I originally envisioned this as a biocontainment mechanism to make working with SARS-CoV-2 safer. However, after consulting with a virologist on campus i realized it could also be useful as a step towards a live attenuated vaccine. My idea is DNA

[Vaccine Google Form Christopher Leichthammer]

Email address	clei...@uwo.ca
Name	Christopher Leichthammer
Country	Canada
Expertise/Background	Background: Biochemistry, Genetics, and Synthetic Biology Expertise: Biocontainment
Hypothesis	I originally envisioned this as a biocontainment mechanism to make working with SARS-CoV-2 safer. However, after consulting with a virologist on campus i realized it could also be useful as a step towards a live attenuated vaccine. My idea is DNA printing a linear version of SARS-CoV-2 or cloning it into an expression vector with an inserted reverse compliment to the sequence of miR-192, a microRNA expressed in human and murine lung epithelial cells. I would then propose expressing it in vitro using RNA polymerase or a kit like mMessage mMachine; Ambion. The resultant viral RNA could then be used to lipofect ferret tracheal or lung epithelial cells. Virions could then be collected from those cells, and then tested on human tracheal/lung epithelial cell cultures and compared to the ferret cell cultures. It would be interesting to see if this strategy works in such a way that only ferret cell cultures support an increased viral load. I assume this could be read out via comparisons of copy number via RT-qPCR. I hypothesize that this would result in a version of SARS-CoV-2 that is unable to replicate in human cells, but should be able to be cultured and harvested from ferret cell lines. Similar strategies are being investigated for HIV using cis-acting siRNAs, but a host derived miRNA would be advantageous because it could be produced easily in a host that doesn't produce a miRNA with high similarity (like ferrets). There is a proof of principle for this strategy for influenza A as a biocontainment mechanism (see useful links), although they didn't test whether it was a potential vaccine. However, the ability to enter human cells using the ACE2 receptor, but the inability to replicate within human cells, could make it useful as a live attenutated vaccine.
Useful links	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3808852/

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