Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

Wang, N.; Yang, K.; Xie, X.; Cui, S.; Pei, X.; Zhao, X.; Hao, D.; Jia, Y.; Yang, G.; Liu, R.; Chen, P.; Dong, W.; Huang, Y.; Lv, X.; Zhang, Z.; Liu, D.

doi:10.64898/2026.01.30.702713

bioRxiv · 10.64898/2026.01.30.702713

Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing

Wang, N.·Yang, K.·Xie, X.·Cui, S.·Pei, X.·Zhao, X.·Hao, D.·Jia, Y.·Yang, G.·Liu, R.·Chen, P.·Dong, W.·Huang, Y.·Lv, X.·Zhang, Z.·Liu, D.

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Abstract

The {gamma}- to {beta}-globin switch is intricately regulated during human ontogeny, and this process is manipulated for therapeutic approaches to treat {beta}-hemoglobinopathies by activating {gamma}-globin expression. Several genetic strategies to reactivate HbF have partially reversed the {gamma}- to {beta}-globin switch and ameliorated the clinical symptoms of {beta}-hemoglobinopathies. However, whether the {gamma}- to {beta}-globin switch can be completely reversed remains unknown. Completely reversing the {gamma}- to {beta}-globin switch requires a thorough redirection of the locus control region (LCR) from interacting with the {beta}-globin gene (HBB) to interacting with the {gamma}-globin gene (HBG). Here, we found that disrupting the KLF1-mediated HBB-LCR interaction by mutating the CACCC motif in HBB leads to the release of the LCR and its retargeting to other {beta}-like globin genes. Moreover, simultaneously disrupting the KLF1-mediated HBB-LCR interaction and the epigenetic repression of HBG by combined editing of the CACCC motif in HBB and the TGACCA motif in HBG reinforces the HBG-LCR interaction, resulting in almost exclusive {gamma}-globin expression while nearly absent {beta}-globin expression, achieving near complete reversal of the {gamma}- to {beta}-globin switch. This finding demonstrates the comprehensive regulation of the {gamma}- to {beta}-globin switch by gene competition and gene silencing mechanisms. This finding also suggests that silenced genes can be fully activated through the redirection of enhancer-promoter contacts and that the specificity of enhancer-promoter contact within chromosomal domains is achieved through the transcription factor clusters binding to enhancers and promoters. Combined editing of the CACCC&TGACCA motifs also offer a more optimal therapeutic strategy for {beta}-hemoglobinopathies.

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Wang, N., Yang, K., Xie, X., Cui, S., Pei, X., Zhao, X., Hao, D., Jia, Y., Yang, G., Liu, R., Chen, P., Dong, W., Huang, Y., Lv, X., Zhang, Z., Liu, D.. 2026-02-01. Near completely reversing the γ- to β-globin switch by enhancer release, retargeting and reinforcing. https://doi.org/10.64898/2026.01.30.702713

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