476. Clonal Tracking of Engineered Hematopoiesis in Vivo in Humans by Insertional Barcoding

Upon gene therapy (GT) for adenosine deaminase (ADA) deficient-SCID and Wiskott-Aldrich Syndrome (WAS), gene-corrected hematopoietic stem/progenitor cells (HSPC) generated a stable genetically engineered hematopoietic system where each vector-marked cell is univocally barcoded by a vector integration site (IS). To study the dynamics of human hematopoietic system, we collected by LAM-PCR+Illumina sequencing 28.539.414 sequence reads corresponding to 89.373 IS tagging clones belonging to 13 different cell types purified from the bone marrow and the peripheral blood of 4 WAS patients up to 48 months after GT. We firstly identified identical IS shared among CD34+ progenitors, and mature Myeloid/Lymphoid cells as markers of the real-time clonal output of vector-marked HSPC clones in vivo. We unraveled the nature and timing of short, intermediate and long term HSPC output showing that CD34+ clones active at 3-6 months after GT are not detectable at later follow up. Distinct waves of HSPC diversity were observed during the first 6-9 months after GT reaching a homeostatic equilibrium only by 12 months. By unsupervised clustering of IS similarities among lineages we unveiled diverse HSPC output towards lymphoid, myeloid and megakaryo-erythroid cells showing hints of a NK cells origin distinct from T and B cells. We exploited IS similarities to infere and test hematopoietic hierarchies by combining conditional probability distributions and static/dynamic graphical models of dependencies. We also estimated by mark-recapture approaches that just few thousands clones (1185-2884) are responsible for the long-term maintenance of the whole genetically engineered hematopoietic system. The ongoing analyses on IS collected from 7 distinct CD34+ subtypes isolated from GT patients will further increase HSPC tracking resolution. To evaluate the preservation of activity by transplanted HSPC we exploited data derived from the IS-based tracking of 4.845 clones in ADA-SCID patients performed for up to 6 years after GT. We showed that identical IS are consistently detected at multiple lineages level even several years after GT. Strikingly, by semi-quantitative PCRs on specific vector-genome junctions we tracked a fluctuating but consistent output of marked HSPC over a period of 5 years without the manifestation of clonal quiescence phases. Since gamma-retroviral vector used in this GT trial actively transduce only replicating cells, this provide the first evidence that HSPC, awaken from dormancy in vitro, can still retain in vivo long-term activity. Overall our work constitute the first molecular tracking of hematopoietic system in humans. The information gathered are crucial for the design of therapeutic approaches for a broad spectrum of hematological diseases and tumors.