Identification of Broadly Applicable Adeno-Associated Virus Vectors by Systematic Comparison of Commonly Used Capsid Variants In Vitro

Abstract

Adeno-associated viruses (AAV) represent highly attractive gene therapy vectors and potent research tools for the modulation of gene expression in animal models or difficult-to-transfect cell cultures. Engineered variants, comprising chimeric, mutated or peptide-inserted capsids, have strongly broadened the utility of AAVs by altering cellular tropism, enabling immune evasion, or increasing transduction efficiency. In this work, the performance of 50 of the most used, predominantly published, AAVs was compared on several primary cells, cell lines and iPSC-derived models from different organs, including adipose tissue, liver, lung, brain, and eyes. To identify the most efficient capsids for each cell type, self-complementary AAVs were standardized by digital PCR, arrayed on 96-well plates, and screened using high-content imaging. To enable best use of the data, all results are also provided in a web app. The utility of one selected AAV variant is further exemplified in a liver fibrosis assay based on primary hepatic stellate cells, where it successfully reversed an siRNA-induced phenotype. Most importantly, our comparative analysis revealed that a sub-selection of only five AAV variants (AAV2.NN, AAV9-SLRSPPS, AAV6.2, AAV6TM and AAV1P5) enabled efficient transduction of all tested cell types and markedly outperformed other well-established capsids, such as AAV2-7m8. These findings suggest that a core panel comprising these five capsid variants is a universally applicable and sufficient tool to identify potent AAVs for gene expression modulation in cellular systems.