Abstract
Abstract Background: Genetic modification of hematopoietic stem cells with retroviral vectors has been associated with clonal expansion and even the induction of T-cell leukemia. Therefore, monitoring the behavior of the transduced cells may reveal critical information related to the viral vector, its application in hematopoietic stem cells and leukemogenesis. We have developed a viral “barcode” that provides a quantitative measure of clonality upon sequence analysis of genomic DNA recovered from transduced cells. In the polyclonal population, sequencing should reveal great variability, yet a decrease in population complexity would a reduce barcode variability. Clonal expansion would be revealed when a single barcode is predominant in the population. Methods: A barcode containing 10 variable sites was produced and inserted in a lentiviral vector, generating a library with up to 1024 possible clones. Virus supernatant encoding the library was produced and used to transduce NIH3T3 cells. gDNA was isolated and the barcode region sequenced (Sanger method) resulting in a chromatogram that reveals the relative contribution of each base at the variable site and, thus, monitors alterations in the cell population. Alternatively, the barcode library was inserted in lentiviral vectors encoding IL2RG (therapeutic gene for the correction of x-linked severe combined immunodeficiency) or LMO2 (an oncogene implicated in the onset of leukemia in X-SCID gene therapy trials) and these viruses were used to transduce mouse hematopoietic progenitor cells before transplant in myeloablated recipient mice. Peripheral blood was collected periodically for analysis. Results: To evaluate the quantitative profile of the technique, a single plasmid clone was isolated and mixed at known proportions with the plasmid library or with a second distinct clone. The barcode region was sequenced and the chromatogram amplitudes used to quantify the amount of the clone present in the mix of plasmids. We have found a correlation of 96% between the obtained and the predicted results. In a tissue culture assay we simulated clonal expansion by mixing cells transduced with a single viral clone with cells transduced with the viral library and monitored the barcode over time, showing that the barcode can reveal the complexity of a cell population. At this time, the transplanted mice are being monitored for the onset of leukemia as revealed by both hematologic exams and evaluation of the barcode. Conclusions: We have shown that common sequencing procedures can be used reliably to quantify the presence of a barcode even in a mixed population of cells. Such a method could be applied to gene therapy approaches in the hematopoietic system as well as used to reveal the behavior of cell populations during leukemogenesis. This work was supported by FAPESP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2843. doi:1538-7445.AM2012-2843
Published Version
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