Highly sensitive and species-specific assay for quantification of human transgene expression levels

During the past few years great efforts have been made to construct and to test human factor VIII (hFVIII) and IX (hFIX) vectors suitable for haemophi lia gene therapy in vivo. However, little is known about the molecular mech anisms of persistence and shut-off of transgene expression in the target or gans after gene transfer using recombinant adenoviral vectors. To evaluate low transgene mRNA levels in different tissues, especially at long times af ter the gene transfer, the common northern blot method is often not sensiti ve enough. For this reason we developed a new, highly sensitive and species -specific method for hFIX mRNA quantification and employed it in mice treat ed with an adenoviral vector (Ad5CMVFIX) expressing human FIX. In addition to its very high sensitivity (lowest detection level=1 fg RNA), the method was shown to be strictly species-specific, since hFIX mRNA signals were nev er detected in untreated mice. In a long-term study of 18 vector-treated mi ce we compared the human FIX:Ag levels in the mouse plasma, the human FIX m RNA levels and human FIX vector DNA concentrations in the mouse liver. We f ound that a slow but continuous decrease of hFIX:Ag levels in mouse plasma was associated with a corresponding decrease of hFIX mRNA levels in the liv er. However, the Ad5CMVFIX vector DNA levels did not decrease to a comparab le degree, suggesting that the decrease of human FIX:Ag levels in mouse pla sma is, to a significant extent, also caused by CMV promotor shut-off and o nly to a minor degree by loss of vector DNA.