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

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ABSTRACT 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 × 10 11 GC] vs. full dose [5.0 × 10 11 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.

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