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1.
Mol Ther ; 2022 Jan 13.
Article in English | MEDLINE | ID: covidwho-1621101

ABSTRACT

accines, clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and urgently needed. Here, we comprehensively illustrate the capacity of Adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin (sh)RNAs (RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antivirals. In cultured monkey cells and in human gut organoids, our most potent vector, called SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24-48 hours. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the COVID-19 pandemic and holds great potential for clinical translation as an original and flexible, preventive or therapeutic antiviral measure.

2.
Viruses ; 12(8)2020 08 07.
Article in English | MEDLINE | ID: covidwho-713633

ABSTRACT

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.


Subject(s)
Betacoronavirus/chemistry , Betacoronavirus/genetics , Clinical Laboratory Techniques/methods , Coronavirus Infections/virology , Pneumonia, Viral/virology , RNA, Viral/isolation & purification , Betacoronavirus/isolation & purification , COVID-19 , COVID-19 Testing , Coronavirus Infections/diagnosis , Humans , Magnetic Phenomena , Molecular Diagnostic Techniques/methods , Nucleic Acid Amplification Techniques/methods , Pandemics , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Reverse Transcription , SARS-CoV-2 , Sensitivity and Specificity
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