Genetic barcode sequencing for screening altered population dynamics of hematopoietic stem cells transduced with lentivirus

Autor(es)

Zanatta, Daniela B. ; Tsujita, Maristela ; Borelli, Primavera ; Aguiar, Rodrigo B. ; Ferrari, Daniel G. ; Strauss, Bryan E.

• Tipo de documento: Artigo Científico
• Fonte: MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT; v. 1, 2014.
• Citações Web of Science: 1

Resumo

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. (AU)

• Processo FAPESP: 09/51386-0 - Avaliação do perfil de segurança associado ao uso de vetores retroviral e lentiviral para terapia gênica no sistema hematopoiético
• Beneficiário: Bryan Eric Strauss
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 07/56495-7 - Desenvolvimento de uma metodologia que permitira monitorar a clonalidade de celulas transduzidas com vetores lentivirais.
• Beneficiário: Daniela Bertolini Zanatta
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 13/25167-5 - Transferência gênica de p19Arf e interferon-beta: delineando a importância de sua combinação em modelos murinos de terapia gênica do câncer
• Beneficiário: Bryan Eric Strauss
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 14/03205-5 - Challenges and Solutions in Cancer Research and Treatment
• Beneficiário: Daniela Bertolini Zanatta
• Modalidade de apoio: Auxílio à Pesquisa - Reunião - Brasil