(W1350) INVESTIGATING THE BIOLOGICAL PROPERTIES OF CIRCULATING HEMATOPOIETIC STEM/PROGENITOR CELLS IN PEDIATRIC SUBJECTS AS NOVEL SOURCE FOR GENE THERAPY.
PhD Student Università Vita-Salute San Raffaele Milan, Italy
Abstract: Autologous Hematopoietic Stem/Progenitor Cells (HSPCs) gene therapy (GT) foresees HSPC collection and ex vivo gene correction before re-infusion in patients to rescue a disease pathophysiology. However, HSPC harvest, through bone marrow (BM) aspirates or leukapheresis after mobilization (MPB), remains technically challenging, particularly in very young patients. Interestingly, we found that pediatric individuals (Ped) during the first years of life showed increased number of physiologically circulating (cHSPC) in the peripheral blood (PB) and might be exploited as target for GT. Nevertheless, pediatric cHSPC biology remains unexplored to this day. We phenotypically, functionally and transcriptionally profiled cHSPC from pediatric healthy donors (HD) in comparison with other clinically-relevant sources including cord blood (CB), BM and MPB. Through multi-parametric immunophenotyping, applied on a cohort of over 100 Ped HD , we observed comparable HSPC composition among cHSPC, CB and MPB with enrichment of primitive HSPC subsets. Furthermore, single sorted primitive Ped cHSPC showed a high differentiation efficiency and multi-lineage hematopoietic production. To gain insight on the molecular features of cHSPC, we generated a Ped HSPC single-cell RNAseq ATLAS exploiting in-house and published datasets of 65,417 HSPC from PB, BM, CB and MPB. Notably, cHSPC were characterized by a lower cycling activity (~ 90% G0/G1 cells), and were enriched in primitive cells in line with phenotypic characterization. We also identified a cluster classified as long-term HSC, expressing gene signatures associated with stemness properties comparably to the other sources. Ped cHSPC can also be efficiently transduced with lentiviral vectors and expanded ex vivo using UM171. Preliminary results suggest that transduced and expanded Ped cHSPC retain multi-lineage differentiation potential in vitro. Ongoing in vivo experiments in mice will allow to estimate the BM-homing and reconstitution properties of pediatric cHSPC as a novel source and cell target for ex vivo and in vivo Gene Therapy strategies.
