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Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

Abstract : Generation and screening of libraries of adeno-associated virus (AAV) variants have emerged as a powerful method for identifying novel capsids for gene therapy applications. For the majority of libraries, vast population diversity requires multi-plexed production, in which a library of inverted terminal repeat (ITR)-containing plasmid variants is transfected together into cells to generate the viral library. This process has the potential to be confounded by cross-packaging and mosaicism, in which particles are comprised of genomes and capsid monomers derived from different library members. Here, we investigate the prevalence of cross-packaging and mosaicism in simplified, minimal libraries using novel assays designed to assess capsid composition and packaging fidelity. We show that AAV library variants are prone to cross-packaging and capsid mosaic formation when produced at high plasmid levels, although to a lesser extent than in a recombi-nant context. We also provide experimental evidence that dilution of input library DNA significantly increases capsid monomer homogeneity and increases capsid:genome correlation in AAV libraries. Lastly, we determine that similar dilution methods yield higher-quality libraries when used for in vivo screens. Together, these findings quantitatively characterized the prevalence of cross-packaging and mosaicism in AAV libraries and established conditions that minimize related noise in subsequent screens.
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Pauline F Schmit, Simon Pacouret, Eric Zinn, Elizabeth Telford, Fotini Nicolaou, et al.. Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries. Molecular Therapy - Methods and Clinical Development, Nature Publishing Group, 2019, 17, pp.107-121. ⟨10.1016/j.omtm.2019.11.014⟩. ⟨inserm-02448091⟩



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