Fig 1: Development of AAV2-hRdCVF and AAV2-hRdCVF/RdCVFL vectors that promote cone survival in vitro. (A) Map of AAV constructs: AAV2-lambda stuffer CMV/CBA-hRdCVF (4703 bp) and AAV2-CMV/CBA-hRdCVF-CMV/CBA-hRdCVFL (4837 bp). ITR, inverted terminal repeat; CMV, cytomegalovirus promoter; CBA, chicken beta actin; BGH, bovine growth hormone. (B) Silver staining of sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) deposits of the prepared AAV2 standard with five increasing amounts (ranging from 0.5 µL to 4 µL) and AAV2-hRdCVF/hRdCVFL (2 µL). M, marker lane. Asterisks indicate the capsid proteins appearing at the expected size: VP1 (87 kDa), VP2 (73 kDa), and VP3 (62 kDa). (C) Transmission electron microscopy image showing the final AAV empty (E) and full (F) particles. (D) For each suspension, the ratio of the percentage of full particles was determined from the acquisition of four separate images taken from the viral vector deposit on the electron microscope grid. Percentages of full capsids among the total AAV particles observed were determined using microscopic analysis with no significant difference across the various AAV preparations. Data represent the mean ± SEM. (E, F) Western blotting analysis revealed the presence of RdCVF and RdCVF/RdCVFL in cell protein extracts from primary porcine RPE cell cultures treated with AAV2-GFP, AAV2-hRdCVF, or AAV2-hRdCVF/hRdCVFL (E) and in corresponding supernatants (F). M, marker lane. (G) Significant cone survival was observed when fed with the supernatant from porcine RPE cultures treated with AAV2-hRdCVF or AAV2-hRdCVF/hRdCVFL compared to two controls (0: chemical defined medium [CDM] or AAV2-GFP). Each n corresponds to the number of technical samples: n = 9 for the control condition, n = 15 for AAV2-GFP, n = 15 for AAV2-hRdCVF, n = 15 for AAV2-hRdCVF/hRdCVFL. Data represent the mean ± SEM. *P < 0.05; **P < 0.01 by two-tailed test. Significance was determined using one-way ANOVA with Dunnett's multiple comparisons test. **P < 0.01 and ***P < 0.001 by two-tailed test. (H) Live/dead assay showing increased chicken cone survival when fed porcine RPE cell supernatants that had been infected with AAV2-hRdCVF or AAV2-hRdCVF/hRdCVFL compared to controls (0: CDM or AAV2-GFP). Scale bar: 20 µm.