From the perspective of a pilot clinical gene therapy trial for Wiskott-Aldrich syndrome (WAS), we implemented a process to produce a lentiviral vector under good manufacturing practices (GMP). The process is based on the transient transfection of 293T cells in Cell Factory stacks, scaled up to harvest 50 liters of viral stock per batch, followed by purification of the vesicular stomatitis virus glycoprotein-pseudotyped particles through several membrane-based and chromatographic steps. The process leads to a 200-fold volume concentration and an approximately 3-log reduction in protein and DNA contaminants. An average yield of 13% of infectious particles was obtained in six full-scale preparations. The final product contained low levels of contaminants such as simian virus 40 large T antigen or E1A sequences originating from producer cells. Titers as high as 2 × 10(9) infectious particles per milliliter were obtained, generating up to 6 × 10(11) infectious particles per batch. The purified WAS vector was biologically active, efficiently expressing the genetic insert in WAS protein-deficient B cell lines and transducing CD34(+) cells. The vector introduced 0.3-1 vector copy per cell on average in CD34(+) cells when used at the concentration of 10(8) infectious particles per milliliter, which is comparable to preclinical preparations. There was no evidence of cellular toxicity. These results show the implementation of large-scale GMP production, purification, and control of advanced HIV-1-derived lentiviral technology. Results obtained with the WAS vector provide the initial manufacturing and quality control benchmarking that should be helpful to further development and clinical applications.

We describe here a clonal approach for efficient and robust construction of recombinant adenoviral genomes that holds certain advantages over existing approaches. Transgenes of interest are cloned into a small, conditionally replicating plasmid containing the left end of a recombinant adenoviral genome, encompassing pIX coding regions. Transformation of this plasmid into recombination-competent Escherichia coli bearing a plasmid containing the right end of a recombinant adenoviral genome, commencing from pIX coding regions, yields a stable co-integrated plasmid encoding a full adenoviral genome, by virtue of shared homology in pIX coding regions contained in both plasmids. The recombination process yielding the full adenoviral plasmid requires only one step, and always results in the formation of only the desired recombinant adenoviral genome. Thus, no screening is required to identify the correct plasmid encoding the desired recombinant adenoviral genome. In addition, the plasmid encoding the right-hand side of the adenoviral genome is itself incapable of producing contaminating adenovirus. We have successfully employed this approach to generate over 200 recombinant adenoviruses, obtaining only the desired recombinant adenoviral species each time. The process is amenable to medium-to-high-throughput parallel construction of adenoviral genomes, and as such should aid efforts aimed towards high-throughput functional annotation of therapeutic gene targets, which aim to leverage the benefits of adenoviruses as gene delivery and expression vectors.