Prep2 cap2

AAV-ophNdi1 (patent no. 10220102), AAV-Ndi1 [39 (link)] and AAV-CAG-EGFP (AAV-EGFP; [47 (link)]) recombinant AAV2/2 viruses were generated as previously described [50 (link)]. Briefly, HEK293 cells were transfected with pAAV-ophNdi1, pAAV-Ndi1 or pAAV-EGFP, pRep2/Cap2 and pHelper (Agilent Technologies, Santa Clara, CA, USA) at a ratio of 1:1:2, using polyethylenimine. At 72 h post-transfection, AAV particles were purified from the clarified lysate by triple caesium gradient ultracentrifugation and dialysed against PBS supplemented with Pluronic F68 (0.001%; [51 (link)]). Genomic titres (vg/mL) were determined by quantitative real-time PCR [52 (link)].

AAV2/2-OPA1 iso 1, AAV2/2-OPA1 iso 7 and AAV2/2-CAG EGFP were prepared by the Farrar group at TCD (Lane et al., 2020 (link)). The two OPA1 cDNAs were cloned into pAAV-MCS (accession no. AF396260.1; Agilent Technologies, Inc., United States).
Recombinant AAV2/2 viruses were generated by helper virus free, triple transfection based on the method described by (Xiao et al., 1998 (link)). Human embryonic kidney cells (accession number CRL-1573; ATCC, United States) were transfected with pAAV-MCS plasmids containing OPA1 iso 1 or 7, pRep2/Cap2 and pHelper (Agilent Technologies, Inc., United States) at a ratio of 1:1:2, using polyethylenimine, as previously described (O’Reilly et al., 2007 (link)). 72 h post-transfection, AAV particles were purified from the clarified lysate by cesium gradient centrifugation. AAV containing fractions were dialyzed against PBS supplemented with Pluronic F68 (0.001%; Bennicelli et al., 2008 (link)). Genomic titers (viral genomes/ml; vg/ml) were determined by quantitative real-time PCR (Rohr et al., 2002 (link)).