Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

Cross-Packaging and Capsid Mosaic Formation in Multiplexed AAV Libraries

Blog Article

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 sweatshirts 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 Warewashing Manual Detergents 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.