GENE-THERAPY FOR CHRONIC MYELOGENOUS LEUKEMIA (CML) - A RETROVIRAL VECTOR THAT RENDERS HEMATOPOIETIC PROGENITORS METHOTREXATE-RESISTANT ANDCML PROGENITORS FUNCTIONALLY NORMAL AND NONTUMORIGENIC IN-VIVO

Chronic myelogenous leukemia (CML) is a malignant disease of the human hematopoietic stem cell caused by the BCR/ ABL gene rearrangement. Th e only curative therapy is allogeneic transplantation. Although autolo gous transplants may prolong survival, most patients relapse because o f disease persisting in the host and in the graft. Continued administr ation of chemotherapy after transplant could reduce the incidence of r elapse provided that the autograft can be protected by transfer of a d rug-resistance gene. However, CML autografts wilt almost certainly con tain malignant stem cells that will also be rendered drug-resistant. T he presence of the BCR/ABL oncoprotein is necessary and sufficient for malignant transformation seen in CIVIL. We thus hypothesized that tra nsfer of a Vector that combines a drug-resistance gene with anti-BCR/A BL antisense (AS) sequences may allow for posttransplant chemotherapy to decrease persistent disease while rendering inadvertently transduce d CML stem and progenitor cells functionally normal. We constructed a retroviral vector, LasBD, that combines the methotrexate (MTX) -resist ant tyrosine-22 dihydrofolate-reductase (tyr22-DHFR) gene and AS seque nces directed at the b3a2 BCR/ABL breakpoint. b3a2 BCR/ABL containing 32D and M07e cells were transduced with LasBD and selected in MTX for 14 days. Expression of the AS sequences reduced BCR/ABL mRNA and p210( BCR/ABL) protein levels by 6- to 10-fold in most cells. This subsequen tly led to the restoration of normal function of BCR/ABL cDNA(+) cells : they grew significantly slower in the presence of interleukin-3 (IL- 3); they underwent apoptotic cell death when cultured without IL-3; an d they had restored expression and function of adhesion receptors. The se effects were specific, because LasBD-containing AS sequences direct ed at the b3a2 BCR/ABL breakpoint did not affect p190(BCR/ABL)-contain ing cells. LasBD also rendered 20% to 30% of primary Ph- and Ph+ CD34( +) cells MTX-resistant and decreased BCR/ABL mRNA levels in MTX resist ant Ph+ CD34(+) cells by 10-fold. Expression of the MTX-resistant DHFR gene and the AS sequences has been stable for at least 1 year in vitr o and for more than 70 days in vivo. Finally, LasBD decreased tumorige nicity of 32D(BCR/ABL) cells in vivo by 3 to 4 logs. In conclusion, th e tyr22-DHFR gene in the LasBD vector can protect normal hematopoietic cells from MTX-mediated toxicity, whereas the AS sequences in LasBD c an suppress expression of the BCR/ABL gene and restore normal function of BCR/ABL cDNA-containing cells. The LasBD vector may therefore prov e to be an extremely useful adjunct in autologous transplantation for CML. (C) 1997 by The American Society of Hematology.