Design and evaluation of small interfering RNAs for the treatment of Severe Acute Respiratory Syndrome-Coronavirus-2

Autor: William Zhang, Aïcha Daher, Anne Gatignol, Robert J. Scarborough
Rok vydání: 2021
Zdroj: McGill Science Undergraduate Research Journal. 16:61-66
ISSN: 1718-0783
1718-0775
Popis: SARS-CoV-2 is the virus responsible for the COVID-19 pandemic. As the 2019 coronavirus disease continues to spread, it will be useful to have as many effective treatment options as possible. This research has the potential to create a siRNA treatment that is safe, effective, and practical in design and administration; 192 siRNAs were designed to target conserved regions of the SARS-CoV-2 genome. The first aim of this study is to confirm, via sequence analysis, that these target sites have remained highly conserved over the course of the pandemic. Multiple sequence alignments were generated for the first half of 30,312 full SARS-CoV-2 genomes, which were averaged and compared with the Wuhan-Hu-1 reference genome. Most target sites maintained a very high level of conservation, suggesting that potential repressor siRNAs could be effective in a majority of infected individuals. To evaluate the efficacy of the 192 test siRNAs, we cloned sections of the SARS-CoV-2 RNA genome into GFP fusion genes. Some of these constructs were transfected in different conditions to set up a screening assay based on GFP expression. Preliminary data on the setup of this GFP reporter assay show that the M, N, E, ORF8, and ORF10 constructs produced a good GFP signal, whereas the S, F1, F2 and F3 constructs did not produce a sufficiently strong GFP signal to detect above background. In a preliminary experiment, we evaluated siRNAs targeting the M, N, and E open reading frames and found some to be efficacious. Future directions for this project include generating alignments of the second half of the SARS-CoV-2 genome for a complete sequence conservation estimate, and cell metabolism assays for supplementing visual observations of siRNA toxicity, optimization of GFP readout, and screening of all designed siRNAs.
Databáze: OpenAIRE
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