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Blood, 15 February 2008, Vol. 111, No. 4, pp. 1866-1875.
Prepublished online as a Blood First Edition Paper on November 8, 2007; DOI 10.1182/blood-2007-04-085506.
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Submitted April 13, 2007
Accepted November 5, 2007
An experimental system for the evaluation of retroviral vector design to diminish the risk for proto-oncogene activation
Byoung Y Ryu, Marguerite V Evans-Galea, John T Gray, David M Bodine, Derek A Persons, and Arthur W. Nienhuis*
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, United States
Genetics and Molecular Biology Branch, National Human Genome Research Institute, Bethesda, MD, United States
* Corresponding author; email: arthur.nienhuis{at}stjude.org.
Pathogenic activation of the LMO2 proto-oncogene by an oncoretroviral vector insertion in a clinical trial for X-linked Severe Combined Immunodeficiency has prompted safety concerns. We utilized an adeno-associated virus vector to achieve targeted insertion of a gammaretroviral long terminal repeat (LTR) driving a GFP expression cassette with flanking loxP sites in a human T-cell line at the precise location of vector integration in one of the X-SCID patients. The LTR-GFP cassette was inserted into the first intron of the LMO2 gene, resulting in strong activation of LMO2. Cre-mediated cassette exchange was used to replace the original LTR-GFP cassette with one flanked by insulator elements leading to a several fold reduction in LMO2 expression. The LTR-GFP cassette was also replaced with a globin gene regulatory cassette which failed to activate the LMO2 gene in lymphoid cells. A gammaretroviral vector with 2 intact LTRs resulted in activation the LMO2 gene when inserted into the first intron but a self-inactivating lentiviral vector with an internal cellular promoter and flanking insulator elements did not activate the LMO2 gene. Thus, this system is useful for comparing the safety profiles of vector cassettes with various regulatory elements with respect to their potential for proto-oncogene activation.
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