Abstract
Purpose :
Adeno-associate virus (AAV) vectors are the gold standard in retinal gene therapy. The major limitation of AAVs is their low genome packaging capacity, which renders them unsuitable for treatment of genes with large coding sequences. One of the most popular approaches to overcome this limitation is the dual hybrid AAV vector (DHAV) approach designed to split the coding sequence into two separate AAV vectors and to reconstitute it on mRNA level via mRNA trans-splicing. Currently, there is an unmet need for convenient and reliable methods to test the reconstitution efficiencies of DHAVs in the retina. To accomplish this goal, we developed a fluorescence-based assay and evaluated it in different cell lines and in mouse retina.
Methods :
For the in vitro assays, the first DHAV contained the 5’ part of split cerulean followed by the elements required for trans-splicing. The second DHAV contained the trans-splicing elements and the 3’ part of split cerulean. In this setting, cerulean fluorescence serves as an indicator for reconstitution. For in vivo experiments, in addition to the elements described above, the first DHAV contained citrine and the second DHAV contained mCherry, both of which serving as expression controls for the individual vectors. All AAVs were produced with the 2/8Y733F capsid variant equipped with the CMV promoter. Reconstitution efficiencies in transfected cell lines (HEK293, 661W and mouse embryonic fibroblasts) were calculated using western blotting and confocal microscopy. For in vivo experiments, C57BL6/J mice were co-injected on postnatal day 21 using titer-matched DHAVs. Retinas were prepared at four weeks post injection and processed for confocal imaging or for western blotting.
Results :
The fluorescence assay was validated in all cell lines. Depending on the different trans-splicing elements used, the reconstitution efficiencies ranged between 0% and 50% and were largely consistent between the individual cell lines. More importantly, the assay could also be validated in vivo and revealed even higher reconstitution efficiencies in the injected retinas than in the single cell lines.
Conclusions :
Our fluorescence assay is suitable for evaluation and optimization of DHAVs in the retina. This assay can be used for evaluation of DHAV-based therapies of retinal disorders caused by mutations in large genes which cannot be treated with conventional AAV-based gene therapy approaches.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.