title: Analysis of the clonal repertoire of gene-corrected cells in gene therapy.
abstract: Gene therapy-based clinical phase I/II studies using integrating retroviral vectors could successfully treat different monogenetic inherited diseases. However, with increased efficiency of this therapy, severe side effects occurred in various gene therapy trials. In all cases, integration of the vector close to or within a proto-oncogene contributed substantially to the development of the malignancies. Thus, the in-depth analysis of integration site patterns is of high importance to uncover potential clonal outgrowth and to assess the safety of gene transfer vectors and gene therapy protocols. The standard and nonrestrictive linear amplification-mediated PCR (nrLAM-PCR) in combination with high-throughput sequencing exhibits technologies that allow to comprehensively analyze the clonal repertoire of gene-corrected cells and to assess the safety of the used vector system at an early stage on the molecular level. It enables clarifying the biological consequences of the vector system on the fate of the transduced cell. Furthermore, the downstream performance of real-time PCR allows a quantitative estimation of the clonality of individual cells and their clonal progeny. Here, we present a guideline that should allow researchers to perform comprehensive integration site analysis in preclinical and clinical studies.
authors.affiliation: Department of Translational Oncology, National Center for Tumor Diseases (NCT) and German Cancer Research Center (DKFZ), Im Neuenheimer Feld 581 and 460, Heidelberg, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Paruzynski
authors.forename: Hanno
authors.initials: H
authors.lastname: Glimm
authors.forename: Manfred
authors.initials: M
authors.lastname: Schmidt
authors.forename: Christof von
authors.initials: Cv
authors.lastname: Kalle
mesh_terms: D002478:Cells, Cultured; D002986:Clinical Trials as Topic; D004247:DNA / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel / Q000379:methods; D017988:Gammaretrovirus / Q000235:genetics* / Q000502:physiology; D015316:Genetic Therapy / Q000009:adverse effects*; D005822:Genetic Vectors; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016254:Mutagenesis, Insertional; D016133:Polymerase Chain Reaction / Q000379:methods; D017410:Practice Guidelines as Topic; D000090063:Proto-Oncogene Mas; D017422:Sequence Analysis, DNA / Q000379:methods; D016662:Virus Integration
chemical_list: C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D004247:DNA