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, we set to identify kinase inhibitors that can increase dual AAV vector transduction. By high throughput screening of a kinase inhibitors library, we identified 3 compounds that increase AAV transduction in vitro, one of which has 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, we performed siRNA-mediated silencing of targeted kinases and identified AURKA and B, PLK1 and PTK2 among those involved in the increase of AAV transduction levels, and we show that kinase inhibitors administration reduces 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 subretinal delivery in mice. Our study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.

High-throughput screening identifies kinase inhibitors that increase dual AAV vectors transduction in vitro and in mouse retina.

Giovannelli P;Castoria G
Investigation
;
2018

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, we set to identify kinase inhibitors that can increase dual AAV vector transduction. By high throughput screening of a kinase inhibitors library, we identified 3 compounds that increase AAV transduction in vitro, one of which has 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, we performed siRNA-mediated silencing of targeted kinases and identified AURKA and B, PLK1 and PTK2 among those involved in the increase of AAV transduction levels, and we show that kinase inhibitors administration reduces 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 subretinal delivery in mice. Our study identifies kinase inhibitors that increase dual and single AAV transduction by modulating AAV entry and post-entry steps.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11591/389835
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