Human IL21 mRNA

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) is the transcription template for all viral mRNAs and is highly stable, making current treatments ineffective in inducing its clearance. Here, we established a mouse model of HBV persistence based on a clinical isolate (referred to as BPS) and identified interleukin 21 (IL-21) as a potent inducer of HBV clearance. Lipid nanoparticle (LNP)-mediated mRNA delivery has been shown to be a highly safe and effective delivery platform. This work explored the applicability and efficacy of the mRNA-LNP platform in IL-21-based HBV therapy. First, LNP-encapsulated mouse IL-21 mRNA (LNP-IL-21) was prepared, characterized, and demonstrated to induce IL-21 expression in vitro and in vivo. Next, LNP-IL-21 was shown to induce clearance of serum and intrahepatic HBV antigens and DNA in two HBV persistence mouse models based on BPS and recombinant cccDNA (rcccDNA), respectively, which was associated with HBV-specific humoral and cellular immune responses. In addition, peripheral blood mononuclear cells from BPS-persistent mice treated with LNP-IL-21 and HBV surface antigen (HBsAg) in vitro induced similar HBV clearance when infused into recipient mice. These results suggest that the IL-21 combined with mRNA-LNP platform represents an effective and promising strategy for the development of novel therapeutics for chronic HBV infection.

Here, researchers used a microfluidic device to mix a lipid mixture containing a fixed ratio of cationic lipids, lipid-anchored PEG, cholesterol, and DSPC with IL-21 mRNA to generate LNP-IL-21 (Figure 1A). In dynamic light scattering (DLS) analysis, the diameter of nanoparticles in the LNP-IL-21 formulation peaked at 125.9-129.9 nm, and the polydispersity index (PDI) was less than 0.2 (Figure 1B). Transmission electron microscopy analysis showed that LNP-IL-21 was a regular spherical or elliptical structure, with diameters mostly in the range of 100-150 nm (Figure 1C), which was consistent with the DLS measurement results. Then, to study the in vitro expression efficiency and cytotoxicity of LNP-IL-21, primary mouse and human hepatocytes were transfected with LNP-IL-21, control LNP, or untreated cells. IL-21 secretion into the culture medium was observed only in cells transfected with LNP-IL-21 (Figure 1D). CCK8 assay showed that LNP-IL-21 transfection had no significant effect on cell viability (Figure 1E). Next, LNP-IL-21 was injected into BALB/c mice via the tail vein to evaluate the expression distribution of IL-21 in vivo. Intracellular IL-21 levels were analyzed using ELISA, and the results showed that in mice injected with LNP-IL-21, the highest expression level of IL-21 was found in the liver tissue, followed by the spleen, and the expression level in the kidney was lower (Figure 1F). In summary, these data indicate that LNP-IL-21 mediates the efficient delivery of IL-21 expression in vitro and in vivo, with a relative preference for liver targeting.

Figure 1. Preparation and characterization of LNP-IL-21. (Shen Z, et al., 2023)