Development of CRISPR-based gene therapy for dominant negative Osteogenesis Imperfecta

Abstract

Osteogenesis imperfecta (OI) is a genetic bone fragility disorder characterised by low-trauma fractures. Most cases arise from COL1A1/COL1A2 mutations, with a smaller proportion caused by defects in genes involved in collagen processing, mineralisation, or chaperone function. Current management reduces fracture risk but does not correct the underlying mutation. CRISPR-Cas9 genome editing offers a potential therapeutic approach by directly targeting disease-causing variants. Chapter 1 reviews OI pathobiology and key advances in CRISPR-Cas9 editing.

Chapter 2 demonstrates proof-of-concept correction of the IFITM5 c.-14C>T mutation (type V OI). A stable mutant cell line was engineered and adenine base editing successfully corrected the variant, establishing a practical pipeline for pre-clinical testing of editing strategies.

Chapter 3 characterises a de novo COL1A1 mutation (c.4323_4342del; Δ20) causing severe type IV OI. The mutation generates a 103-amino acid readthrough in the α1 C-terminal pro-peptide, consistent with a dominant-negative mechanism. Patient-derived cells showed altered osteogenic gene expression, ER enlargement, and misfolded collagen. A Col1a1Δ20/+ mouse model exhibited abnormal bone microarchitecture, reduced biomechanical strength, altered bone cell histomorphometry, and disrupted collagen organisation.

Chapter 4 develops an allele-specific CRISPR therapy targeting the Δ20 allele. An engineered COL1A1Δ20 cell line enabled screening of disruption strategies; an SaCas9 approach showed strong on-target activity and specificity in vitro. Edited cells persisted in bone following osteotropic AAV delivery, but in vivo disruption and phenotypic rescue were not detected, indicating delivery limitations.

Overall, this thesis establishes CRISPR-Cas9 as a powerful platform for OI disease modelling and therapeutic development, while highlighting key translational barriers and future directions (Chapter 5).