We have now performed experiments in HT29 and HepG2 cells to test mitigation/synthetic lethality between…
Week 2 progress update
Fewer than two weeks after starting our experiments aimed at assessing genetic pathways mediating host toxicity of the SARS-CoV2 drugs remdesivir and hydroxychloroquine (HCQ), we already have our first experiment finished. Here’s an update on our progress this week:
- Minsun collected RNA from our 4 cell lines given doses of remdesivir and HCQ that we determined not to yield cell death within 8-24 hours (our two RNA-seq harvest timepoints) but high enough to affect cell physiology and gene expression (as determined by their toxicity starting 48 hours after dosing). RNA yields (listed in 033020_Rem_HCQ_RNAseq_experiment) revealed lower RNA concentrations in many drug treatment conditions, especially after 24 hours, suggesting that the doses we gave did affect cell physiology. Whether we hit the “Goldilocks” zone of a high enough dose to measure gene expression changes but not so high to induce massive pro-apoptotic gene expression remains to be seen, so we may have to repeat some conditions.
- Minsun performed Lexogen Quantseq 3’ RNA-seq prep (this is a robust kit that we have used successfully in the past) on 24 samples, and we ran Nextseq on them. The run finished yesterday.
- Grace Yeo from David Gifford’s lab, a long-time collaborator of ours, is currently processing the data. We will upload it when it’s ready and begin analysis, hopefully within 1-2 days.
- Minsun and Ersin have now titrated the lentiCRISPRv2 Brunello library on our four cell lines, identifying a dose that should yield ~50% infection for 4*10^7 cells for each cell line.
- We are currently expanding the four cell lines and expect to perform lentiviral transduction tomorrow (Sunday, April 5). We have developed a screening protocol (to be uploaded soon) that should allow us to complete the cell culture phase of the CRISPR screen within 2 weeks.
- We are in the midst of a second drug titration (protocols uploaded below) that will determine a dose of remdesivir and HCQ for each cell line that leads to 50% cell survival within a 4-6 day time window. This titration will finish by Monday such that we will have the final drug concentrations well before we begin dosing the screening plates with drug by the end of this coming week.
Establishing a scalable mammalian cell fluorescence-based assay for SARS-CoV2 RdRP activity
- Mia has designed cloning protocols for an assay in which we hope to enable measurement of SARS-CoV2 RdRP replication activity through GFP fluorescence. She will post information on this assay soon.
- We have ordered SARS-CoV2 viral genomic RNA from ATCC which they recently made available. Once that ships, we can begin cloning.
- We have also been working with our neighboring lab, Steve Elledge’s lab, who have cloned all SARS-CoV2 ORFs and have graciously allowed us to use them for establishing this assay.
- If we can successfully establish this assay, it
has promise in the following areas:
- Evaluating remdesivir-resistant mutations in SARS-CoV2 RdRP that may arise during treatment of SARS-CoV2 patients.
- Screening and evaluating other drugs with RdRP-inhibiting activity without the need for a BSL3 live virus assay.
- Understanding host genes that interact with SARS-CoV2 RdRP which may help us identify markers and mechanisms of genetic susceptibility and resistance to SARS-CoV2 infection and remdesivir treatment.