Phelper vector

Briefly, a suspension of HEK293 cells (ECACC 85120602) was transfected with pAAV expression plasmid (ND4 or GFP), pHelper Vector (Part No. 340202, Cell Biolabs Inc., San Diego, CA, USA), and pAAV-RC6 Vector (Part No. VPK-426) with a molar ratio of 2:2:5, respectively. Then, pAAV-GFP plasmid (Cat. number VP-401, CellBiolabs, San Diego, CA, USA) was used to produce AAV6-GFP vector. To produce an AAV vector for allotopic expression of ND4, the pAAV-COX8-ND4 plasmid, expressing the ND4 gene with a COX8 mitochondrial localisation signal at the 5′ end under the control of the CMV promoter, was used (Supplementary Figure S1). DNA (1.5 µg per 1 million cells) was mixed with PEI MAX (Linear polymer, MW 40’000, Polysciences Inc., Warrington, PA, USA) at a mass ratio of 1:5, respectively, to a final volume of 5%. After transfection, the cells were cultivated in BalanCD HEK293 medium (Irvine Scientific, Santa Ana, CA, USA) at 37 °C, 5% CO₂, and 100 rpm for 120 h. The cells were then lysed (0.05% Tween-20), nuclease-treated, and centrifuged at 3000× g for 10 min, and the supernatant was filtered by 0.22 μm membranes. The concentrate was then purified by affinity chromatography (AAVX, Thermo Fisher Scientific). The number of viral genomes was determined by qPCR. The obtained sample was subsequently filtered by a 0.22 μm syringe filter (TPP 99722).