Induced pluripotent stem cell-based modeling of hemolytic anemia in patients with compound heterozygous KLF1 mutations reveals defective erythroid differentiation

BACKGROUND: Transfusion-dependent hemolytic anemia caused by compound heterozygosity due to mutations in the erythroid Krüppel-like factor 1 (KLF1) gene is a rare and severe blood disorder. The clinical manifestations of the patient are mainly related to erythroid cells. Moreover, the roles of the identified KLF1 mutations in the pathophysiology of this disease remain unclear due to the lack of an appropriate study model. The advent of genome editing technology combined with the generation of patient-specific induced pluripotent stem cells (iPSCs) may provide a better understanding of the molecular mechanisms underlying this disease in an in vitro system and offer a novel therapeutic approach in the future. METHODS: KLF1-mutant iPSCs were generated from patients with compound heterozygosity of KLF1 mutations, and the mutation was corrected through the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system together with a single-stranded oligodeoxynucleotide donor template (ssODN). The obtained iPSC lines were differentiated towards erythroid cells, and the disease-related phenotypes were examined. RESULTS: Erythroid cells derived from KLF1-mutated iPSCs had lower proliferative capacity, showed delayed maturation, and expressed lower level of the KLF1-related gene, CD44. These results were consistent with some of the phenotypes observed in the patients. After CRISPR/Cas9 gene editing, the corrected iPSCs retained pluripotency, exhibited a normal karyotype, and had undetectable off-target mutations. Importantly, some of the defects were partially restored after genetic correction of the KLF1 gene. CONCLUSIONS: KLF1-iPSCs presented disease-related phenotypes of compound heterozygous KLF1 mutations, which could be mediated by gene editing through CRISPR/Cas9 and ssODN. This study offers a useful strategy for studying the underlying disease mechanisms of rare diseases, which could be applied to the development of novel treatments for inherited blood disorders in the future.