Identifying the dominant variant of a cis-double-variant in COL1A1 from an osteogenesis imperfecta patient and developing gene therapies.

Osteogenesis imperfecta (OI) is a hereditary skeletal disorder that is mainly caused by variants in COL1A1/2. So far, no specific treatment has been developed to correct its underlying etiology. We aimed to gain a better understanding of the pathological mechanisms of OI and develop gene editing techniques to correct OI-causing variants. A de novel cis-double-variant c.[175C>T;187T>A] in COL1A1 was identified from a 5-year-old OI patient by whole exome sequencing. Three peptide nucleic acids (PNAs) were designed and then transfected patient-derived fibroblasts. PNA2 affected the translational strand and induced an optimal interfering effect at 0.25 μM concentration, proved by Sanger sequencing, qPCR, Western blot, and immunostaining. Additionally, induced pluripotent stem cells (iPSCs) were cultured from patient-derived fibroblasts. Clones of iPSCs with c.187T>A variant and those with both variants largely restored their osteogenic capacities after CRISPR/Cas9 gene editing, which corrected the variants. Importantly, correcting c.187T>A variant alone in CRISPR-edited iPSCs was sufficient to alleviate OI phenotypes, as indicated by increased levels of COL1A1, COL1A2, ALP mRNAs, and COL1A1 protein. Our findings suggest that c.187T>A is the dominant variant of cis-double-variant in COL1A1 that led to OI, and PNA interference and CRISPR/Cas9 gene editing may be new therapeutic tools for OI treatment.