Donor insertion into <i>CX3CR1</i> 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.
Authors
- Guillermo Guenechea-Amurrio
- Pilar Muñoz (ORCID: https://orcid.org/0000-0002-7985-9859)
- Araceli Aguilar-González (ORCID: https://orcid.org/0000-0002-2960-8672)
- Francisco Martı́n (ORCID: https://orcid.org/0000-0003-1961-8612)
- Manuel A.F.V. Gonçalves (ORCID: https://orcid.org/0000-0002-8111-1073)
- Carla Fuster‐García (ORCID: https://orcid.org/0000-0002-4209-7248)
- Francisco J. Molina-Estévez (ORCID: https://orcid.org/0000-0001-9565-2199)
- Iris Ramos-Hernández (ORCID: https://orcid.org/0000-0001-8351-3693)
- Francisco J. Sánchez‐Luque (ORCID: https://orcid.org/0000-0003-1138-6124)
- Toni Cathomen (ORCID: https://orcid.org/0000-0002-7757-4630)
- María L Lozano-Vinagre
Institutions
- University of Freiburg (DE)
- Universidad de Granada (ES)
- National Academies of Sciences, Engineering, and Medicine (US)
- Leiden University Medical Center (NL)
- University Medical Center Freiburg (DE)
- Institute for Transfusion Medicine (US)
- Centre for Biomedical Network Research on Rare Diseases (ES)
- Instituto de Investigación Biosanitaria de Granada (ES)
- Pfizer-University of Granada-Junta de Andalucía Centre for Genomics and Oncological Research (ES)
- Instituto de Parasitología y Biomedicina "López - Neyra" (ES)
- Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (ES)
- Universidad Autónoma de Madrid (ES)
Publication Details
- Journal
- Nucleic Acids Research
- Published
- 2025-04-22
- DOI
- https://doi.org/10.1093/nar/gkaf344
- Citations
- 2
- Primary Topic
- Virus-based gene therapy research
- Type
- article
- Field-Weighted Citation Impact
- 3.63
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