Fig. 1: An enhancer discovery pipeline for gene therapy applications.

A one-shot approach for screening and functionally validating erythroid specific enhancers for lineage-specific induction of transgene expression at therapeutic levels in gene therapy viral vectors. Briefly, 5393 DNase I hypersensitive sites (DHS) activated de novo during ex vivo human erythropoiesis were selected and broken down into ~3 198bp-long tiles each, comprising a library of total ~15 k elements. The tiles were then cloned into a clinically relevant GFP reporter lentiviral vector and HUDEP-2 cells were transduced at MOI (Multiplicity of infection) <1. The cells were then sorted by flow cytometry into 3 bins based on GFP expression. DNA libraries were constructed from each bin and read counts were assigned to each tile as function of GFP expression. Top performing tiles were then mapped to their full-size DHSs which were then in turn cloned into a therapeutic vector where the candidate elements were assessed based on their ability to achieve phenotypic correction of β-thalassemia patient donor derived erythroid cells. MFI: Mean Fluorescence Intensity.