Resistance to modulation of in vivo gene expression by genomic editing

Abstract

Enhancer editing is widely proposed as a strategy to modulate gene dosage therapeutically. EKLF/KLF1, a master erythroid transcription factor, is an attractive target because haploinsufficiency is benign and associated with hereditary persistence of fetal hemoglobin (HPFH). Here we test whether enhancer disruption can recapitulate this state by targeting two KLF1 regulatory elements, EHS1 and INT1. EHS1 editing reduces KLF1 expression and increases γ-globin, and combined INT1/EHS1 targeting produces additional increases in some samples, although responses are variable. In xenotransplantation models, edited cells engraft and retain indels but fail to sustain consistent γ-globin induction. Epigenetic profiling across the KLF1 genetic locus reveals a remarkable resistance to loss of hypersensitive sites. These results establish a key principle: the transcriptional consequences of enhancer editing are strongly constrained by local genomic architecture. Attempts to mimic KLF1 haploinsufficiency by non-coding indels are blunted by dense regulatory architecture, enhancer redundancy, and long-range interaction. Thus, it may be difficult to develop KLF1 as a target for gene therapy for hemoglobinopathies until tools for recapitulating natural mutations via heterozygous edits are developed. Our findings underscore the observation that disruption of non-coding elements may not always recapitulate the biology of haploinsufficiency from heterozygous loss-of-function mutations. Structural and epigenetic properties of the human KLF1 locus constrain the in vivo effects of efficient editing at its non-coding regulatory elements, providing a cautionary tale for the design of functional enhancer modifications.