Abstract
Purpose :
Stargardt disease is an inherited, chronic, and progressive retinal dystrophy caused by mutations in the ABCA4 gene resulting in the most common form of juvenile macular degeneration, for which there are currently no therapeutic options. The ABCA4 coding sequence is 6,822 nucleotides in length and therefore exceeds the packaging capacity of a typical AAV capsid. To enable delivery of large genes, several dual AAV vector strategies have been employed, including homologous recombination, trans-splicing, and intein-mediated protein splicing, but each method has advantages and limitations. To overcome these hurdles, we have created a dual AAV approach that allows efficient recombination of N-terminal and C-terminal ABCA4 fragments by exploiting Cre recombinase.
Methods :
The first AAV vector encodes the N-terminal region of the ABCA4 gene and also expresses Cre recombinase via a self-cleaving T2A peptide. The second AAV vector encodes the C-terminal region of the ABCA4 gene. Cre recombinase recognizes LoxP sites inserted into each ABCA4 construct and combines the N- and C-terminal fragments resulting in a full-size ABCA4 expression cassette that will exist episomally within the cell. During the same recombination process, the Cre recombinase gene loses its promoter and inactivates itself.
Results :
We have demonstrated that infection in tissue culture with a 1:1 ratio of AAV.ABCA_N and AAV.ABCA_C vectors results in efficient ABCA4 coding sequence reconstitution and full-length ABCA4 protein production within 48 hours. Tissue culture cells infected with ABCA_N and ABCA_C vectors lacking Cre recombinase show no detectable full-length ABCA4 protein after up to 120 hours.
Conclusions :
Full-length ABCA4 protein can be efficiently reconstituted from two independent AAV vectors by utilizing Cre recombinase. Future studies exploring Cre-mediated ABCA4 reconstitution in vivo and the ability of dual AAV-delivered ABCA4 to clear lipofuscin build-up will be essential milestones to move this early-stage program towards the clinic.
This is a 2021 ARVO Annual Meeting abstract.