Generation of adenovirus vectors devoid of all viral genes by recombination between inverted repeats.

The starting point for the presented study was an observation made with first-generation adenovirus (Ad) vectors that contained fragments of Ad5 DNA, specifically the vaI (23) or the precursor to the terminal protein (pTP) (26) genes, inserted into the E1 region. The presence of these sequences in addition to the corresponding endogenous gene resulted in the appearance of two viral bands with different buoyant density in CsCl gradients after ultracentrifugation of lysates from infected 293 cells. This phenomenon was interesting, considering the unique mechanism by which the adenovirus replicates and the functional potential of repetitive sequences to mediate genetic rearrangements. The genomes of Ad2 and Ad5 are double-stranded, linear DNA molecules, approximately 35 kb in length with an inverted terminal repeat sequence (ITR) of 102 bp on each end. Numerous studies in cell-free systems and in infected cells have established that Ad DNA replication takes place in two steps (reviewed in references 2 and 37). In the first stage, DNA synthesis is initiated by pTP. pTP binds as a heterodimer with the Ad polymerase (Pol) to specific sites within the ITRs. Ad DNA replication begins at both ends of the linear genome, resulting in a daughter strand that is synthesized in the 5′ to 3′ direction, displacing the parental strand with the same polarity. Three nonexclusive mechanisms are proposed for the second step, the replication of the displaced parental strand. (i) Displaced single strands can form partial duplexes by base pairing of the ITRs on which a second round of DNA synthesis may be initiated (22, 36). (ii) When two oppositely moving displacement forks meet, the two parental strands can no longer be held together and therefore separate, resulting in partially duplex and partially single-stranded molecules; the synthesis is then completed on the displaced parental strand (21). (iii) Displaced strands, with opposite polarity resulting from initiation at two different molecular ends, can renature to form a double-stranded daughter molecule (37). Elongation of DNA synthesis requires only DNA binding protein (DBP) and Pol. With 20 to 30 bp being synthesized per second, Ad elongation is relatively slow compared to that in the eukaryotic replication systems (which synthesize ∼500 bp/s). DBP may stabilize the formation of the panhandle structure and the interstrand renaturation process (39). Repetitive sequences are a common feature of prokaryotic and eukaryotic genomes. Direct repeats (DR) and inverted repeats (IR) are associated with DNA recombination processes (5, 20, 29). Furthermore, it is thought that IR-induced DNA secondary structures cause pausing of replication by DNA polymerases and reverse transcriptases, resulting in genetic alterations (1, 7, 12, 13, 19, 38). The unique Ad replication strategy, involving single-stranded replication intermediates, prompted us to investigate in detail whether repetitive homologous sequences inserted into the Ad vector genome would affect replication of viral DNA or whether it would induce genomic rearrangements. In these studies, we have found that, as a result of the replication of E1-deleted Ad vectors containing IR flanking a transgene cassette, a small viral genome is efficiently formed and packaged. These genomes were devoid of all Ad genes. Particles containing this small genome could be separated from virions with full-length genomes by ultracentrifugation in CsCl gradients. In addition to having interesting virological aspects, this finding has practical importance for Ad vector development. Ads have a number of properties that make them attractive vehicles for gene transfer. These include highly efficient mechanisms of gene transfer to a large variety of cell types in vivo and the easy production of purified virus at high titers. Highly efficient transduction is mediated by the capsid and core proteins involved in cell attachment and by internalization, endosomal lysis, and nuclear import. Most Ad vectors used for in vivo gene transfer are deleted for E1 genes. The major limitation associated with these E1-deleted vectors has been in short-term expression in vivo, due to the development of immune responses to expressed viral proteins which result in toxicity and viral clearance. In order to overcome some of these problems, Ad vectors have been developed from which almost the entire Ad genome has been deleted. These include vectors with “gutless,” almost full-length genomes, which have been shown to mediate stable transgene expression in vivo (32), as well as encapsidated Ad minichromosomes with genomes of ∼13 kb, which also have successfully been used for gene transfer in vitro and in vivo (8, 17, 18). Both vector systems require helper viruses and several serial passages for production. As an application of our finding that IRs can mediate predictable genetic rearrangements within Ad genomes, we demonstrate here the efficient and straightforward production of new vectors representing small Ad genomes devoid of all viral genes, which are packaged into functional Ad capsids.