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Implications for mechanisms of insertional oncogenesis in gene therapy
B.S. 2002, Massachusetts Institute of Technology
Thesis Advisor: Hua Zhu, Ph.D.
Department: Microbiology and Molecular Genetics
ICPH 1st Floor Auditorium
Wednesday, May 13, 2009
The first unambiguous successes at human gene therapy took place in France and the UK with the treatment of infants with X-SCID. However, insertional oncogenesis developed in patients in both protocols, resulting from the dysregulation of proto-oncogenes at sites of retroviral vector integration. In this work, we use gene expression and an in vitro model to determine the risk of retroviral vector-mediated insertional mutagenesis and assay the safety of vector modifications.
Gammaretrovirus-based vectors are known to target actively transcribing genes. Therefore, assessing transcriptional activity of genes in target cells can be indicative of the integration profile. We find that the differences in culture conditions used in the French and British clinical trials induced differences in gene expression profiles, which could explain differences in the integration profiles reported from cells obtained from patients in those trials. On the other hand, functional similarities were apparent among unique genes activated by both conditions, which may explain the occurrence of leukemia in both trials. In addition, retroviral transduction experiments suggested that both the activation of expression and constitutively high levels of expression of target genes might be factors in the susceptibility of those genes to retroviral vector integration.
Following retroviral vector integration in the host genome, the prevention of proto-oncogene dysregulation may avoid induction of leukemia. Using a model for proto-oncogene dysregulation, we investigated if a self-inactivating (SIN) vector reduced proto-oncogene activation. We could not demonstrate a significant decrease in proto-oncogene activation using SIN vectors, indicating that additional vector modifications are necessary, likely including careful selection of the internal vector promoter. This work may result in improved safety for future gene transfer protocols.