Cluster of Excellence - “From Regenerative Biology to Reconstructive Therapy” Special Lecture : Luca Biasco Gene Therapy Program Dana-Farber/Boston Children’s Cancer and Blood Disorders Center „Clonal Tracking of Human Hematopoiesis Through Integration Sites Analysis “ Wednesday April 19th, 2017 Lecture Hall G, time 5 pm c.t. Host: Prof. Axel Schambach, Tel.: 5170

Abstract Cluster of Excellence - “From Regenerative Biology to Reconstructive Therapy” Abstract Retroviral/lentiviral vectors are widely used for efficiently and stably delivering therapeutic genes to mature T cells and hematopoietic stem/progenitor cells (HSPC) upon ex vivo gene therapy (GT). As viral integrations occur in a semi-random fashion, upon gene correction each transduced cells becomes univocally tagged by an individual insertion site (IS) that can be monitored for safety through LAM-PCR + high throughput sequencing. Our group has been focusing on using IS as molecular barcodes to address basic biology questions on the fate of hematopoietic clones in vivo in humans. We comprehensively studied human T-cells dynamics analyzing samples from adenosine deaminase deficient-SCID patients treated with HSPC or T cell GT. By tracing >1700 individual clones we discovered that engineered T memory stem cells can functionally last for decades in human beings in absence of supply by hematopoietic progenitors. We more recently tracked in vivo in humans the dynamics of hematopoietic reconstitution after HSPC GT in Wiskott Aldrich syndrome patients. Analyzing more than 89.000 clones belonging to 15 hematopoietic populations we described repopulating waves, populations’ dynamics and hierarchical relationships among lineages. We are further expanding this analysis collecting more than 10.000 IS from 7 different HSPC subtypes isolated from the bone marrow of GT patients, to address survival and activity of primitive and committed hematopoietic progenitors directly in vivo in humans. Concomitantly, we are characterizing vector-host interaction in vitro on the HSPC and T cell products uncovering specific insertional features marking each cell subtype upon in vitro manipulation. These studies are expanding our understanding of the biology of engineered human hematopoiesis.