Submitted September 2, 2008
Accepted March 6, 2009
Long-term polyclonal and multilineage engraftment of MGMTP140K gene-modified dog hematopoietic cells in primary and secondary recipients
Brian C. Beard, Reeteka Sud, Kirsten A. Keyser, Christina Ironside, Tobias Neff, Sabine Gerull, Grant D. Trobridge, and Hans-Peter Kiem*
Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, United States
Department of Medicine and Pathology, University of Washington, Seattle, WA, United States
* Corresponding author; email: hkiem{at}fhcrc.org.
Overexpression of methylguanine methyltransferase P140K (MGMTP140K) has been successfully used for in vivo selection and chemoprotection in mouse and large animal studies and has promise for autologous and allogeneic gene therapy. We examined the long-term safety of MGMTP140K selection in a clinically relevant dog model. Based on the association of provirus integration and proto-oncogene activation leading to leukemia in the X-linked immunodeficiency (SCID-X1) trial, we focused our analysis on the distribution of retrovirus integration sites (RIS) relative to proto-oncogene transcription start sites (TSS). We analyzed RIS near proto-oncogene TSS before (n=157) and after (n=129) chemotherapy in dogs that received MGMTP140K gene-modified cells and identified no overall increase of RIS near proto-oncogenes TSS following chemotherapy. We also wanted to determine if in vivo selected cells retained fundamental characteristics of hematopoietic stem cells (HSC). To that end, we performed secondary transplantation of MGMTP140K gene-modified cells following in vivo selection in DLA-matched dogs. Gene-modified cells achieved multilineage repopulation, and we identified the same gene-modified clone in both dogs >800 and 900 days after transplantation. These data suggest that MGMTP140K selection is well tolerated and should allow clinically for selection of gene-corrected cells in genetic or infectious diseases or chemoprotection for treatment of malignancy.
