The observed down-regulation of ACE2 gene expression by several tested extracts of new C. sativa cultivars is a novel and crucial finding. Our results establish a foundation for further in-depth analyses of the effects of C. sativa on the molecular etiology and pathogenesis of COVID-19 and other viral diseases wherein the viruses use the ACE2 receptor as a molecular gateway. If these results are further confirmed, these high-CBD cannabis extracts can be used to develop prevention strategies directed at lowering ACE2 levels in high-risk gateway tissues. ACE2 level modulation is of particular importance since it appears to change throughout disease progression, and some studies show that ACE2 is essential for lung function in animal models of SARS [18, 19]. It would also be important to test the effects of C. sativa lines on other receptors involved in SARS-CoV-2 entry for their anti-inflammatory potential.

Furthermore, cannabis has over 100 phytocannabinoids [20], of which the main ones are Δ⁹-THC and CBD [21]. Cannabis possesses many minor cannabinoids, such as cannabigerol (CBG) and CBN, and numerous terpenes. Terpenes are responsible for variations in scent and may act synergistically with cannabinoids, with the potential to strongly enhance cannabinoid effects. Terpenes and minor cannabinoids are responsible for the ‘entourage effect’ [21], whereby whole plant extracts have more pronounced biological effects than individual cannabinoids. Here, we did not find a strong correlation between CBD levels and the observed down-regulation of ACE2 and TMPRSS2. Hence, we may indeed be observing the entourage effect in action, and the effects of extracts on ACE2 expression may not necessarily be attributed to CBD. In the future, it would be important to identify the cannabinoids and terpenes responsible for these observed effects, although, based on the entourage effect, one could predict that whole flower extracts may be more potent than single compounds [22, 23]. Here, we noted that the effects of extracts were more pronounced than those of CBD or CBN that alone did not affect ACE2 levels (Figure 6). In the future, it would be important to undertake further studies of other isolate cannabinoids and terpenes and determine their biological effects in well-defined conditions, as well as conduct reconstruction experiment to identify the key active ingredients or combinations.

Moreover, a notable aspect of our study is that, while the molar amounts and ratios of the major cannabinoids (THC and CBD) were similar between the analyzed extracts (Table 1), not all extracts were equally effective. Some produced undesired molecular effects on the levels of the ACE2 gene and protein. This finding emphasizes that different medical cannabis cultivar may have different effects, and each cannabis cultivar has to be analyzed in detail to identify the ones that are the most potent.

Another intriguing observation is the potential tissue-specificity of cannabis effects. Interestingly, extract #45 upregulated ACE2 expression in EpiOral tissues, but down-regulated it in the EpiIntestinal and EpiAirway tissues. Overall, tissue specificity of cannabis effects needs to be further explored in detail.

Within this study, extracts were applied via media to model medical delivery, such as local oral cavity applications, encapsulated extracts and dosed oils, and inhalers or nebulizers. Therefore, our results cannot be extrapolated to the effects of cannabis smoking. Moreover, in light of recent findings showing that tobacco smoking increases ACE2 levels and exacerbates the clinical outcomes of COVID-19 [24], the effects of cannabis smoking on the levels of ACE2 expression should be carefully investigated. Interestingly, animal model studies have shown that CBD decreased inflammation and improved lung function in murine models of acute lung injury [25]. In addition, a recent article suggested that CBD can be a useful addition to current COVID-19 protocols [25].

Most importantly, seven active cultivars have less than 0.3% total THC and therefore can be classified as hemp CBD in Canada and the USA, allowing for easy implementation.

However, our study has limitations. Our initial experiments were conducted using two replicates per condition. While it is feasible to conduct a differential expression study with only two replicates, the capability to detect true differentially expressed genes (DEGs) is reduced. Indeed, a recent study by Lamarre et al. (2018) [26] analyzed the impact of the number of replicates on the number of DEGs. For a library size of 15–20 million reads, which is consistent with that of the current study, Lamarre et al. detected 14,000 DEGs. The DEG number increased by about 1,000 with every increase in the number of replicates, and the DEG increase rate slowed down after five replicates were reached. A power analysis presented in the same study showed that, with a sequencing depth of 15–20 million reads, one would expect to detect 80%–90% DEGs with a two-replicate design, depending on the level of expression (low, medium, high). Lamarre et al.’s specificity and sensitivity analysis, which compared four different DEG detection tools (including DESeq2, which was used in the present study), showed a 40%–50% true positive rate (TPR), 0.2%–2% false-positive rate (FPR) and 0.01 FDR [26]. Moreover, Lamarre and colleagues performed their analyses using individual organisms, as opposed to the 3D tissues in our case. We expect the biological variance between 3D tissue samples to be lower compared with separate plants or animals, resulting in a further reduction of the replicate number required to reach the same statistical power [26]. However, we do acknowledge the limited statistical power of our experiment; the addition of replicates would expand the list of DEGs and enhance the ability to detect significant genes at fewer fold changes.

To substantiate our data we, therefore, conducted additional experiments using EpiAirway, EpiAirway FT, and EpiOral tissues with three replicates per group, and further confirmed the effectiveness of several novel cannabis cultivars in modulation of the ACE2 expression. Nonetheless, future studies are needed to establish the precise mechanisms of action of high-CBD cannabis extracts, their tissue specificity, and the effects on ACE2, given the new knowledge on the tissue and disease stage specificity of ACE 2 expression.

Our original experiments were designed to screen the biological activities of novel cannabis extracts in 3D human tissue models. They allowed us to pinpoint the important effects of cannabis on the levels of ACE2 and TMPRSS2 expression. Further studies are needed and being undertaken to link the levels of ACE2 and inflammation. Indeed, higher ACE2 expression after SARS-CoV-2 infection is correlated with a cytokine surge in patients [8], which is thought to occur via activation of the NLRP3 inflammasome [27]. ACE2 levels are affected by age [14], and ACE2 blockers have an anti-aging potential [28]. Furthermore, linking ACE2 levels, inflammation and aging is extremely important, as aging is an inflammation-related condition. Inflamm-aging, a certain level of chronic inflammation that develops with advanced age, increases the rate of biological aging and underlies numerous age-related diseases [29]. Inflamm-aging and macroph-aging, a related phenomenon that involves senescence-related changes in macrophages, and their contributions to COVID-19 need to be further investigated [30].