Correction of intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing.

Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, Intron one inversion (Inv1) completely splits the gene into two parts and disrupts the transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of coding region. We hypothesized that genetic manipulation of to add a promoter and exon one before the exon two could restore the expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs). The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs. Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.