CRISPR/Cas9 genome editing for a correction of LMNA p.H222P mutation using human-induced pluripotent stem cells (hiPSCs)

Abstract

Cardiomyopathy caused by A-type lamins gene ( LMNA ) mutations is associated with an early development of heart failure and a poor prognosis. Gene editing is a promising approach to cure these monogenic disorders. CRISPR/Cas9-based strategies have recently allowed an in vivo correction of mutations leading to Duchenne muscular dystrophy. We aim to develop a CRISPR/Cas9-based gene editing therapy of the LMNA p.H222P mutation, which leads to Emery Dreifuss muscular dystrophy. The development and testing of this therapy are carried out on patient-specific induced pluripotent stem cells (iPSCs). We first compared the efficiency of several guide RNAs for two Cas9 enzymes (NmCas9 and SpCas9) in HEK cells and in iPSCs. For the correction, asymmetric single-stranded donor matrices were designed by taking 36 bp on the PAM-distal side and 91 bp on the PAM-proximal side, either on the targeting or on the non-targeting strand. Silent mutations were added to prevent edited DNA recutting and allow the identification of the edited strand. In HEK cells, SpCas9 generated double-strand breaks with higher efficiencies than NmCas9. Two guide RNAs for SpCas9 were therefore selected to be tested in iPSCs carrying the LMNA p.H222P mutation: one with higher specificity (G1), and another with higher efficiency (G2). IPSCs nucleofection with the donor matrix on the targeting strand and either G1 or G2 resulted in 48 and 9 clones respectively. This may be due to a higher double-strand cut efficiency for G2 associated with a lower homologous direct repair (HDR) efficiency. Only one clone issued from G1 had efficiently integrated the donor matrix. To improve HDR efficiency, the matrix on the non-targeting strand as well as the addition of small molecules are currently being tested. We succeeded to obtain one corrected clone using our gene-editing strategy. This will allow us to assess the impact of the LMNA p.H222P mutation by comparing the characteristics of cardiomyocytes derived from mutated iPSCs to their isogenic control. However, the correction needs optimization to increase HDR efficiency before being translated in vivo.