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
- 1Centre for Regenerative Medicine and Stem Cell Research, Aga Khan University, Karachi, Pakistan
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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BibTeX
@misc{hussain2026prime,
title = {{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}},
author = {i. hussain and K. Mumtaz and M. Javed and S. Fatima and H. Zahra and A. Alam and M. Jameel and N. A. Malik and F. Syed and F. U. Rehman and S. Kirmani and A. Fatima and A. A. Mian},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.22.753453},
url = {https://www.biorxiv.org/content/10.64898/2026.09.22.753453v1},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - 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
AU - i. hussain
AU - K. Mumtaz
AU - M. Javed
AU - S. Fatima
AU - H. Zahra
AU - A. Alam
AU - M. Jameel
AU - N. A. Malik
AU - F. Syed
AU - F. U. Rehman
AU - S. Kirmani
AU - A. Fatima
AU - A. A. Mian
PY - 2026
DA - 2026/09/22
PB - bioRxiv
DO - 10.64898/2026.09.22.753453
UR - https://www.biorxiv.org/content/10.64898/2026.09.22.753453v1
AB - 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.
ER -