CRISPR Activation Enhances In Vitro Potency of AAV Vectors Driven by Tissue-Specific Promoters

• Main Authors: Devin S. McDougald, Thu T. Duong, Katherine C. Palozola, Anson Marsh, Tyler E. Papp, Jason A. Mills, Shangzhen Zhou, Jean Bennett
• Format: Article
• Language: English
• Published: Elsevier 2019-06-01
• Series: Molecular Therapy: Methods & Clinical Development
• Online Access: http://www.sciencedirect.com/science/article/pii/S2329050119300312

Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements. Keywords: AAV, CRISPR, Cas9, dCas9, retina, photoreceptor