Coding Region-Specific Destabilization of mRNA Transcripts Attenuates Expression from Retroviral Vectors Containing Class 1 Aldehyde Dehydrogenase cDNAs

Abstract

Class 1 aldehyde dehydrogenases (ALDH-1) function as drug resistance gene products by catalyzing the irreversible conversion of aldophosphamide, an active metabolite of cyclophosphamide, to an inert compound. Because the dose-limiting toxicity of cyclophosphamide is myelosuppression, retrovirus-mediated transfer of ALDH-1 to bone marrow cells has been proposed as a protective strategy. Here we show that expression of ALDH-1 vectors was problematic due to low levels of ALDH-1 mRNA accumulation. A number of vectors containing several different ALDH-1 cDNAs were introduced into a variety of different cell lines either by transfection or transduction. Detectable ALDH-1 protein and enzyme activity was only seen in one transfected cell clone. Cells transduced with ALDH-1 retroviral vectors had no detectable protein expression and very low levels of ALDH-1 mRNA. Analogous vectors containing other drug resistance cDNAs led to much higher levels of steady-state mRNA. The mRNA half-life from ALDH-1 vectors was less than 2 hr suggesting that vector-derived mRNAs were destabilized by ALDH-1 coding sequences. These results suggest that methods which increase the stability of ALDH-1 mRNAs will be important for increased drug resistance in retrovirally transduced hematopoietic cells.

Overview summary

Protection against cyclophosphamide-induced myelosuppression in vivo will require significant expression of ALDH-1 following gene transfer into hematopoietic cells. Here we show that expression of ALDH-1 cDNAs in vitro is limited due to mRNA instability. This expression problem is specific for ALDH-1 and is not evident with dihydrofolate reductase (DHFR) or MDR-1 cDNAs expressed from identical vectors. Study of a variety of cell types transduced or transfected with several different ALDH-1 retroviral vectors showed that the majority of clones expressed extremely low to undetectable levels of ALDH-1 mRNA. Modification of the ALDH-1 cDNA coding region will likely be necessary to prevent the rapid degradation of its nascent mRNA and thus to enable future gene therapy applications.