Prime Editing Corrects the HBB Codon 8/9 (+G) Mutation in Patient-Derived Induced Pluripotent Stem Cells and Restores β-Globin Expression in iPSC-Derived Erythroid Cells

hussain, i.; Mumtaz, K.; Javed, M.; Fatima, S.; Zahra, H.; Alam, A.; Jameel, M.; Malik, N. A.; Syed, F.; Rehman, F. U.; Kirmani, S.; Fatima, A.; Mian, A. A.

doi:10.64898/2026.09.22.753453

bioRxiv · 10.64898/2026.09.22.753453

Prime Editing Corrects the HBB Codon 8/9 (+G) Mutation in Patient-Derived Induced Pluripotent Stem Cells and Restores β-Globin Expression in iPSC-Derived Erythroid Cells

hussain, i.·Mumtaz, K.·Javed, M.·Fatima, S.·Zahra, H.·Alam, A.·Jameel, M.·Malik, N. A.·Syed, F.·Rehman, F. U.·Kirmani, S.·Fatima, A.·Mian, A. A.

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Abstract

Homozygosity for the HBB codon 8/9 (+G) frameshift (c.27dup (p.Ser10ValfsTer14)) causes transfusion-dependent {beta}-thalassaemia and is common in South Asia. Prime editing can reverse this insertion without double-strand breaks or donor DNA, but its efficiency depends on pegRNA design. We derived Sendai-reprogrammed iPSCs from a homozygous patient, optimised PEmax editing (spacer, pegRNA extension, secondary nick, MLH1dn) and compared patient, PEmax-treated and control iPSC-derived erythroid cells by flow cytometry, colony assays, RT-qPCR, western blotting and cation-exchange HPLC. Patient iPSCs had a normal 46,XY karyotype, expressed pluripotency markers, formed all three germ layers and were Sendai-free by passage 15. A PAM-disrupting spacer with a +68 nicking sgRNA gave the highest intended-edit frequency 8.4%; MLH1dn added little. PEmax-treated cultures matured and formed colonies more like control than patient cultures, restored HBB transcript (about 4-fold control iPSC-derived cells) and detectable {beta}-globin protein, and contained an HPLC fraction consistent with HbA ( undetectable in patient cells). Fetal haemoglobin (about 80%) and embryonic globin remained predominant. Prime editing corrects HBB c.27dupG in patient iPSCs and restores {beta}-globin expression, with partial restoration of adult haemoglobin in a fetal/embryonic-type erythroid background. Validation in additional donors, haematopoietic stem cells and in vivo is required.

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hussain, i., Mumtaz, K., Javed, M., Fatima, S., Zahra, H., Alam, A., Jameel, M., Malik, N. A., Syed, F., Rehman, F. U., Kirmani, S., Fatima, A., Mian, A. A.. 2026-09-24. Prime Editing Corrects the HBB Codon 8/9 (+G) Mutation in Patient-Derived Induced Pluripotent Stem Cells and Restores β-Globin Expression in iPSC-Derived Erythroid Cells. https://doi.org/10.64898/2026.09.22.753453

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