Donor insertion into CX3CR1 allows epigenetic modulation of a constitutive promoter on hematopoietic stem cells and its activation upon myeloid differentiation.

To improve ex vivo gene therapy strategies involving hematopoietic stem and progenitor cells (HSPCs), we propose a novel knock-in strategy (named KI-Ep) aiming to achieve transgene regulation of the inserted cassette through the acquisition of naturally occurring epigenetic marks. Based on this hypothesis, we selected CX3CR1 (a myeloid-specific gene presenting a poised histone signature on primitive HSPCs) as safe harbor to generate KI-Ep HSPCs. We demonstrated that, unlike the expression pattern achieved with lentiviral vectors (LVs), the insertion of a constitutive expression cassette into the intron 1 of the CX3CR1 locus (CX3CR1-I) in HSPCs resulted in very low expression levels in the more primitive HSPCs but, crucially, strong expression in HSPC-differentiated populations (especially myeloid cells), both in vitro and in vivo. Furthermore, we showed that the promoter of the expression cassette inserted into CX3CR1-I acquired epigenetic marks associated with poised genes during the HSPC stage. These marks transitioned to activated histone states upon KI-Ep HSPCs differentiation. In summary, here, we introduce the KI-Ep concept which enables the epigenetic modulation of the inserted transgene during the HSPCs stem cell stages and its subsequent activation upon differentiation.