Harnessing rAAVs as viral vectors for therapeutic transgene delivery still requires improvements in yields and specificity to lower vector doses, and therefore manufacturing cost, as well as to improve patient safety. To this end, our research is focused on developing novel technologies to ensure manufacturing of high yielding rAAV particles using transient transfection, as well as enhancing features of rAAV vectors that act on the overall size of packaged material and specificity of delivery. Here we present our state-of-the art approach to design new helper plasmids (phelpers) with the aim of improving both the infectiosity (TU/mL) and the quality (full/empty ratio) of the viral particle obtained from suspension cultures. We took the opportunity to exploit our proprietary DNA assembly method technology to explore the synergies of multiple genetic features modularly assembled in synthetic plasmids. Comparison of the biological activity of several versions of rationally designed pHelpers led us to identify the optimal configuration able to outperform existing helper plasmids in every tested bioproduction conditions. Our expertise in DNA plasmid design and assembly together with our scalable transfection solutions for rAAV manufacturing gives us the potential to improve both productivity and specificity of gene therapy products.

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