Nikoletta Psatha, Pavel Sova, Grigorios Georgolopoulos, Kiriaki Paschoudi, Mineo Iwata et al.
Gene expression during cell development and differentiation is orchestrated by distal regulatory elements that precisely modulate cell selective gene activity. Gene therapy vectors leverage these elements for precise spatiotemporal transgene expression. Here, we develop a one-shot approach to screen candidate regulatory sequences from large-scale epigenomics data for programmable transgene expression within gene therapy viral vectors. We assess a library of 15,000 short sequences derived from developmentally active elements during erythropoiesis using a clinically relevant reporter vector. These elements display a gradient of transcriptional enhancer activity in erythroid cells, with high cell type restriction and developmental stage specificity. Finally, replacing the canonical β-globin μLCR with a compact enhancer in a β-thalassemia lentiviral vector successfully corrects the thalassemic phenotype in patient-derived hematopoietic and stem and progenitor cells (HSPCs), while increasing viral titers and cell transducibility. Our approach provides further insights into enhancer biology with wider implications for human gene therapy.
Nikoletta Psatha, Pavel Sova, Grigorios Georgolopoulos, Kiriaki Paschoudi, Mineo Iwata, Jordan Bloom, Tatyana Ulyanova, Hao Wang, Alexandra Kirtsou, Ninos-Ioannis Vasiloudis, Matthew S. Wilken, John A. Stamatoyannopoulos, Evangelia Yannaki, Thalia Papayanopoulou, George Stamatoyannopoulos, Jeff Vierstra. Large-scale discovery of potent, compact and erythroid specific enhancers for gene therapy vectors. 2025-05-09. https://doi.org/10.1038/s41467-025-59235-x
A pooled follow-up library of 205 full-size DHS sequences selected from the tile screen was cloned in the same GFP lentiviral screening vector and assayed in WT HUDEP-2 cells by GFP-bin sorting. The processed table contains the author-reported per-element activity scores, hg38 coordinates, and full genomic sequences supplied in Supplementary Data 4.
A 14,999-element library of overlapping tiles from 5,393 erythroid DNase-I hypersensitive sites was cloned upstream of a 169-bp minimal human β-globin promoter driving GFP in an integrating lentiviral vector. WT HUDEP-2 cells were transduced at MOI 0.4, sorted five days later into GFP-low, medium, and high bins, and the integrated tile DNA was sequenced to estimate enhancer activity.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 11 files (ZIP)GSE252159_family.soft.gzGSE252159_tile_counts_per_GFP_bin.txt.gzREADME.mdsample_map.tsvsource_data.xlsxsupplementary_data_1_motif_enrichment.xlsxsupplementary_data_2_tile_library.xlsxsupplementary_data_3_motif_enrichment_selected.xlsxsupplementary_data_4_full_length_205_library.xlsxsupplementary_data_5_individual_40_enhancers.xlsxsupplementary_data_6_TALEN_sequences.xlsx