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

Generating cell-based models of FGFR3-related diseases (#212)

Julian FH Chu 1 , Alexandra O'Donohue 1 , Samantha Ginn 2 3 , Aaron Schindeler 1 3
  1. Bioengineering and Molecular Medicine Laboratory, The Children’s Hospital at Westmead, Westmead, NSW, Australia
  2. Gene Therapy Unit, the Children's Medical Research Institute, Westmead, NSW, Australia
  3. Faculty of Medicine and Health, The University of Sydney, Sydney, NSW, Australia

Background: Fibroblast growth factor receptor 3 (FGFR3) is a critical factor in mammalian growth and development. Several well-defined mutations in FGFR3 are associated with human disease. Achondroplasia (ACH) or dwarfism is an autosomal dominant disease associated with short stature. The most common causative mutations for ACH are c.1138G>A and c.1138G>C, which make up >90% of all cases. Thanatophoric dysplasia type 1 and 2 (TDI/TDII) are also caused by mutations in FGFR3 but are more severe and result in neonatal death. All reported cases of TDII are caused by a c.1948A>G mutation. These common mutations represent ideal gene therapy targets they are base editable using emerging CRISPR technology.

Aim: Generate cell models featuring ACH and TDII mutations using CRISPR/Cas9 editing.

Methods: CRISPR gene editing strategies were designed using CHOPCHOP v3 and Integrated DNA Technology systems. Oligonucleotides (Sigma-Aldrich) encoding for specific sgRNAs were subcloned into CRISPR plasmids (Addgene) and long oligos were made incorporating c.1138G>A and c.1948A>G mutations. Cell pools and stable lines were generated using HEK293T cells (CellBank Australia). Sequencing of constructs and gDNA was performed by AGRF and analysed using the ICE CRISPR Analysis Tool (Synthego). 

Results: Expression plasmids with subcloned FGFR3-specific sgRNAs were confirmed by sequencing. Both dsDNA cutting and dual nickase vectors were produced. CRISPR constructs were introduced by lipofection and used to create cell pools. No-long-oligo controls demonstrated a lack of HDR. Approaches with high HDR rates were then used to generate clonal cell lines and also confirmed by sequencing. CRISPR-BE constructs for therapeutic repair were designed and subcloned in parallel.

Discussion: Generation of these cell lines is an important first step towards validating future gene therapy approaches. While plasmid delivery is not practicable as a gene therapy treatment, the CRISPR-BE methodology can be adapted using adeno-associated viral vector (AAV) delivery systems targeting musculoskeletal tissues available in-house.