Hardwiring tissue-specific AAV transduction in mice through engineered receptor expression

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TL;DR

This paper describes the SELECTIV mouse system, which achieves tissue-specific AAV transduction by overexpressing the AAV receptor (AAVR) in target cells while knocking out endogenous Aavr. The system enables efficient and precise transgene expression in hard-to-transduce cell types like muscle stem cells and specific retinal layers, and can be paired with existing Cre lines for flexible targeting. This approach offers a rapid, cost-effective alternative to traditional transgenic models for biomedical research.

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**Background:** Adeno-associated virus (AAV) vectors are widely used for in vivo gene delivery in research and therapy, but their utility is limited by poor transduction of certain cell types and lack of tissue specificity. The AAV receptor (AAVR, KIAA0319L) is essential for transduction by most AAV serotypes. This study aimed to develop a system to control AAV transduction by regulating AAVR expression, enabling efficient and cell-type-specific gene delivery in mice without generating new transgenic lines for each gene.

**Methods:** The authors created SELECTIV mice carrying a Cre-inducible cassette encoding mouse AAVR fused to mCherry and spCas9, inserted into the H11 locus. Crossing with Cre-driver lines allows AAVR overexpression in specific cell types. SELECTIV-WB (whole-body overexpression) mice were generated by crossing with E2A-Cre. SELECTIV-KO mice were produced by breeding SELECTIV mice onto an Aavr knockout background, so that only Cre-expressing cells overexpress AAVR while all other cells lack endogenous AAVR. The system was tested with five Cre lines: E2A-Cre (whole-body), Pax7-CreER^T2^ (muscle stem cells), Myh6-Cre (heart), Alb-Cre (liver), and Chat-Cre (cholinergic neurons). Transduction was assessed using AAV vectors encoding luciferase or GFP, delivered locally (intramuscular, intravitreal) or systemically (intravenous). Outcomes included in vivo imaging, ex vivo luciferase assays, flow cytometry, fluorescence microscopy, and PET/CT tracking of radiolabeled AAV9.

**Key Results:** In SELECTIV-WB mouse embryonic fibroblasts (MEFs), AAVR overexpression increased transduction by AAV2 (fold-change not clearly reported), AAV8 (P<1×10^−15), and AAV9 (P<1×10^−15), but not AAV4 (AAVR-independent). In vivo, intramuscular AAV2-luciferase injection in SELECTIV-WB mice showed significantly higher luciferase activity at days 4 and 7 (P=0.034 and P=0.0090, respectively), with sustained expression up to 120 days post-injection. In muscle stem cells (MuSCs), SELECTIV-Pax7^CE^ mice with tamoxifen-induced AAVR overexpression showed a >4-fold increase in MuSC transduction after intramuscular AAV2-GFP (14–26% GFP+ vs 3–5.5% in controls, P=0.013). After systemic AAV9-GFP delivery, MuSC transduction increased 36-fold in tibialis anterior (0.13% to 4.7%) and 30-fold in diaphragm (0.18% to 5.3%) (P=0.0286 for both). In the retina, intravitreal AAV2-GFP injection in SELECTIV-WB mice resulted in significantly higher photoreceptor layer transduction (220.11±155.97 vs 87.80±39.93 GFP+ cells, P=0.0032), while RGC layer transduction was lower (52.94±20.46 vs 102.80±27.96, P=3.7×10^−6). In SELECTIV-KO-Myh6 mice (heart-specific AAVR), systemic AAV9-luciferase led to near 10-fold higher heart and lung luciferase activity compared to controls, while liver and muscle activity were reduced to near background levels (liver P=9.0×10^−6, heart P=7.9×10^−5, lung P=0.00051, muscle P=1.0×10^−5). PET/CT with ^64^Cu-AAV9 showed increased heart accumulation (12 %ID/g vs 2.4 %ID/g in controls, P=0.012) and decreased liver accumulation (P=0.0053) in SELECTIV-KO-Myh6 mice. Similarly, SELECTIV-KO-Alb mice (liver-specific AAVR) showed robust liver transduction with significant reductions in heart (225-fold), lung (3.7-fold), and muscle (230-fold). In the brain, SELECTIV-KO-Chat mice (cholinergic neuron-specific AAVR) showed highly localized GFP expression in the pons and medulla after systemic AAV-PHP.eB-GFP, with no detectable transduction in cerebral cortex, hippocampus, or liver.

**Clinical Implications:** The SELECTIV system provides a versatile platform for tissue-specific AAV-mediated gene delivery in mice, enabling efficient transduction of previously refractory cell types (e.g., MuSCs, photoreceptors) and near-complete elimination of off-target transduction. This approach can accelerate preclinical testing of gene therapies, CRISPR-based genome editing, and studies of gene function in specific cell populations. The system is compatible with existing AAV serotypes and Cre-driver lines, offering a cost-effective and flexible alternative to generating new transgenic mouse lines for each gene of interest. Potential applications include modeling genetic diseases, testing gene replacement or editing strategies, and studying cell-type-specific biology in neuroscience, muscle biology, and ophthalmology.