The ability of recombinant adeno-associated virus (AAV) to transduce cells with a marker gene in vitro was found to be substantially increased by the presence of adenovirus. Transfection experiments with adenovirus genomic DNA suggest that this increase is not facilitated by adenovirus-mediated viral uptake but is instead dependent on adenovirus gene expression. Using various adenovirus mutants, we were able to map this function to early-region E4 open reading frame 6. Plasmid expression of open reading frame 6 protein in cells infected with recombinant AAV increased transduction between 100-and 1,000-fold. The increase in transduction was not dependent on the recombinant AAV gene cassette but instead appeared to involve an immediate early step of the AAV life cycle. Chemical and physical agents that have been shown to induce helper-free replication of wild-type AAV were also able to stimulate recombinant AAV transduction, suggesting that the phenomenon might affect AAV DNA replication. Further experiments showed that viral uncoating was not affected and that the rate-limiting step involved synthesis of a second strand on the single-stranded genomic AAV DNA. These data suggest that the adenovirus E4 region, as well as chemical and physical agents, can play an essential role in an immediate-early step of the AAV life cycle, specifically in second-strand synthesis, and have important implications for the use of AAV vectors in gene therapy protocols.

We demonstrate the rapid and reliable quantification of the recombinant, but not in the wild-type AAV-2 prepphysical AAV-2 (adeno-associated virus type 2) particles arations. Moreover, additional expression of VP proteins via a novel ELISA based on a monoclonal antibody which during rAAV production was found to result in an excessive selectively recognizes assembled AAV-2 capsids. Titration capsid formation, whilst yielding only minor increases in of a variety of recombinant AAV-2 (rAAV) preparations DNA-containing or transducing rAAV particles. We conrevealed that at least 80% of all particles were empty, comclude that encapsidation of viral genomes rather than cappared with a maximum of 50% in wild-type AAV-2 stocks, sid assembly can be limiting for rAAV production, provided indicating that the recombinant genomes were less that a critical level of VP expression is maintained. The efficiently encapsidated. This finding was confirmed upon feasibility of quantifying AAV-2 capsid numbers via the titration of CsCl gradient fractions from recombinant and ELISA allows determination of physical to DNA-containing wild-type AAV-2 stocks. ELISA-based measurement of or infectious particle ratios. These are important paracapsid numbers revealed a large number of physical parmeters which should help to optimize and standardize the ticles with low densities corresponding to empty capsids in production and application of recombinant AAV-2.Keywords: AAV-2; recombinant AAV-2; AAV-2 titration; AAV-2 ELISA Gene therapy vectors derived from the human parvovirus AAV-2 (adeno-associated virus type 2) have gained attention owing to a unique combination of attractive features. Wild-type AAV-2 is nonpathogenic in humans and naturally defective, requiring coinfection with a helpervirus (eg adenovirus) for a productive infection. 1 Recombinant AAV-2 can infect both dividing and nondividing cells in vitro and in vivo [2][3][4][5][6] and have the potential for sitespecific integration into chromosome 19.7-9 Long-term expression of heterologous genes transduced by rAAV has been observed in a variety of human cells and tissues, such as muscle, 10,11 lung, 12 central nervous system, 13-15 retina 16 and liver. 17 An important prerequisite for the testing of AAV-2 vectors in preclinical and clinical studies is the accurate and reliable titration of the recombinant virus particles. Precise information on rAAV titers is not only crucial for the careful planning and execution of such studies, but also for comparing results amongst laboratories. In brief, presently available methods for rAAV titration can be divided into biological and physical assays. Biological assays rely on infection of cultured cells followed by events that depend on the biological functionality of the 18-20 These two types of assay yield titers of infectious or transducing particles, respectively. In contrast, physical methods are independent of biological functions of the recombinant viruses. Typically, viral DNA is extracted from the rAAV particles via enzymatic digestion ...

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