Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration

We have developed a new helper adenovirus (Ad) based on serotype 2, Ad2LCBc CARP, for use in the Cre/loxP system (Parks et al. Proc Natl Acad Sci USA, 1996; 93: 13565-13570) to generate Ad vectors deleted of all protein coding sequences (helper-dependent Ad vectors (hdAd)). A comparison of Ad2LC8cCAR P and our original helper Virus (based on serotype 5, Ad5LC8cluc) showed th at the two helper viruses amplified hdAd with a similar efficiency, and res ulted in a similar yield and purify after large-scale preparation of vector In vitro, the resulting hdAd2 had a similar transduction efficiency and ex pression kinetics of transgene (beta-gal) as the hdAdS. An important featur e of the helper-dependent system is that all virion components, except the virion DNA, derive from the helper virus. Consequently, vectors produced wi th help from Ad2LC8cCARP were not neutralized by antibodies against Ad5, an d vectors produced with Ad5 helper were resistant to neutralizing antibodie s against Ad2. Analysis of transgene expression in mouse liver after intrav enous injection of the Ad2-based hdAd showed that the vector could efficien tly transduce the liver, and produce high levels of a foreign transgene, si milar to those expressed by the hdAd generated with the Ad5 helper virus. M ice immunized with hdAd2 produced Ad2-neutralizing antibodies, which did no t crossreact with hdAdS. To determine if successful repeat Ad vector admini stration could be achieved by sequential use of alternative Ad serotypes, w e injected mice with hdAd2 (hSEAP) followed 3 months later by a lacZ-expres sing hdAd of either the same or different serotype. Repeated administration of hdAd2 resulted in a 30- to 100-fold reduction in transgene expression c ompared with naive animals. In contrast no decrease in transgene expression was observed when the second vector was of a different serotype. These res ults demonstrate that effective vector readministration can be achieved by the sequential use of hdAds based on alternative serotypes.