Abstract: This invention provides novel replication-defective adenoviral vectors comprising an adenoviral genome in which the protein IX gene, preferably under the control of its own promoter, is in an inverted orientation relative to the direction of transcription of the native protein IX gene at a location where the protein IX gene normally resides, for production of replication-competent adenovirus (RCA) free, or substantially RCA-free, adenovirus preparations. Said vector preferably encodes a gene of interest. The invention relates to viral particles, host cells and compositions comprising said adenoviral vector. This invention further relates a method for propagating adenovirus preparations, free, or substantially free, of replication-competent adenovirus (RCA) particles, from host cells comprising vectors of this invention, for use to treat a subject suffering from a disease or disorder or to prevent a subject from getting a disease or disorder, such as cancer.