Cannabidiol Inhibits SARS-CoV-2 Replication through Induction of the Host ER Stress and Innate Immune Responses

The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1a RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time. Overall design: Lung aveolar A549 cells were stably overexpressed with human angiotensin converting enzyme 2 (ACE2) protein and seeded at 10,000 cells per well in a 96-well plate. Cannabidiol or vehicle were added together to the cells. Cannabidiol (Cayman Chemical, 90080) was dissolved in a 10mM stock solution with DMSO (Sigma-Aldrich, D2650-100mL). Final concentration of CBD was 10 µM. The virus stock was then removed and replaced with fresh 2% FBS DMEM media with drug. The cells were incubated for another 24 hours before total RNA extraction using the NucleoSpin 96 RNA kit (Takarabio, 740709). Three independent biological replicates were performed per experimental condition, with 12 total RNA samples. RNA sample quality check, library construction, and sequencing were performed by the University of Chicago Genomics Facility following standard protocols. The average RNA Integrity Score was 8.9. All 12 samples were sequenced in two runs by a NovaSeq 6000 sequencer to generate paired-end 100bp reads. For each sample, the raw FASTQ files from two flow cells were combined before downstream processing. Cannabidivarin (CBDV) was isolated from the hemp oil using centrifugal partition chromatography (CPC), a countercurrent separation technique, and a biphasic liquid-liquid solvent system. Identical experimentation to the above CBD experiment was then performed. The average RNA Integrity Score was also 8.9. RNA-seq data for both experiments were analyzed separately using a local Galaxy 20.05 instance for the following steps. Quality and adapter trimming were performed on the raw sequencing reads using Trim Galore! 0.6.3. The reads were mapped to both the human genome (UCSC hg19 with GENCODE annotation) and the SARS-COV-2 genome (NCBI Assembly ASM985889v3 with Ensembl annotation) using RNA STAR 2.7.5b. The resulting mapped reads from each sample were counted by featureCounts 1.6.4 for per gene read counts. The raw counts were analyzed for differential expression between experimental conditions using DESeq2 1.22.1.