Cell viability assay
1 × 104 primary mouse or human hepatocytes were cultured in 96-well plate and transfected with 0.025-0.1 μg of LNP-IL-21 or LNP, or left untreated. Cell viability was assessed at day 1-4 post transfection with using a Cell Counting Kit-8 kit (Dojindo, Japan) according to the manufacturer’s instructions.
RNA sequencing analysis
BALB/c mouse was injected with LNP or 2 × 1011 genome equivalents (geq) of AAV in 200 μl PBS via tail vein, or left untreated. At 3 days post injection, RNA extracted form liver tissues was subjected to library construction and deep sequencing on Illumina NovaSeq 6000 by Novogene Biotechnology Co. Ltd. (Shanghai, China). FPKM, fragments per kilobase of transcript per million mapped reads, was used to estimate gene expression levels. Differential expression analysis of two groups was performed using the DESeq2 R package (1.20.0). Differentially expressed genes (DEGs) with a fold change ≥ 2 and a false discovery rate (FDR) < 0.05 were identified and presented in Table S1 and S2 .