The availability of efficient and nontoxic gene delivery technologies is fundamental to the translation of therapeutic concepts into clinical practice by gene transfer. High-capacity adenoviral (HC-Ad) vectors are characterized by the ability to transduce cells in vitro and in vivo with more than 30 kb of nonviral DNA. This quality allows simultaneous gene transfer of several expression cassettes, large promoters, and some genes in their natural genomic context. Because all viral coding sequences are removed from these vectors, safety is considerably improved compared with previous-generation adenoviral vectors.