Abstract
Vectors based on adeno-associated viruses (AAVs) are promising therapeutic modalities used in gene therapy. Robust cell-based assays that demonstrate and quantify the potency of AAV vectors in expressing their transgene are needed for clinical development. However, many AAV clinical serotypes poorly transduce cells in vitro and often contain cell-type-specific promoters inactive in commonly used cell lines. Here, we enhance the efficiency of in vitro AAV transduction by overexpressing the AAV receptor (AAVR/KIAA0319L), preventing transcriptional silencing by the HUSH complex, and using CRISPR activation (CRISPRa) to drive transgene expression. For the latter, we utilized guide RNAs targeting the conserved AAV2 inverted terminal repeat (ITR) sequence present in most AAV transfer vectors. Using this strategy, we engineered cell lines that showed marked increases in transduction by AAV vectors across a wide range of clinically relevant serotypes and containing cell-type-specific promoters. These improvements enabled the efficient determination of AAV functional titers (also referred to as transducing titers), which can be used to robustly monitor potency across diverse AAV preparations. The strongly enhanced susceptibility of these cell lines to transduction by a variety of divergent AAV vectors could facilitate the development of standardized in vitro quantitative assays for AAV-based gene therapy products.