Editing aberrant splice sites efficiently restores β-globin expression in β-thalassemia

Area of Science:

• Genetics
• Molecular Biology
• Hematology

Background:

• Thalassemias are genetic blood disorders caused by mutations in beta-globin genes.
• Current treatments are limited and often require lifelong management.
• Hematopoietic stem cell (HSC) gene editing presents a potential curative approach for beta-thalassemia.

Purpose of the Study:

• To evaluate the efficacy of CRISPR-Cas9 and CRISPR-Cas12a/Cpf1 gene editing systems in correcting beta-thalassemia mutations.
• To demonstrate the restoration of functional beta-globin expression in edited hematopoietic stem and progenitor cells (HSPCs).
• To assess the potential of splice site disruption as a therapeutic strategy for beta-thalassemia.

Main Methods:

• Targeting specific splice site mutations (IVS1-110G>A and IVS2-654C>T) in beta-thalassemia using Cas9 and Cas12a/Cpf1 ribonucleoprotein (RNP) complexes.
• Utilizing primary CD34+ HSPCs from beta-thalassemia patients.
• Analyzing erythroid progeny for aberrant splicing reversal and beta-globin expression restoration.

Main Results:

• High efficiency and penetrance of therapeutic edits were achieved with both Cas9 and Cas12a/Cpf1 RNPs.
• Edited HSPCs generated erythroid cells with corrected splicing patterns.
• Restored beta-globin expression was observed in the erythroid progeny of edited cells.

Conclusions:

• Allelic disruption of aberrant splice sites is a robust strategy for correcting beta-thalassemia mutations.
• This gene editing approach holds promise for treating a significant portion of transfusion-dependent beta-thalassemia patients.
• The findings support the use of currently available gene-editing technology for therapeutic applications in beta-thalassemia.