1:Cell Technology Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; 2:Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain; 3:Scientific IT Unit, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT), Madrid, Spain; 4:Viralgen, San Sebastian, Spain

Gene editing strategies through homology directed repair (HDR) have shown great promise for the treatment of hematologic diseases. However, due to the homogeneous modification of the cell genome in HDR gene editing, clonality studies of edited cells cannot be carried out. DNA barcoding offers a powerful approach for labeling individual cells to allow individual tracking of cell progeny. Here, we describe a barcoding donor template strategy to track the clonal outputs of gene edited human CD34⁺ cells.

First, we developed a barcode adenoasociated vector serotype 6 library including a semi-random 65bp sequence oligonucleotide carrying the required sequences to knock-in the PKLR locus. This system allows the tracking of up to 10⁵ different clones. We used this barcode library to study gene editing conditions, addressing both gene editing efficacy and clonality of modified hematopoietic progenitors. Two different culture media in combination or not with StemRegenin 1 (SR-1) were tested. A high in vitro targeting efficiency, up to 60% targeted hematopoietic progenitors was obtained. When these edited hematopoietic progenitors was transplanted into immunodeficient mice, gene editing efficiencies were slightly reduced. Analysis of clonal output in vivo showed up to 300 individually marked HSPCs, with 9 to 12 unique HSPCs clones per mouse responsible for 90% of the human hematopoietic engraftment. No evidence of progressive clonal dominance in any of the conditions tested was observed

Overall, DNA barcode is a powerful approach to study hematopoietic clonality of gene edited HSPCs and to optimize gene editing procedures to make it clinically applicable.