Lung-targeted lipid nanoparticle-delivered siUSP33 attenuates SARS-CoV-2 replication and virulence by promoting envelope degradation

As a structural protein of SARS-CoV-2, the E protein not only plays a key role in the formation of viral particles but also forms ion channels and has pathogenic functions, including triggering cell death and inflammatory responses. The stability of E proteins is controlled by the host ubiquitin-proteasome system. By screening human deubiquitinases, we found that ubiquitin-specific protease 33 (USP33) could enhance the stability of E proteins depending on its deubiquitinase activity, thereby promoting viral replication. In the absence of USP33, E proteins are rapidly degraded, leading to a reduced viral load and inflammation. Using lipid nanoparticle (LNP) encapsulation of siUSP33 by adjusting the lipid components (ionizable cationic lipids), siUSP33 was successfully delivered to mouse lung tissues, rapidly reducing USP33 expression in the lungs and maintaining knockdown for at least 14 d, effectively suppressing viral replication and virulence. This method of delivery allows efficient targeting of the lungs and a response to acute infections without long-term USP33 deficiency. Our research, based on the deubiquitination mechanism of USP33 on the E protein, demonstrates that LNP-mediated siRNA delivery targeting USP33 plays a role in antiviral and anti-inflammatory responses, offering a novel strategy for the prevention and treatment of SARS-CoV-2. Overall design: As the article outlines, USP33 serves as a versatile E3 ligase within the host organism. To more effectively illustrate that no significant side effects were observed in mice treated with LNP-siUSP33, we procured blood samples from each cohort on both the first and fourth day following administration. The strategy here is based on the fact that in vivo delivery of LNP reaches its peak on the day immediately after injection, while the optimal efficiency for USP33 knockdown occurs four days post-injection. These samples were subsequently examined for changes in the transcriptome. Furthermore, conventional cationic lipids such as DOTAP (trimethyl-2,3-dioloyl-propylammonium bromide) have a propensity to aggregate with negatively charged serum proteins, resulting in swift elimination of lipid nanoparticles by the immune system and a markedly reduced half-life in vivo. This aggregation also heightens the risk of adverse effects, including hemolysis. To surmount these obstacles, ionizable cationic lipids with pKa values ranging from 6.0 to 7.0 have been engineered. These ionizable lipid LNPs (iLNPs) adeptly encapsulate nucleic acids while mitigating toxicity (PMID: 28412170). Consequently, the enhanced LNP employed in this study is crafted to address the side effects of traditional cationic lipids. Our approach, which involves utilizing ionizable cationic lipids, notably Dlin-MC3-DMA, to encapsulate siRNA and incorporating an extra cationic lipid (DOTAP), proved effective in diminishing side effects. This LNP system preserves a relatively neutral surface charge at physiological pH levels. We also ensured its diameter remained under 140 nm to minimize the binding of LNP to fibrinogen and the subsequent clot formation (PMID: 38394670). Moreover, to more rigorously validate its efficacy, we collected peripheral blood from mice across various treatment groups on both day 1 and day 4 for transcriptomic analysis.