Bone Abstracts

Objectives: While local gene therapy for bone applications has shown some success in preclinical models, systemic delivery of transgenes to the skeleton remains a considerable challenge. Viral vectors such as adeno-associated viruses (AAVs) have great potential as vectors for systemic transgene delivery and may be adapted for emerging gene editing technologies. Furthermore, AAV vectors can have high efficiency, low immunogenicity, and selective tropism towards different tissues.

Methods: In this study, we screened 18 natural and engineered recombinant AAV variants. AAVs constitutively expressing Cre-recombinase (under a CAG promoter) were assessed using a murine fracture model using the Ai9 reporter mouse line (Cre-dependent tdTomato expression). AAVs were injected into fractures at time of surgery, and transduction efficiency assessed after two weeks. Transduced osteoblasts were detected using fluorescent alkaline phosphatase staining. Next, a systemic delivery model was performed using tail vein injection, which compared variants AAV2, AAV8, and AAV-DJ. Additionally, Cre expression was restricted using bone cell specific promoters Sp7 and Col2.3. Transduction of bone, and other tissues including brain, heart, lung, liver, spleen and kidney were examined after two weeks.

Results: AAV8, AAV9, and AAV-DJ were able to yield robust tdTomato expression within the healing fracture callus at the study end point. AAV8 and AAV-DJ showed the highest transduction of osteoblastic cells within the callus, which were then used for the systemic delivery. Following tail vein injection, AAV8 was able to transduce cells within the skeleton. Restriction of reporter expression to bone cells was facilitated by constructs utilising the Sp7 and Col2.3 promoters, with Sp7 providing the greatest specificity. At moderate doses (5 × 10¹¹/mouse or ~2.5 × 10¹³/kg) high levels of tdTomato+ osteoblasts and osteocytes were observed throughout the long bones.

Conclusion: We have identified AAV variants with a high tropism for murine bone cells, and vectors for high efficiency in vivo gene delivery to bone. The AAV8-Sp7-Cre vector has significant practical applications for inducing gene deletion in floxed mouse models in post-natal bone. Future work will validate AAV vectors that enable targeted CRISPR gene editing and deletion that have relevance to the treatment of genetic bone disease.

Disclosure: AS and DL have received funding support from Amgen, Novartis AG, Celgene Corp, and N8 Medical for research unrelated to this study. CJM has received funding support from Alexion, Novartis AG for research unrelated to this study.