Abstract 21177: Seamless Gene Correction of LDLR in Familial Hypercholesterolemia Induced Pluripotent Stem Cells Mediated by CRISPR/Cas9 and PiggyBac

Abstract

Introduction: Familial hypercholesterolemia (FH) is an autosomal disorder, characterized by elevated circulating lipoproteins and high risk of premature coronary heart disease. Mutations in low-density lipoprotein receptor gene (LDLR) is one of the major causes. Induced pluripotent stem cell (iPSC) technology helps us generate cell model with patient-specific genetic background. In this study, we aimed to correct LDLR mutation in iPSCs reprogrammed from an FH patient, using CRISPR/Cas9 and piggyBac . Methods: Patient-specific iPSCs were generated from an FH patient with heterozygous duplication of “TGCTGGC” (c.2108_2114dup, p.Ala705fsX14) in exon 14 of LDLR. CRISPR/Cas9 targeting the mutation site and piggyBac were employed to correct the mutation via homologous recombination. Corrected iPSCs were subjected to karyotype analysis, and a series of tests of pluripotency, then differentiated into hepatocyte-like cells (iHeps) to assess the LDLR function in vitro and in vivo . Results and conclusions: We designed two sgRNAs which showed high cutting efficiency in the target site. Co-delivery of CRISPR/Cas9 and piggyBac in FH iPSCs facilitates the integration of donor constructs in the target site with high efficiency. Then, the drug selection cassette was removed by hyPBase expression vectors. After two rounds of genetic manipulation, we achieved a seamless genetic correction in iPSCs, which showed retain full pluripotency and normal karyotypes. In addition, the corrected iPSCs can be differentiated into functional iHeps efficiently, and these iHeps showed restored function of LDLR, characterized by recovery of LDL receptor expression levels and the capacity of LDL uptake. Our study highlights the feasibility of using CRISRP/Cas9 and piggyBac to generate seamless, genetically corrected patient-specific iPSCs, and functional iHeps. These genetically corrected cells are not only ideal models for disease modeling, but also the potential source for autologous cell replacement therapies. Acknowledgment: This work was supported by the Shenzhen Science and Technology Council Basic Research program (JCYJ20150331142757383), the Hong Kong Research Grant Council Theme Based Research Scheme (T12-705/11).