Osteogenesis imperfecta (also known as brittle bone disease or OI) is a debilitating bone fragility disease often leading to frequent fractures. Although numerous genes are reported to be involved in the pathogenesis of OI, 85-90% of OI cases are attributed to mutations in the genes COL1A1 and COL1A2, encoding for the α1 and α2 chains of type 1 collagen, respectively. We report a patient mutation featuring a 20bp deletion (∆20) in COL1A1, producing a readthrough in the C-terminal pro-peptide of the α1 chain of type 1 collagen. While this mutation is a candidate for gene therapy, these therapeutic strategies require distinct cell and animal models for pre-clinical testing.
Using CRISPR gene editing, we have developed a HEK293T cell line harbouring the ∆20 mutation. An SpCas9 nickase approach with homology directed repair (HDR) yielded a 73.6% editing efficiency, with 26.4% cells incorporating other DNA edits. In parallel, we used CRISPR to generate a genetically modified mouse line harbouring an analogous ∆20 mutation. With evidence for ∆20 homozygosity being lethal, the bone phenotype of ∆20 heterogeneous mice (∆20/+) was characterized by MicroCT. Both trabecular and cortical bone parameters were reduced in the ∆20/+ male and female mice including trabecular BV/TV (-24.6% males, -26.9% females p < 0.05) and cortical thickness (-19.7% males, -12.4% females p < 0.05). Predicted bone strength (polar moment of inertia) was less, which will be confirmed by mechanical testing.
These models will enable further understanding of the OI disease pathobiology in C-terminal pro-peptide mutations, whilst also serving as in vitro and in vivo testing platforms for CRISPR-based gene therapies for disease rescue.