Abstract
Background:
β-thalassemia and sickle cell disease (SCD) are severe monogenic disorders resulting from mutations in the β-globin gene (HBB), contributing to a substantial global disease burden. β-thalassemia arises from point mutations or small deletions that impair β-globin production, leading to α/β-chain imbalance and ineffective erythropoiesis. SCD is caused by a single nucleotide substitution (Glu6Val), resulting in hemoglobin S polymerization, vaso-occlusion, and hemolytic anemia. While allogeneic hematopoietic stem cell transplantation (HSCT) offers a potential cure, its clinical applicability is limited by donor availability, immunologic risks, and transplant-associated complications. Reactivation of fetal hemoglobin (HbF) via targeted gene editing has emerged as a promising therapeutic strategy.
Aims:
We investigated the functional role of the BCL11A ZnF4 domain in fetal globin silencing and assessed the impact of its targeted disruption in primary and patient-specific stem cell models of β-thalassemia and SCD. Additionally, we developed a targeted delivery system for gene-editing components to improve translational potential.
Methods:
In silico analysis and literature review identified ZnF4 as a critical BCL11A domain mediating γ-globin repression. Using CRISPR-Cas9, we specifically disrupted the ZnF4 domain in CD34+ hematopoietic stem/progenitor cells (HSPCs) isolated from β-thalassemia patients. Post-editing, cells were assessed for HbF induction, γ-globin expression, and erythroid differentiation via colony-forming assays and flow cytometry.
Concurrently, we established integration-free patient-derived induced pluripotent stem cell (iPSC) models using Sendai virus reprogramming of fibroblasts harboring common HBB mutations: IVS1-5 (G>C), FSC 8/9 (+G), and Glu6Val (SCD). A two-stage differentiation protocol directed iPSCs into hematopoietic (CD34+/CD45+) and erythroid (CD71+/CD235a+) lineages under serum-free conditions.
We further designed a novel CD34-targeting peptide to facilitate targeted delivery of gene-editing tools. Liposomal and exosomal vectors conjugated with this peptide were evaluated for cell-specific uptake and HbF induction in vitro and in vivo.
Results:
CRISPR-Cas9-mediated editing of BCL11A ZnF4 in patient-derived HSCs resulted in a robust increase in HbF expression (p < 0.001), without affecting essential erythroid target genes. Edited HSCs retained multilineage differentiation potential and showed enhanced γ-globin expression in erythroid colonies. Comparable results were observed in iPSC-derived HSPCs, confirming the consistency and reproducibility of the ZnF4 targeting strategy across different stem cell platforms.
In vivo validation using a humanized mouse model confirmed increased HbF levels post-editing. The CD34-targeted liposome/exosome delivery system achieved efficient, selective delivery of gene-editing components, leading to increased hemoglobin production and minimal off-target effects.
Conclusion:
Our findings underscore the therapeutic potential of targeting BCL11A's ZnF4 domain for HbF induction in β-thalassemia and SCD. This study represents the first successful generation of gene-edited, patient-derived iPSC models in Pakistan, coupled with a novel, clinically translatable delivery platform. These results pave the way for personalized, gene-editing-based therapies for hemoglobinopathies.
