High-Throughput Screening Identifies Kinase Inhibitors That Increase Dual Adeno-Associated Viral Vector TransductionIn Vitroand in Mouse Retina

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.

Seeing the Light after 25 Years of Retinal Gene Therapy

“…This result also confirms that co-infection by multiple AAV vectors is favored in the subretinal space of larger animals, as observed for dual AAV [45]. In addition, strategies that can improve single or multiple AAV transduction, such as the combined delivery of kinase inhibitors with AAV [47], may help to bring multiple AAV-based strategies to the required efficacy to move from bench to bedside.…”

Hair Cell Transduction Efficiency of Single- and Dual-AAV Serotypes in Adult Murine Cochleae

“…Although the co-transduction efficiency of dual vectors in retina and neurons has been investigated, the dualvector co-transduction rate in the inner ear has not been analyzed in adult mice. 15 To identify AAV serotypes that transduce both mature murine IHCs and OHCs, we first selected five AAV serotypes, AAV1, AAV2, AAV8, AAV9, and Anc80, to inject into the cochlea using the RWM+CF technique. All vectors carried a CMV-driven EGFP transgene and the woodchuck hepatitis post-transcriptional virus regulatory element (WPRE) cassette.…”

Full-length ATP7B reconstituted through protein trans-splicing corrects Wilson disease in mice

“…Serotype 8 AAV vectors were produced by triple transfection of HEK293 cells as previously described. 37 For Southern blot analysis, DNA was extracted from 6Â10 10 viral particles measured as genome copies (gc) for AAV2/8-HLP-5 0 ATP7B-N-intein, and 1.2Â10 11 gc for AAV2/8-HLP-C-intein-3 0 ATP7B, and for AAV2/8-HLP-full-length ATP7B. To digest unpackaged genomes, the vector solution was incubated with 30 mL of DNase I (Roche) in a total volume of 300 mL, containing 50 mM Tris (pH 7.5) and 1 mM MgCl 2 for 2 h at 37 C. The DNase was then inactivated with 50 mM EDTA, followed by incubation at 50 C for 1 h with Proteinase K and 2.5% N-lauryl-sarcosyl solution to lyse the capsids.…”