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 numberCRL-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)).
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
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