Generation and Genetic Correction of USH2A c.2299delG Mutation in Patient-Derived Induced Pluripotent Stem Cells.

Xuezhong Liu,Wenliang Zhu,Nicholas Gosstola,Anna M Clark,Derek Dykxhoorn,Justin Lillywhite,Zaohua Huang,Jun Yang,Colin T Maguire,Zheng-Yi Chen

doi:10.3390/genes12060805

Abstract

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.

Highlights

Usher syndrome (USH) is a devastating, clinically and genetically heterogenous disorder characterized by a triad of clinical phenotypes—sensorineural deafness, vision loss, and vestibular dysfunction [1,2]
The advantages of using peripheral blood mononuclear cells (PBMCs) for the derivation of induced pluripotent stem cells (iPSCs) include the ease of blood collection, PBMC extraction, storage, and processing for downstream applications, while skin punch biopsies are more invasive with potential complications from infection, bleeding, and scaring
The USH2A proteins of human and mouse share a similar structural organization, which includes a large extracellular domain composed of LamGL, LamNT, EGF-Lam, LamG, and FN3 motif repeats, a membrane-spanning domain, and an intracellular PDZ-binding C terminus (Figure 1) [28]

Summary

Introduction

Usher syndrome (USH) is a devastating, clinically and genetically heterogenous disorder characterized by a triad of clinical phenotypes—sensorineural deafness, vision loss, and vestibular dysfunction [1,2]. One important application of stem cells for deafness treatment is to use patient-derived induced pluripotent stem cells (iPSCs) to model the impact of specific genetic variants on disease pathogenesis. This approach helps in deciphering underlying pathogenic mechanisms and testing cutting edge gene editing methods including CRISPR/Cas to develop treatment modalities for the identified genetic defect [16,17,18]. We generated a patient-derived iPSC line carrying c.2299delG mutation and successfully corrected this mutation using CRISPR/Cas9-mediated homology-directed repair (HDR). The generation of patient-derived human iPSCs with pathogenic, compound heterozygous USH2A variants (c.1256G > T; p.Cys419Phe) and (c.2299delG; p.Glu767Serfs*21), 2. The protocol can be quantified and standardized among different biomedical labs and institutions

Methods

Results

Conclusion