Oral Presentation 33rd ASM of the Australian & New Zealand Bone & Mineral Society 2023

What a knock-out! Recombinant adeno-associated viruses for generation of post-natal bone knockouts (#68)

Alexandra K O'Donohue 1 , Ya Xiao 2 , Lucinda R Lee 1 , Timothy Schofield 1 , Tegan L Cheng 1 , Craig Munns 3 4 , Paul A Baldock 2 , Aaron Schindeler 1 5
  1. Bioengineering & Molecular Medicine Laboratory, The Children’s Hospital at Westmead and the Westmead Institute for Medical Research, Sydney, NSW, Australia
  2. Bone Division, Garvan Institute for Medical Research, Darlinghurst, NSW, Australia
  3. Child Health Research Centre, Faculty of Medicine, The University of Queensland, Brisbane, QLD, Australia
  4. Department of Endocrinology and Diabetes, Queensland Children’s Hospital, Brisbane, QLD, Australia
  5. The Children’s Hospital at Westmead Clinical School, The University of Sydney, Sydney, NSW, Australia

Background: Generating conditional gene knockout mice using traditional technologies can be challenging and costly. Such models are of high utility in bone research as many global gene knockouts can be embryonic lethal or yield confounding phenotypes. Our group engineered a bone-targeted recombinant adeno-associated viral vector (AAV8-Sp7-Cre) and speculated it enable bone-selective gene deletion without a need for crossbreeding with Cre-strains. This study aimed to show proof-of-principle in a mouse possessing a conditional allele for the Sclerostin (Sost) gene, which is a critical factor in the negative regulation of bone mass.

Methods: 8-week-old Sostflox/flox were systemically injected with AAV8-Sp7-Cre (5×1011 vg/mouse) or saline controls. After 6 weeks, detailed bone analysis was performed via microCT, biomechanical testing, and bone tissue histology. Ai9 fluorescent Cre-reporter mice were dosed in parallel to verify the specificity and longevity of gene editing.

Results: MicroCT analysis of Sostflox/flox:AAV-Cre mice confirmed a functional effect on bone mass, with an increase of 22% in the bone volume of the vertebrae (p<0.01), translating to a 17% increase in compressive strength (p<0.01). Significant alterations were also seen in the bone microarchitecture and were associated with a +25% increase in the mineral apposition rate. Immunohistochemistry for sclerostin protein and analysis of AAV8-Sp7-Cre mediated recombination in an Ai9 fluorescent reporter mouse model showed specificity and efficiency in osteoblasts and later osteocytes.

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Discussion: Sostflox/flox:AAV-Cre mice showed a high bone mass phenotype and increased bone anabolism consistent with prior reports of Sost null mice. This technology represents a streamlined and versatile approach to generate conditional bone knockout mice that could be applied to a range of floxed mouse strains. Our future research aims to develop AAV8-Sp7-CRISPR gene editing vectors able to disrupt or repair wild-type alleles.