miRNA Pathway Alteration in Response to Non-Coding RNA Delivery in Viral Vector-Based Gene Therapy.

Gene therapy is widely used to treat incurable disorders and has become a routine procedure in clinical practice. Since viruses can exhibit specific tropisms, effectively penetrate the cell, and are easy to use, most gene therapy approaches are based on viral delivery of genetic material. However, viral vectors have some disadvantages, such as immune response and cytotoxicity induced by a disturbance of cell metabolism, including miRNA pathways that are an important part of transcription regulation. Therefore, any viral-based gene therapy approach involves the evaluation of side effects and safety. It is possible for such effects to be caused either by the viral vectors themselves or by the delivered genetic material. Many gene therapy techniques use non-coding RNA delivery as an effective agent for gene expression regulation, with the risk of cellular miRNA pathways being affected due to the nature of the non-coding RNAs. This review describes the effect of viral vector entry and non-coding RNA delivery by these vectors on miRNA signaling pathways.

The Tissue Distribution of SARS-CoV-2 in Transgenic Mice With Inducible Ubiquitous Expression of hACE2.

The novel coronavirus disease COVID-19 has become one of the most socially significant infections. One of the main models for COVID-19 pathogenesis study and anti-COVID-19 drug development is laboratory animals sensitive to the virus. Herein, we report SARS-CoV-2 infection in novel transgenic mice conditionally expressing human ACE2 (hACE2), with a focus on viral distribution after intranasal inoculation. Transgenic mice carrying hACE2 under the floxed STOP cassette [(hACE2-LoxP(STOP)] were mated with two types of Cre-ERT2 strains (UBC-Cre and Rosa-Cre). The resulting offspring with temporal control of transgene expression were treated with tamoxifen to induce the removal of the floxed STOP cassette, which prevented hACE2 expression. Before and after intranasal inoculation, the mice were weighed and clinically examined. On Days 5 and 10, the mice were sacrificed for isolation of internal organs and the further assessment of SARS-CoV-2 distribution. Intranasal SARS-CoV-2 inoculation in hACE2-LoxP(STOP)×UBC-Cre offspring resulted in weight loss and death in 6 out of 8 mice. Immunostaining and focus formation assays revealed the most significant viral load in the lung, brain, heart and intestine samples. In contrast, hACE2-LoxP(STOP) × Rosa-Cre offspring easily tolerated the infection, and SARS-CoV-2 was detected only in the brain and lungs, whereas other studied tissues had null or negligible levels of the virus. Histological examination revealed severe alterations in the lungs, and mild changes were observed in the brain tissues. Notably, no changes were observed in mice without tamoxifen treatment. Thus, this novel murine model with the Cre-dependent activation of hACE2 provides a useful and safe tool for COVID-19 studies.

Deletion of BST2 Cytoplasmic and Transmembrane N-Terminal Domains Results in SARS-CoV, SARS-CoV-2, and Influenza Virus Production Suppression in a Vero Cell Line.

SARS-CoV-2, which emerged in Wuhan (China), has become a great worldwide problem in 2020 and has led to more than 1,000,000 deaths worldwide. Many laboratories are searching for ways to fight this pandemic. We studied the action of the cellular antiviral protein tetherin, which is encoded by the BST2 gene. We deleted the transmembrane domain-encoding part of the gene in the Vero cell line. The transmembrane domain is a target for virus-antagonizing proteins. We showed a decrease in SARS-CoV-2 in cells with deleted transmembrane BST2 domains compared to the initial Vero cell line. Similar results were obtained for SARS-CoV and avian influenza virus. This finding may help the development of antiviral therapies competitively targeting the transmembrane domain of tetherin with viral-antagonizing proteins.