Recombinant AAV serotype PHP.eB was generated by a triple transfection of the HEK293T/17 cell line using polyethylenimine (PEI) transfection reagent and the 3 following plasmids: pAAV-CMV-eGFP-H1-shRNA (expressing shRNAs targeting CAG repeats or scrambled, sequences given in Figure 1 B and Figure 4 A), pUCmini-iCAP-PHP.eB [18 (link)] (encoding the AAV serotype PHP.eB capsid), and pHelper (Agilent, Santa Clara, CA, USA) (encoding the adenovirus helper functions). After 48 h of transfection, rAAV vectors were harvested from the cell lysate and treated with Benzonase (Merck Millipore 101697, Molsheim, France) at 100 U/mL. They were further purified by gradient ultra-centrifugation with Iodixanol (OptiprepTM density gradient medium, Sigma-Aldrich, Saint-Quentin-Fallavier, France)) followed by dialysis and concentration against Dulbecco’s Phosphate Buffered Saline (DPBS) (Sigma-Aldrich) using centrifugal filters (Amicon Ultra-15 Centrifugal Filter Devices 100K, Merck Millipore). Viral titers were quantified by real-time PCR using the LightCycler480 SYBR Green I Master (Roche Diagnostics, Meylan, France) and primers targeting the eGFP sequence. Titers were expressed as viral genome copies per milliliter (vgc/mL).
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