Protection and Selection for Gene Therapy in the Haemopoietic System





Geofff Margison, Leslie J. Fairbairn

Cancer Research UK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK



Myelotoxicity is a frequent complicating factor in anti-cancer chemotherapy. We have been developing a gene therapy approach to addressing this, using retroviral transfer and expression of the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT) to bone marrow stem and progenitor cells. Using a mutant version of the protein, MGMTP140K, which is resistant to clinically useful inactivators of the wild-type protein, we have shown that this provides inactivator-insensitive protection to bone marrow in vivo. In view of these data we are proposing to conduct a clinical trial of MGMTP140K-based chemoprotection in patients receiving inactivator/alkylating agent combination for treatment of solid tumours.

Our early data show a clear survival and in vivo selection advantage for cells expressing mutant MGMT. To further enhance this, we have investigated the utility of the homeobox domain protein, HOXB4 to augment in vivo selection. We have developed vectors co-expressing both HOXB4 and MGMTP140K and have used these to transduce murine bone marrow cells. Using a competitive in vivo transplantation/chemoselection model we have shown that HoxB4-expressing cells have a competitive repopulation advantage over either mock-transduced cells or those expressing MGMTP140K alone. Following exposure of animals to the combination of inactivator/O6-alkylating agent, however, cells co-expressing both MGMTP140K and HOXB4 show a clear and profound selection advantage over those expressing either selective marker alone.

The mechanism by which HOXB4 confers an engraftment and selective advantage on repopulating cells is as yet undefined. We have transduced a murine, multipotent haemopoietic cell line, FDCP-mix, with a HOXB4-expressing retroviral vector. Analysis of the differentiation potential of control and HOXB4-expressing FDCP-mix reveals a profound differentiation delay in the latter. This may explain some of the properties of HOXB4 in facilitating increased engraftment of repopulating stem cells. However, a constitutively expressed differentiation delay may also represent an increased risk of leukaemic transformation. Therefore we have developed a tamoxifen-inducible version of HOXB4 (HOXB4-TxR). In mice transplanted with bone marrow transduced with HOXB4-TxR only low levels of transduced cells were detected in the peripheral circulation after haemopoietic recovery when no tamoxifen citrate was added to their drinking water. However, in those animals that received tamoxifen citrate a significantly higher level of transduced cells engrafted. These data suggest that it may be possible to achieve a pharmacologically regulated selection advantage in repopulating haemopoietic stem cells using gene therapy.




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