In-Depth Optimization and Systematic Evaluation of a Recombinant AAV-Based Hepatic-Targeted Gene Knockdown Murine...

Precise gene-edited animal models are essential for preclinical liver research but are typically time-consuming and costly. Here, a liver-specific knockdown mouse model was developed based on a recombinant AAV (rAAV) tool, which is rapid and economical, with optimized delivery methods and systematically evaluated knockdown efficiency. Hepatic ischemia/reperfusion (IR) injury-related genes (Btg2, Flrt3, Klf4) were screened through GEO data mining. To knock down these genes, rAAV vectors featuring a liver-specific promoter TBG669, Firefly luciferase (Fluc) for real-time tracking, and miR30-based regulatory elements were engineered. Delivery protocols were optimized through comparative analysis of reporting systems (GFP vs. Fluc), administration routes (tail vein vs. dorsal penile vein), and doses (half [2.5 × 10¹¹ GC] vs. full dose [5.0 × 10¹¹ GC]). Safety and knockdown efficiency were systematically assessed using histopathology, serum biochemistry, qRT-PCR, and western blot analysis both in bulk liver tissue and single-cell subsets. The optimized rAAV system achieved 100% hepatic infection rates with minimal off-target expression. Dorsal penile vein injection simplified delivery without compromising efficacy. Full-dose administration produced stronger bioluminescence by Week 4, with no adverse effects on organ function or histology. For knockdown efficiencies, Btg2 and Flrt3 mRNA and protein levels were reduced by > 50%, whereas Klf4 exhibited moderate suppression in bulk liver tissue. In single-cell subsets, the three genes were all effectively knocked down in Kupffer cells, followed by hepatocytes and LSECs. A time- and cost-efficient rAAV-based hepatic gene knockdown murine model was established, enabling precise investigation of liver disease mechanisms and therapeutic development.