Donor insertion into CX3CR1 allows epigenetic modulation of a constitutive promoter on hematopoietic stem cells and its activation upon myeloid differentiation
Year: 2025
Type: article
Abstract: 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.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkaf344
Institutions: Universidad de Granada, Instituto de Investigación Biosanitaria de Granada, Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research, Fundación para la Investigación Biosanitaria de Andalucía Oriental - Alejandro Otero, University of Freiburg, University Medical Center Freiburg, Centre for Biomedical Network Research on Rare Diseases, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas, Instituto de Investigación Sanitaria Fundación Jiménez Díaz, Consejo Superior de Investigaciones Científicas, Instituto de Parasitología y Biomedicina "López - Neyra", Leiden University Medical Center
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,992
Funders: Canadian Institute of Steel Construction, Ministerio de Ciencia, Innovación y Universidades, European Cooperation in Science and Technology, Centro para el Desarrollo Tecnológico Industrial, Federación Española de Enfermedades Raras, Ministerio de Ciencia e Innovación, Leids Universitair Medisch Centrum, Universidad de Granada, Spark Therapeutics, Instituto de Salud Carlos III, European Regional Development Fund, NextGenerationEU
Awards: UiTRUCKs: Células T CARs universales e inducibles de cuarta generación para inmunoterapia de linfomas refractarios., G1668313643, G2466573103, G3519466985, G353479501, G4252420834, G5316918490, G6423621450, G6515544267, Optimización de la edición genómica para su aplicación a terapia génica de enfermedades monogénicas, Herramientas mejoradas para terapia celular-génica de la enfermedad de Pompe
OpenAlex ID: W4409904530