Osteogenesis Imperfecta (OI) is a genetic bone fragility condition associated with frequent fractures. 85-90% of cases are attributed to mutations in COL1A1 and COL1A2, which encode for the α1 and α2 chains in type 1 collagen respectively. Our group has conceptualised a CRISPR-based gene therapy to ameliorate the impact of dominant negative OI. A prototypical patient mutation was selected, featuring a 20 bp deletion in COL1A1 (∆20) that results in a frameshift and 79 amino acid readthrough in the C-terminal pro-peptide of the α1 chain. This is associated with severe disease. It is hypothesized that CRISPR/Cas9 non-homologous end joining (NHEJ) could induce a frameshift that abrogates the pathogenic readthrough and allow normal collagen folding.
Firstly, HEK293T cells possessing the human mutation (∆20) was generated using a SpCas9 nickase and homology directed repair (HDR). As a therapeutic approach, SaCas9 and a novel guide were expressed in ∆20 cells to induce NHEJ. Editing was achieved in 79% of mutant alleles, with -1 frameshifts shifting the sequence back to the normal reading frame. In parallel, we generated a homologous mutant mouse model (Col1a1∆20/+). This mouse showed lower bone parameters in the tibiae and spine by microCT and biomechanical testing consistent with an OI phenotype. This mouse is an ideal target for preclinical testing of an AAV8-SaCas9-sgRNA∆20 vector designed for in vivo rescue.
In conclusion, this work represents a novel and effective pipeline for the preclinical development of CRISPR gene therapies for OI and may be suitable for n of 1 trialling on the affected patient in the future. This may be transformational as current pharmacotherapies yield increase bone mass but do not cannot address defective bone quality caused by collagen mutations.