Chemoprotective gene transfer II:: Multilineage in vivo protection of haemopoiesis against the effects of an antitumour agent by expression of a mutant human O6-alkylguanine-DNA alkyltransferase

Murine bone marrow cells were transduced ex vivo with a retrovirus encoding an O-6-benzylguanine (O-6-beG) insensitive, double mutant form of human DNA repair protein O-6-alkylguanine-DNA alkyltransferase (hATPA/GA) In animals reconstituted with the transduced bone marrow about 50% of cells in the multipotent spleen colony-forming cells (CFU-S) and lineage restricted granulocyte-macrophage (GM-CFC) haemopoietic progenitor populations were found to be carrying the transgene and this correlated with the frequency of bone marrow cells and spleen colonies which stained positive hATPA/GA by immunocytochemistry. Expression of hATPA/GA was associated with significant in vivo protection of both CFU-S (P=0.001) and GM-CFC (P<0.024) against the toxicity of the antitumour methylating. agent, temozolomide, given in combination with O-6-beG. Expression of hATPA/GA also led to a reduction in the frequency of combined O-6-beG/temozolomide-induced micronuclei seen in polychromatic erythrocytes (P<0.003). This study is the first to demonstrate in vivo protection of multipotent haemopoietic progenitors against the toxic and clastogenic effects of an O-6-alkylating agent in the presence of O-6-beG. It also represents the first report of reduced clastogenesis as a consequence of expression of an O-6-beG-resistant ATase. In the accompanying article we report hATPA/GA-mediated resistance of human CD34(+) haemopoietic progenitors to combined O-6-beG/O-6-alkylating agent toxicity. Together these two reports suggest that a gene therapy strategy whereby protection of normal haemopoietic tissue: may be combined with O-6-beG-mediated tumour sensitisation may be efficacious in achieving an increase in therapeutic index.