Poster Presentation 29th Australian and New Zealand Bone and Mineral Society Annual Scientific Meeting 2019

Generating a toolbox for screening genetic variants associated with bone fragility disorders (#170)

Alexandra O'Donohue 1 2 , Anousha Rahimzada 1 3 , Lucinda Lee 1 2 , Craig Munns 4 , Andrew Biggin 2 4 , Aaron Schindeler 1 2
  1. Bioengineering & Molecular Medicine Laboratory, The Children's Hospital at Westmead, Sydney, New South Wales, Australia
  2. Discipline of Child & Adolescent Health, The University of Sydney, Sydney, New South Wales, Australia
  3. Applied Medical Science, The University of Sydney, Sydney, New South Wales, Australia
  4. Institute of Endocrinology and Diabetes, The Children's Hospital at Westmead, Sydney, New South Wales, Australia

Background: Validating the role of gene variants in disease pathogenesis remains challenging, and the discovery of such variants is increasing with the accessibility of sequencing technology. This is particularly pertinent in osteogenesis imperfecta, where uncertain putative mutations can impact family planning, clinical management, and future gene therapy. We are developing a streamlined CRISPR-based system for characterising these variants of unknown significance (VUSs) using in vitro and in vivo assays for bone.

Aims: (1) To create a bank of human mesenchymal stem cell (MSC) lines with mutations in genes associated with bone fragility; (2) To functionally assess gene-edited MSCs following their osteoblastic differentiation.

Methods: Patient VUSs were screened using the gnomAD and PolyPhen2 databases to prioritise likely pathogenic mutations and used to guide a priority gene list. Knockout and base-editing guides were generated using crispr.mit.edu and ligated into CRISPR-Cas9 plasmids. Constructs were screened using the T7 E1 assay in HEK293 cells to confirm mutational efficiency.

Results: Examination of clinical VUSs in a cohort of 120 bone fragility disorder patients identified LRP5, BMP1, LEPRE1, PLOD2, and FKBP10 as high priority genes. CRISPR-Cas9 strategies were designed and constructs based on the PX459 plasmid (Addgene) were generated by sub-cloning short oligonucleotides encoding specific sgRNA sequences. Initial work using the human foetal osteoblast line (hFOB1.19) revealed the cell line to be slow-growing and unsuitable for CRISPR gene editing. This led to the adoption of a 2-step process where CRISPR constructs are validated in HEK293 and functionally tested in ASC52telo MSCs using biochemical assays and an in vivo xenograft assay.

Discussion: This approach is currently being validated and the goal is to streamline the process for use in a NATA-accredited hospital laboratory within 2 years. This platform has considerable utility for human diagnostics, and analogous methods are certainly possible for conditions outside of bone.