Prevention of Transcriptional γ-globin Gene Silencing by Inducing
The Hereditary Persistence of Fetal Hemoglobin Point Mutation
Using Chimeraplast-Mediated Gene Targeting
Hemoglobin F (HbF) augmentation is considered a clinically beneficial phenomenon in β-hemoglobinopathies.
Prevention of γ-globin gene silencing, inspired by the hereditary persistence of fetal hemoglobin, may be a suitable strategy
to upregulate HbF expression in these patients. Therefore, our objective was to assess the potential feasibility of induced
Materials and Methods
In this experimental study, human peripheral blood-derived hematopoietic stem cells (HSCs) and the K562 cell line were differentiated to erythroid cells. Erythroid maturation was examined using cell morphology parameters and flow cytometry analysis of CD235a expression. A synthesised chimeraplast was transfected to differentiating cells. The efficiency of chimeraplast delivery into target cells was assessed by flow cytometry. Restriction-fragment length polymorphism and DNA sequencing verified oligonucleotide-directed mutagenesis. Gene conversion frequency and globin genes expression was quantified through Allele specific-quantitaive polymerase chain reaction (AS-qPCR) and quantitative-PCR respectively.
Increase in CD235a-expressing cells along with observations made for different stages of erythroid maturation
confirmed erythroid differentiation in HSCs and K562 cells.
Our results suggest the effectiveness of chimeraplasty in induction of the mutation of interest in both
EPCs and K562 cells. We also demonstrate that the single nucleotide promoter variant was able to significantly inhibit