Abstract: Friedreich’s ataxia (FRDA or FA) is a rare autosomal recessive neurodegenerative disorder caused by guanine-adenine-adenine (GAA) repeat expansions in the first intron of the frataxin (FXN) gene. To address this underlying genetic cause, we developed paired prime editors (twinPE) to precisely delete GAA repeats. Using a 38-bp attachment site of bacteria (attB) sequence as the reverse transcription template (RTT), we achieved a precise 1.2-kb deletion with attB insertion in HEK293T cells with a 48.0% editing efficiency. We further delivered two synthetic pegRNAs and PE6 mRNA into GM03816 FRDA fibroblasts and GM23404, as well as GM23913 patient-derived induced pluripotent stem cells (iPSCs) via electroporation, achieving average editing rates of 13.3%, 40.6%, and 25.0%, respectively. In both patient-derived iPSCs, twinPE successfully restored FXN expression, resulting in a ∼2-fold increase in mRNA levels, relative to non-treated controls, demonstrating a promising functional correction of gene expression. Importantly, NGS analysis revealed that >95% of all edits were precise, with minimal indels across all tested cell types. We directly delivered twinPE to iPSC-derived motor neurons (iMNs) using virus-like particles (VLPs), achieving 12.6% editing. In summary, our results demonstrate that twinPE enables efficient and precise deletion of GAA repeats across multiple cell types, including iMNs. The restoration of FXN mRNA in iPSCs represents a promising functional recovery, establishing a foundation for twinPE-mediated genome editing as a potential therapeutic strategy for FRDA.
Tuesday, September 29, 2026
TwinPE delivered as RNA or VLP enables precise deletion of GAA repeats in the FXN gene
Xuntao Zhou, Thomas C. Todeschini, Tianxiong Yu, Runyuan Wang, Bin Liu, Annie Collins, Whitney Welch, Aditya Valji Ansodaria, Zhiping Weng, Scot A. Wolfe, Jonathan K. Watts, Erik J. Sontheimer, Wen Xue, TwinPE delivered as RNA or VLP enables precise deletion of GAA repeats in the FXN gene, Cell Insight, 2026, 100368, ISSN 2772-8927, doi:10.1016/j.cellin.2026.100368.
