Anti‐CovidMicroRNA Therapy Blocksthe Expression of the SpikeGeneof SARS‐CoV‐2

Abstract

Emerging viral diseases have increased in recent decades. In December 2019, an epidemic with low respiratory infections emerged in Wuhan, China. The disease, Covid‐19 was found to be caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). As of October 14, 2021, WHO has confirmed greater than 239,000,000 global cases, nearly 5 million deaths worldwide, including greater than 720,000 in the USA. Fortunately, a vaccine has been approved and distributed, yet there are no approved therapeutics for infected individuals, and the threat of emerging vaccine‐resistant strains remain. From advances in biotechnology, the genome and structure of SARS‐CoV‐2 is known. Three proteins are anchored in the viral envelope, Spike (S), Envelope (E), and Membrane (M), which is linked to the Nucleocapsid (N) protein connecting to the viral RNA genome. Our lab is developing an innovative therapy that delivers multiple therapeutic microRNAs to block the expression of distinct Covid viral proteins. The design of the anti‐Covid microRNAs 1) mimics human microRNA cluster 17‐92a structural stability, 2) forms guide‐RNA substrates for the RNA induced silencing complex, and 3) are complementary to specific regions of the SARS‐CoV‐2 RNA genome without off‐targets effects in the human genome. Twenty‐one microRNA sequences were designed to target the S gene, six for N, two for M, and one for E. These were cloned into our microRNA‐17‐92 therapy vector which expresses six distinct anti‐Covid RNA therapeutics simultaneously. We have transfected the S gene into our tissue culture model to measure the efficacy of the anti‐Covid microRNA therapy to down‐regulate the S gene expression. In our preliminary experiments we show a significant 2.5‐fold reduction in the relative abundance of the Spike mRNA in the treated cells (p < 0.05). We are currently testing additional therapies and verifying changes in spike protein levels. Next steps are to examine the secondary structure of our RNA therapy using SHAPE‐MAP to optimize RNA therapeutic stability in comparison to the stable structure based on the human gene, microRNA Cluster 17‐92a.