Ancestral library identifies conserved reprogrammable liver motif on AAV capsid.

Gene therapy is emerging as a modality in 21st-century medicine. Adeno-associated viral (AAV) gene transfer is a leading technology to achieve efficient and durable expression of a therapeutic transgene. However, the structural complexity of the capsid has constrained efforts to engineer the particle toward improved clinical safety and efficacy. Here, we generate a curated library of barcoded AAVs with mutations across a variety of functionally relevant motifs. We then screen this library in vitro and in vivo in mice and nonhuman primates, enabling a broad, multiparametric assessment of every vector within the library. Among the results, we note a single residue that modulates liver transduction across all interrogated models while preserving transduction in heart and skeletal muscles. Moreover, we find that this mutation can be grafted into AAV9 and leads to profound liver detargeting while retaining muscle transduction-a finding potentially relevant to preventing hepatoxicities seen in clinical studies.

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries.

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 multiplexed 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 recombinant 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.