Functional In Vitro Assessment of rAAV-Delivered Retinol Dehydrogenase 12 (RDH12) Activity.

Gene replacement therapy can be used for the treatment of hereditary retinopathies, such as retinol dehydrogenase 12 (RDH12)-associated Leber congenital amaurosis 13 (LCA13); however, the lack of animal models accurately mimicking the human disease phenotype requires the initial in vitro confirmation of therapy efficacy. Two synthetic serotypes (2.7m8 and PHP.S) of adeno-associated virus (AAV) were tested against the natural serotypes (5 and 9) with the aim of increasing the transduction efficiency and delivery of the green fluorescent protein (GFP) in HEK293 and ARPE-19 cells. The three most efficient serotypes were then used for the delivery of RDH12, followed by the assessment of its functional activity in the transduced cells. In the in vitro test system, a cassette encoding GFP and the wild-type (wt) RDH12 was delivered into ARPE-19 and HEK293 cells by rAAV 5, PHP.S, and 7m8 at 30K and 60K VG/cell. RDH12 mutants pThr155Ile (RDH12mut) and Met1* (RDH12sc) were used to mimic the RDH12-associated pathology. Transduction efficiency and protein expression were assessed by flow cytometry, fluorescence microscopy, and Western blotting. Percentages of AAV7m8-transduced GFP+ cells 1.5- and 6.4-times higher were observed as compared to AAV5 and AAV.PHP.S, respectively. 4-hydroxynonenal (4-HNE), more toxic to the cells with dysfunctional RDH12, was used on cells expressing the three RDH12wt versions. Following treatment with 100 μM 4-HNE, 2.6 (AAV5) and 8.8 (AAV7m8) times more cells co-expressing RDH12wt and GFP were alive as compared to the cells expressing only GFP. The number of live RDH12wt-expressing cells was also 32 and 9.6 times higher than that of RDH12sc-expressing cells and the negative control (NC), respectively. The developed approach enables the functional assessment of RDH12 replacement therapy only in rAAV-transduced cells and demonstrates that rAAV7m8 is the most efficient serotype for this purpose.