title: Genetic barcode sequencing for screening altered population dynamics of hematopoietic stem cells transduced with lentivirus.
abstract: Insertional mutagenesis has been associated with malignant cell transformation in gene therapy protocols, leading to discussions about vector security. Therefore, clonal analysis is important for the assessment of vector safety and its impact on patient health. Here, we report a unique approach to assess dynamic changes in clonality of lentivirus transduced cells upon Sanger sequence analysis of a specially designed genetic barcode. In our approach, changes in the electropherogram peaks are measured and compared between successive time points, revealing alteration in the cell population. After in vitro validation, barcoded lentiviral libraries carrying IL2RG or LMO2 transgenes, or empty vector were used to transduce mouse hematopoietic (ckit+) stem cells, which were subsequently transplanted in recipient mice. We found that neither the empty nor IL2RG encoding vector had an effect on cell dynamics. In sharp contrast, the LMO2 oncogene was associated with altered cell dynamics even though hematologic counts remained unchanged, suggesting that the barcode could reveal changes in cell populations not observed by the frontline clinical assay. We describe a simple and sensitive method for the analysis of clonality, which could be easily used by any laboratory for the assessment of cellular behavior upon lentiviral transduction.
authors.affiliation: Center for Translational Investigation in Oncology/LIM 24, Cancer Institute of Sao Paulo, School of Medicine, University of Sao Paulo , Sao Paulo, Brazil.
authors.forename: Daniela B
authors.initials: DB
authors.lastname: Zanatta
authors.affiliation: Department of Clinical and Toxicologic Analyses, School of Pharmaceutical Sciences, University of Sao Paulo , Sao Paulo, Brazil.
authors.forename: Maristela
authors.initials: M
authors.lastname: Tsujita
authors.affiliation: Department of Clinical and Toxicologic Analyses, School of Pharmaceutical Sciences, University of Sao Paulo , Sao Paulo, Brazil.
authors.forename: Primavera
authors.initials: P
authors.lastname: Borelli
authors.affiliation: Center for Translational Investigation in Oncology/LIM 24, Cancer Institute of Sao Paulo, School of Medicine, University of Sao Paulo , Sao Paulo, Brazil.
authors.forename: Rodrigo B
authors.initials: RB
authors.lastname: Aguiar
authors.affiliation: Natural Computing Laboratory, School of Computing and Informatics, Mackenzie University , Sao Paulo, Brazil.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Ferrari
authors.affiliation: Center for Translational Investigation in Oncology/LIM 24, Cancer Institute of Sao Paulo, School of Medicine, University of Sao Paulo , Sao Paulo, Brazil.
authors.forename: Bryan E
authors.initials: BE
authors.lastname: Strauss