Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors

We present a systematic comparison of three modules that enhance expression from retroviral gene transfer vectors at a posttranscriptional level: (i) splice signals (SS) that create an intron in the 5' untranslated region; (i i) constitutive RNA transport elements (CTE), originally discovered in D-ty pe retroviruses; and (iii) the posttranscriptional regulatory element of wo odchuck hepatitis virus (WPRE). Here we show that enhancement of expression depends not only on the specific element, but also on the gene of interest , implying context-dependent activity of the RNA elements. Interestingly, d ifferent results were obtained for genes that normally require or do not re quire such control elements. Expression of the HIV-1 gag-protease gene, whi ch normally depends on the viral export factor Rev, was strongly enhanced b y an oligomeric CTE, while WPRE had only a marginal effect. On the other ha nd, both CIE and WPRE compensated for the lack of an intron in the expressi on of human P-globin. In this case, the strongest stimulation of RNA produc tion was observed when functional SS were combined with the WPRE. Both CTE and, in particular, WPRE also enhanced expression of cDNAs that do not norm ally require any such element (green fluorescent protein, human multidrug r esistance-1). In this study, functional SS and WPRE acted in an additive ma nner, resulting in a 10-fold higher level of expression. Our results indica te that the described modules act on different levels of RNA processing, tr ansport, and translation and that the correct choice of a posttranscription al enhancer configuration depends on the type of cDNA to be expressed.