Targeted postnatal knockout of Sclerostin using a bone-targeted adeno-associated viral vector increases bone anabolism and decreases canalicular density.

Abstract

The creation of murine gene knockout models to study bone gene functions often requires the resource intensive crossbreeding of Cre transgenic and gene-floxed strains. The developmental versus postnatal roles of genes can be difficult to discern in such models. For example, embryonic deletion of the Sclerostin (Sost) gene establishes a high-bone mass phenotype in neonatal mice that may impact on future bone growth. To generate a postnatal skeletal knockout of Sost in adult mice, this study used a single injection of a bone-targeted recombinant adeno-associated virus (rAAV) vector. 8-week-old Sostflox/flox mice were injected with saline (control) or a single injection containing 5×1011 vg AAV8-Sp7-Cre vector. Ai9 fluorescent Cre reporter mice were dosed in parallel to confirm targeting efficiency. After 6weeks, detailed bone analysis was performed via microCT, biomechanical testing, and bone histology on vertebral and long bone specimens. The AAV8-Sp7-Cre vector induced widespread persistent recombination in the bone compartment. Regional microCT analyses revealed significant increases in bone with vector treatment. In the L3 vertebrae, Sostflox/flox:AAV-Cre showed a 22% increase in bone volume and 21% in trabecular bone fraction compared to controls; this translated to a 17% increase in compressive strength. In the tibiae, Sostflox/flox:AAV-Cre led to small but statistically significant increases in cortical bone volume and thickness. These were consistent with a 25% increase in mineral apposition rate, but this did not translate into increased four-point bending strength. Ploton silver nitrate stain on histological sections revealed an unexpected increase in canalicular density associated with Sost ablation. This report demonstrates a proof-of-concept that the AAV8-Sp7-Cre vector can efficiently produce postnatal skeletal knockout mice using gene-floxed strains. This technology has the potential for broad utility in the bone field with existing conditional lines. These data also confirm an important postnatal role for Sost in regulating bone homeostasis, consistent with prior studies using neutralizing Sclerostin antibodies, and highlights a novel role of Sost in canalicular remodeling.