1) OSR refined an HSC expansion protocol supporting a net HSCs maintenance during ex vivo culture, though lentiviral transduction adds stress that may impact HSC function if non-optimised protocols are used. Long-term grafts deriving from ex vivo expanded HSCs have a highly polyclonal composition, which is encouraging for clinical translation.
Homology-driven repair (HDR) has been successfully established for gene editing in the expansion culture setup and lipid nanoparticles (LNPs) resulted beneficial in improving HDR efficiency and stress response.
Integrating base (BE) and prime editing (PE) techniques in the HSC expansion protocol resulted promising with a slightly higher editing efficiency using LNPs.
2) UNAV developed a comprehensive multiome dataset to explore HSC subpopulations and novel regulatory features within CD34+ cells isolated from bone marrow and mobilized peripheral blood. UNAV and CIEMAT established standardized protocols for isolating, culturing, and modifying CD34+ cells, ensuring consistent multiomic assessments, with further experiments planned. OSR contributed multiome data from ex vivo expanded cells, which are being integrated with the data from UNAV. Gene editing experiments targeting the AAVS1 locus and initial single-cell sequencing studies are currently underway.
3) UKLFR developed a comprehensive safety pipeline to assess CRISPR-based editing in HSC expansion, highlighting the persistence of chromosomal risks despite double-nickase use. OSR found that HDR-based engineering induces a senescence-like response in HSPCs, involving p53 and inflammation, necessitating mitigation strategies. Ex vivo culture triggers p38 MAPK activation, increasing ROS and proliferative stress, which can be alleviated by p38 inhibition to enhance self-renewal. The team aims to test DNA damage response (DDR) and senescence modulators, optimize genome editing base and prime editors, and evaluate genotoxicity risks.
4) CIEMAT significantly advanced studies on editing approaches in Fanconi Anemia (FA) and Diamond Blackfan Anemia (DBA). They applied PE and BE to identify the most suitable approaches for future treatments. Ongoing research aims to develop precise and effective therapies to address their underlying genetic causes. CHUV has tested novel, potentially less toxic strategies for engraftment of genetically-engineered HSCs, showing promising potential for further refinement. Progress was also made in HSC-based gene therapies for cancer, focusing on tumor infiltrating macrophage-specific loci driving the expression of antitumor genes. These advances will guide further development in the coming year.
5) NBM-FMS established a long-term gene therapy data repository within EGA. To support education and engagement within X-PAND, video resources have been expanded to cover key topics like Gene Therapy for Multiple Myeloma and Deep Learning. The team made significant progress in refining the HSC fingerprint, with consistent findings across hematopoietic compartments. Multiomic profiling confirmed the ability to distinguish cells carrying transferred genes, replicating in vitro expression patterns observed in vivo. Efforts to map HSC gene regulatory networks are ongoing, with a strong focus on CellOracle-based analyses to identify key transcriptional elements involved in haematopoiesis. The team continues to enhance open-source tools and guidelines. (i) The GitHub repository for single-cell multiome and CITE-seq pipelines is regularly updated to improve usability and reproducibility. (ii) The newly improved GeneSetCluster2.0 tool has been designed to facilitate functional analysis in single-cell data.
