Adeno-associated virus is an integrating DNA parvovirus with the potential to be an important vehicle for somatic gene therapy. A potential barrier, however, is the low transduction efficiencies of recombinant adenoassociated virus (rAAV) vectors. We show in this report that adenovirus dramatically enhances rAAV transduction in vitro in a way that is dependent on expression of early region 1 and 4 (E1 and E4, respectively) genes and directly proportional to the appearance of double-stranded replicative forms of the rAAV genome. Expression of the open reading frame 6 protein from E4 in the absence of E1 accomplished a similar but attenuated effect. The helper activity of adenovirus E1 and E4 for rAAV gene transfer was similarly demonstrated in vivo by using murine models of liver-and lung-directed gene therapy. Our data indicate that conversion of a single-stranded rAAV genome to a duplex intermediate limits transduction and usefulness for gene therapy.

Recombinant adenoviruses are being developed for gene therapy of inherited disorders such as cystic fibrosis because they efficiently transduce recombinant genes into nondividing cells in vivo. First generation recombinant adenoviruses, rendered defective by deletion of sequences spanning E1a and E1b, express low levels of early and late viral genes that activate destructive cellular immune responses. Current strategies for improving recombinant adenoviruses attempt to inactivate other essential genes through deletion and growth in new packaging cell lines or incorporation of temperature sensitive mutations which allow propagation of the virus in available packaging cell lines at permissive temperatures. We describe in this report a new type of recombinant adenovirus that is deleted of all viral open reading frames. This recombinant (called delta-rAd), which contains only the essential cis elements (i.e., ITRs and contiguous packaging sequence), is propagated in 293 cells in the presence of E1-deleted helper virus. Concatamers of the monomeric vector genome were passaged and capable of transduction. The delta-rAd genome is packaged into virions that sediment at a lower density than the helper virus in cesium gradients forming the basis for a purification protocol. A fully deleted recombinant adenovirus that expresses human cystic fibrosis transmembrane conductance regulator was produced and used to transduce human airway epithelial cells derived from a cystic fibrosis patient. Packaging and propagation of a fully deleted adenovirus is an important step toward the development of a safer vector. Improved production and purification strategies need to be developed before this new vector system can be evaluated in vivo.

Adenovirus and adeno-associated virus (AAV) are eukaryotic DNA viruses being developed as vectors for human gene therapy. The strengths of each system have been exploited in a novel vector that is based on an adenovirus-AAV hybrid virus incorporated into a plasmid-based molecular conjugate. Efficient rescue and replication of the recombinant AAV genome in this hybrid required transient expression of rep. This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the hybrid virus through a polylysine bridge. The resulting particle is an attractive vehicle for gene therapy because it is easily manufactured and capable of efficiently transducing cells with the end result being rescue and replication of the recombinant AAV genome. This particle is also useful in the production of recombinant AAV resulting in yields 10-fold greater than that achieved with transfection-based protocols.

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