Usher syndrome (USH) is the leading cause of inherited combined hearing and vision loss. As an autosomal recessive trait, it affects 15,000 people in the United States alone and is responsible for ~21% of inherited blindness and 3 to 6% of early childhood deafness. Approximately 2/3 of the patients with Usher syndrome suffer from USH2, of whom 85% have mutations in the USH2A gene. Patients affected by USH2 suffer from congenital bilateral progressive sensorineural hearing loss and retinitis pigmentosa which leads to progressive loss of vision. To study the molecular mechanisms of this disease and develop a gene therapy strategy, we generated human induced pluripotent stem cells (iPSCs) from peripheral blood mononuclear cells (PBMCs) obtained from a patient carrying compound heterozygous variants of USH2A c.2299delG and c.1256G>T and the patient's healthy sibling. The pluripotency and stability were confirmed by pluripotency cell specific marker expression and molecular karyotyping. Subsequent CRISPR/Cas9 genome editing using a homology repair template was used to successfully correct the USH2A c.2299delG mutation back to normal c.2299G in the generated patient iPSCs to create an isogenic pair of lines. Importantly, this manuscript describes the first use of the recombinant Cas9 and synthetic gRNA ribonucleoprotein complex approach to correct the USH2A c.2299delG without additional genetic effects in patient-derived iPSCs, an approach that is amenable for therapeutic genome editing. This work lays a solid foundation for future ex vivo and in vivo gene therapy investigations and these patient's iPSCs also provide an unlimited resource for disease modeling and mechanistic studies.
Keywords: CRISPR/Cas9 gene therapy; USH2A; patient-derived induced pluripotent stem cells.