Bone- and muscle-targeted adeno-associated viral vectors enable tissue-selective vitamin D receptor knockdown in mice

Abstract Vitamin D receptor (VDR) regulates musculoskeletal biology, but its adult, tissue-specific roles are difficult to resolve with germline or conventional conditional knockouts. We developed recombinant adeno-associated viral vectors (rAAVs) to drive Cre recombinase selectively in bone or muscle and used them to delete Vdr postnatally in Vdr fl/fl mice. To engineer a muscle-selective vector, we screened AAV9 constructs carrying candidate muscle promoters and identified tMCKΔ63 as the most selective promoter. Packaging this cassette in the myotropic AAVMYO capsid further reduced off-target skeletal expression while preserving strong muscle transduction. Local intramuscular delivery of AAVMYO–tMCKΔ63 enabled unilateral targeting with minimal systemic spread. In parallel, a bone-selective AAV8-Sp7 vector supported skeletal delivery. These vectors produced tissue-restricted Vdr deletion in Vdr muscle AAV and Vdr bone AAV mice. Muscle-targeted VDR loss reduced grip strength (–9.27%, p < 0.01) and endurance (–16.58%, p < 0.05). Bone-targeted deletion caused modest but significant skeletal changes, including increased cortical thickness ( + 7%, p < 0.05) and higher vertebral stiffness ( + 27%, p < 0.001), without effects on body weight or tibial strength. This scalable, crossbreeding-independent strategy enables compartment-specific functional studies in floxed models, including genes with embryonic lethality or complex tissue interactions. It also provides a general framework for iterative capsid-promoter optimization to maximize specificity in vivo across diverse tissues.