Effects of stuffer DNA on transgene expression from helper-dependent adenovirus vectors

We have analyzed transgene (lacZ) expression from a first-generation adenov irus (Ad) vector in comparison to helper-dependent (hd) Ads deleted for var ious portions of the viral coding sequences and generated by using the Cre/ loxP helper-dependent system (R.J. Parks et al., Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996). An hd vector deleted for approximately 70% of the A d genome (AdRP1001) provided levels and durations of transgene expression s imilar to those of a control first generation Ad vector containing an ident ical expression cassette. Deletion of all Ad sequences from the hdAd and re placement with a similar to 22-kb fragment of lambda DNA resulted in a decr ease in the level and duration of lacZ expression which could not be revers ed by the inclusion of a matrix attachment region. However, substitution of the lambda stuffer in the fully deleted hdAd with sequences from the human hypoxanthine-guanine phosphoribosyltransferase gene resulted in significan tly improved transgene expression. In vitro assays for cytotoxic T lymphocy tes (CTL) directed against putative peptides encoded by the vector backbone showed that, although CTL were generated against the vector containing the lambda DNA, no such CTL were generated against the vector containing the h ypoxanthine-guanine phosphoribosyltransferase (HPRT) sequences. Surprisingl y, the rate of loss of the HPRT- and lambda-containing vectors from mouse l iver was similar, despite the differences in expression kinetics, indicatin g that the lambda stuffer-directed CTL were inefficient at eliminating the transduced cells. Thus, the nature of the DNA backbone of hdAds can have im portant effects on the functioning of the vector. Since most fully deleted vectors require "stuffer" DNA as part of the vector backbone to maintain op timum vector size, these observations must be taken into account in the des ign of hdAd vectors.