Abstract

In vitro gene transfer of CDD protects murine and human hematopoietic cells from cytarabine (araC) and gemcitabine induced toxicity. Thus CDD represents a drug resistance gene with considerable clinical potential. We now have examined CDD gene transfer in a bone marrow transplant model with C57Bl/6 mice using the retroviral vectors SF91-CDD-IRES-EGFP and SFβ1-EGFP (control).

Protection from araC was investigated in three experiments. Gene transfer efficiency was analysed by EGFP expression in the peripheral blood after hematopoietic reconstitution. In these experiments 42±4%, 31±1% and 69±2% EGFP+ granulocytes and 9±1%, 10±1% and 36±3% EGFP+ lymphocytes were detected following SF91-CDD-IRES-EGFP transduction (n=5–6 for each experiment). After araC application (500mg/kg; 1xdaily; d1-4; i.p.) substantial protection of granulocytes and thrombocytes was observed. Following the first treatment cycle nadir counts [x103/μl] were 2.9±0.6 vs. 0.7±0.1, 1.1±0.2 vs. 0.5±0.1 and 2.3±0.3 vs. 0.7±0.2 for granulocytes and 510±148 vs. 80±9, 165±48 vs. 70±42 and 728±133 vs. 85±22 for thrombocytes, respectively (SF91-CDD-IRES-EGFP vs. control; all differences significant at p<0.01 for granulocytes and p<0.05 for thrombocytes). Similar protection was noted during repetitive araC treatment cycles. However, no significant enrichment of CDD transduced cells following araC application was seen and lack of selection was confirmed in two additional experiments utilizing low efficiency gene transfer (<5% and <12% EGFP+ granulocytes or lymphocytes throughout the experiment). No major toxicity of CDD-expression in hematopoietic cells was noted and steady levels of gene marked cells were seen for up to six months in primary and at 5 weeks in secondary recipients. Substantial protection of hematopoiesis by CDD overexpression also was observed for gemcitabine. Mice receiving CDD transduced cells and exposed to 250mg/kg gemcitabine (1xdaily i.p., d1-2), showed granulocyte and thrombocyte nadirs [x103/μl] of 2.4±0.6 vs. 1.4±0.2 and 416±62 vs. 277±97 (SF91-CDD-IRES-EGFP vs. control). When a third day of gemcitabine treatment was added, all mice in the control group died within 4 days while animals receiving CDD-transduced bone marrow survived with nadir counts [x103/μl] of 0.8±0.1 granulocytes and 1130±292 thrombocytes [n=3]. Again, no enrichment of transduced cells was observed.

In summary, our data demonstrate efficient protection from araC and gemcitabine hematotoxicity by CDD overexpression. This protective effect was on progenitor rather than stem cells, as no selection of CDD-expressing cells was demonstrated. Thus, a promising approach to utilize CDD clinically may be the combination with a selectable drug resistance gene such as MGMT or MDR-1 to allow enrichment of transduced cells while at the same time protecting against additional drugs.

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