A HELPER-DEPENDENT ADENOVIRUS VECTOR SYSTEM - REMOVAL OF HELPER VIRUSBY CRE-MEDIATED EXCISION OF THE VIRAL PACKAGING SIGNAL

Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to th e induction of a host immune response against transduced cells. We hav e developed a helper-dependent packaging system for production of vect ors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals flanked by loxP sites so that in 2 93 cells that stably express the Cre recombinase (293Cre), the packagi ng signal was efficiently excised, rendering the helper virus genome u npackageable. However, the helper virus DNA was replicated at normal l evels and could thus express all of the functions necessary in trans f or replication and packaging of a vector genome containing the appropr iate cis-acting elements. Serial passage of the vector in helper virus -infected 293Cre cells resulted in an approximate to 10-fold increase in vector titer per passage. The vector could be partially separated f rom residual helper virus by cesium chloride buoyant density centrifug ation. Large scale preparations of vector yielded semi-purified stocks of approximate to 10(10) transducing virions/ml, with <0.01% contamin ation by the E1-deleted helper virus. This system should have great ut ility for the generation of adenovirus-based vectors with increased cl oning capacity, increased safety and reduced immunogenicity.