant TBSV population. These results demonstrate that the recombination of TBSV to remove nonviral fragments is influenced by silencing and the type of inserts.

scholarly journals Defective Interfering RNA Hinders the Activity of a Tombusvirus-Encoded Posttranscriptional Gene Silencing Suppressor

2005 ◽  
Vol 79 (1) ◽  
pp. 450-457 ◽  
Author(s):  
Zoltán Havelda ◽  
Csaba Hornyik ◽  
Anna Válóczi ◽  
József Burgyán
Keyword(s):  
Gene Silencing ◽  
Viral Infections ◽  
Rna Viruses ◽  
Helper Virus ◽  
Small Interfering Rnas ◽  
Posttranscriptional Gene Silencing ◽  
Virus Rna ◽  
Defective Interfering Rna ◽  
Interfering Rna

ABSTRACT Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.

scholarly journals Effect of base modifications on structure, thermodynamic stability, and gene silencing activity of short interfering RNA

RNA ◽  
2007 ◽  
Vol 13 (8) ◽  
pp. 1301-1316 ◽  
Author(s):  
K. Sipa ◽  
E. Sochacka ◽  
J. Kazmierczak-Baranska ◽  
M. Maszewska ◽  
M. Janicka ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Thermodynamic Stability ◽  
Short Interfering Rna ◽  
Base Modifications ◽  
Interfering Rna

Biocompatible, chimeric peptide-condensed supramolecular nanoparticles for tumor cell-specific siRNA delivery and gene silencing

2014 ◽  
Vol 50 (58) ◽  
pp. 7806-7809 ◽  
Author(s):  
Hangxiang Wang ◽  
Wei Chen ◽  
Haiyang Xie ◽  
Xuyong Wei ◽  
Shengyong Yin ◽  
...  
Keyword(s):  
Tumor Cell ◽  
Gene Silencing ◽  
Delivery System ◽  
Self Assembly ◽  
Sirna Delivery ◽  
Single Step ◽  
Chimeric Peptide ◽  
Supramolecular Nanoparticles ◽  
Sirna Delivery System

A practical and tumor cell-specific siRNA delivery system was developedviasingle-step self-assembly of an arginine-rich chimeric peptide with siRNA.

Targeted Gene Silencing by Small Interfering RNA-Based Knock-Down Technology

2004 ◽  
Vol 5 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Bentham Science Publisher Zhang J. ◽  
Bentham Science Publisher Hua Z.C.
Keyword(s):  
Gene Silencing ◽  
Small Interfering Rna ◽  
Knock Down ◽  
Interfering Rna

scholarly journals Expression of Interferon-γ by a Coronavirus Defective-Interfering RNA Vector and Its Effect on Viral Replication, Spread, and Pathogenicity

Virology ◽  
1997 ◽  
Vol 233 (2) ◽  
pp. 327-338 ◽  
Author(s):  
Xuming Zhang ◽  
David R. Hinton ◽  
Daniel J. Cua ◽  
Stephen A. Stohlman ◽  
Michael M.C. Lai
Keyword(s):  
Viral Replication ◽  
Interferon Γ ◽  
Defective Interfering Rna ◽  
Interfering Rna

A defective interfering RNA that contains a mosaic of a plant virus genome

Cell ◽  
1987 ◽  
Vol 51 (3) ◽  
pp. 427-433 ◽  
Author(s):  
Bradley I. Hillman ◽  
James C. Carrington ◽  
Thomas J. Morris
Keyword(s):  
Plant Virus ◽  
Virus Genome ◽  
Defective Interfering Rna ◽  
Interfering Rna

scholarly journals Suppression of eNOS-derived superoxide by caveolin-1: a biopterin-dependent mechanism

2011 ◽  
Vol 301 (3) ◽  
pp. H903-H911 ◽  
Author(s):  
Kanchana Karuppiah ◽  
Lawrence J. Druhan ◽  
Chun-an Chen ◽  
Travis Smith ◽  
Jay L. Zweier ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Oxidase Activity ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Caveolin 1 ◽  
Calcium Calmodulin Dependent ◽  
Enos Uncoupling ◽  
Nadph Oxidation ◽  
Interfering Rna

In the vasculature, nitric oxide (NO) is generated by endothelial NO synthase (eNOS) in a calcium/calmodulin-dependent reaction. In the absence of the requisite eNOS cofactor tetrahydrobiopterin (BH4), NADPH oxidation is uncoupled from NO generation, leading to the production of superoxide. Although this phenomenon is apparent with purified enzyme, cellular studies suggest that formation of the BH4 oxidation product, dihydrobiopterin, is the molecular trigger for eNOS uncoupling rather than BH4 depletion alone. In the current study, we investigated the effects of both BH4 depletion and oxidation on eNOS-derived superoxide production in endothelial cells in an attempt to elucidate the molecular mechanisms regulating eNOS oxidase activity. Results demonstrated that pharmacological depletion of endothelial BH4 does not result in eNOS oxidase activity, whereas BH4 oxidation gave rise to significant eNOS-oxidase activity. These findings suggest that the endothelium possesses regulatory mechanisms, which prevent eNOS oxidase activity from pterin-free eNOS. Using a combination of gene silencing and pharmacological approaches, we demonstrate that eNOS-caveolin-1 association is increased under conditions of reduced pterin bioavailability and that this sequestration serves to suppress eNOS uncoupling. Using small interfering RNA approaches, we demonstrate that caveolin-1 gene silencing increases eNOS oxidase activity to 85% of that observed under conditions of BH4 oxidation. Moreover, when caveolin-1 silencing was combined with a pharmacological inhibitor of AKT, BH4 depletion increased eNOS-derived superoxide to 165% of that observed with BH4 oxidation. This study identifies a critical role of caveolin-1 in the regulation of eNOS uncoupling and provides new insight into the mechanisms through which disease-associated changes in caveolin-1 expression may contribute to endothelial dysfunction.

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