1. ¹Gene Therapy, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
2. ²Carcinogenesis Groups, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester M20 4BX, UK
3. ³Terry Fox Laboratory, British Columbia Cancer Agency, Vancouver, BC V5Z 1L3, Canada

Correspondence: Leslie J. Fairbairn, Gene Therapy, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK. Fax: (+44) 0161 446 3109. E-mail: lfairbairn@picr.man.ac.uk

Received 21 January 2004; Accepted 15 July 2004.

Abstract

To attain therapeutic levels of gene-modified hematopoietic stem cells, it may be necessary in the majority of disorders to provide an in vivo selective advantage that facilitates the expansion of their numbers. A popular strategy to achieve in vivo selection has been to employ drug selection while coexpressing a transgene that conveys chemoresistance, such as O⁶-methylguanine-DNA-methyltransferase (MGMT). An alternate approach is to confer an enhanced proliferative potential upon gene-modified hematopoietic stem cells through the delivery of the homeobox transcription factor HOXB4. By developing a novel tricistronic retroviral vector, we have facilitated the simultaneous coexpression of a mutant version of MGMT and HOXB4 in retrovirally transduced bone marrow. Using an in vivo competitive repopulation assay, we demonstrate that primary bone marrow cells containing this construct show enhanced reconstitution following transplant and improved selection subsequent to chemotherapeutic challenge in comparison to cells expressing either HOXB4 or MGMT alone. This selection advantage was evident even when HOXB4/MGMT-coexpressing cells were infused along with a large excess of unmodified cells. We propose that this selection cassette may facilitate the in vivo expansion of gene-modified hematopoietic stem cells at a level in excess of previous strategies.