1. High-Throughput Screening Identifies Kinase Inhibitors That Increase Dual Adeno-Associated Viral Vector Transduction In Vitro and in Mouse Retina.
- Author
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Maddalena A, Dell'Aquila F, Giovannelli P, Tiberi P, Wanderlingh LG, Montefusco S, Tornabene P, Iodice C, Visconte F, Carissimo A, Medina DL, Castoria G, and Auricchio A
- Subjects
- Animals, Aurora Kinase A antagonists & inhibitors, Aurora Kinase A genetics, Cell Cycle Proteins antagonists & inhibitors, Cell Cycle Proteins genetics, Dependovirus genetics, Focal Adhesion Kinase 1 antagonists & inhibitors, Focal Adhesion Kinase 1 genetics, Gene Expression Regulation drug effects, Genetic Vectors therapeutic use, High-Throughput Screening Assays, Humans, Mice, Photoreceptor Cells drug effects, Photoreceptor Cells virology, Protein Serine-Threonine Kinases antagonists & inhibitors, Protein Serine-Threonine Kinases genetics, Proto-Oncogene Proteins antagonists & inhibitors, Proto-Oncogene Proteins genetics, Retina pathology, Retina virology, Polo-Like Kinase 1, Genetic Therapy, Genetic Vectors drug effects, Protein Kinase Inhibitors administration & dosage, Retina metabolism, Transduction, Genetic
- Abstract
Retinal gene therapy based on adeno-associated viral (AAV) vectors is safe and efficient in humans. The low intrinsic DNA transfer capacity of AAV has been expanded by dual vectors where a large expression cassette is split in two halves independently packaged in two AAV vectors. Dual AAV transduction efficiency, however, is greatly reduced compared to that obtained with a single vector. As AAV intracellular trafficking and processing are negatively affected by phosphorylation, this study set to identify kinase inhibitors that can increase dual AAV vector transduction. By high-throughput screening of a kinase inhibitors library, three compounds were identified that increase AAV transduction in vitro, one of which has a higher effect on dual than on single AAV vectors. Importantly, the transduction enhancement is exerted on various AAV serotypes and is not transgene dependent. As kinase inhibitors are promiscuous, siRNA-mediated silencing of targeted kinases was performed, and AURKA and B, PLK1, and PTK2 were among those involved in the increase of AAV transduction levels. The study shows that kinase inhibitor administration reduces AAV serotype 2 (AAV2) capsid phosphorylation and increases the activity of DNA-repair pathways involved in AAV DNA processing. Importantly, the kinase inhibitor PF-00562271 improves dual AAV8 transduction in photoreceptors following sub-retinal delivery in mice. The study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.
- Published
- 2018
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