Expression of a novel double-mutant dihydrofolate reductase-cytidine deaminase fusion gene confers resistance to both methotrexate and cytosine arabinoside

A novel fusion gene consisting of the open reading frame of a double-mutant (Phe22-Ser31) dihydrofolate reductase (dmDHFR) cDNA fused to the open read ing frame of cytidine deaminase (CD) was constructed and characterized for the purpose of conferring simultaneous resistance to methotrexate (MTX) and cytosine arabinoside (ara-C), The kinetic properties of purified recombina nt dmDHFR-CD fusion protein were compared with those of purified CD and dmD HFR. The fusion protein was found to retain enzymatic properties of both dm DHFR and CD, in that the K-m and K-cat values of purified dmDHFR-CD protein were found to be virtually identical to those of CD and dmDHFR alone, Retr ovirus-mediated expression of dmDHFR-CD in NIH 3T3 cells conferred signific ant resistance (10- to 12-fold) against MTX and ara-C, compared with mock- and single gene-infected cells and the level of resistance obtained was sim ilar to that of cells expressing both CD and dmDHFR from a retroviral bicis tronic vector. Infection of mouse bone marrow cells with the dmDHFR-CD cons truct also showed high levels of resistance to MTX and ara-C in a CFU-GM as say. This fusion protein confers resistance to two antineoplastic agents th at differ in their mechanism of action, and may be useful in the design of gene transfer strategies for protection of target cells against multiple dr ugs. Since high-dose ara-C and MTX are used in the treatment of lymphomas, this vector may be of value in protecting human hematopoietic progenitor ce lls from the toxicity of these antimetabolites.