Nanoengineered 3D culture substrate enables superior long-term and polyclonal engraftment of genetically engineered hematopoietic stem and progenitor cells

Ex vivo culture of hematopoietic stem and progenitor cells is crucial for gene therapy applications but inadvertently triggers detrimental cellular responses, potentially threatening clinical success. In this study, we employ a biocompatible 3D scaffold with cell-scale resolution to provide mechanical support during ex vivo manipulation. This innovative 3D culture system improves multi-lineage differentiation and engraftment capacity by leveraging mechanobiological control over nuclear morphology, cytoskeleton organization, metabolism, and DNA integrity. Notably, 3D culture enables efficient genetic engineering across multiple platforms, including long-range gene editing, base- and prime-editing, and lentiviral gene addition. Moreover, this scaffold increases the clonal output and the long-term persistence of genetically engineered cells in xenotransplantation experiments and outperforms traditional 2D culture in a clinical protocol for Wiskott-Aldrich Syndrome. Overall, we propose a transformative approach to enhance the efficacy and safety of hematopoietic stem cell-based gene therapies within emerging and pre-existing clinical workflows.