500. Triple AAV Vectors Expand AAV Cargo Capacity in the Retina

We have recently shown that dual AAV vectors expand AAV cargo capacity in the retina up to 9kb thus allowing their application to the therapy of inherited blinding conditions like Stargardt disease or Usher syndrome type IB (USHIB). However, for several other inherited blinding conditions the size of the coding sequence to be transferred would not fit into dual AAV vectors and would require a triple AAV vector system. We have generated triple AAV vectors encoding a reporter EGFP-DsRed fusion protein under the transcriptional control of the ubiquitous CMV promoter. The expression cassette was split in three parts and packaged in three distinct AAV vectors. In order to favour the directional concatemerization upon co-infection, highly recombinogenic sequences were added to the constructs which would then be eliminated from the mature transcript by the inclusion of flanking splicing signals (Figure 1). Triple AAV 2/2 vectors were used to infect HEK293 cells in vitro and AAV 2/8 vectors in vivo by subretinal administration in C57Bl/6 mice. Direct fluorescence analysis showed EGFP and DsRed colocalization in HEK293 cells; full length protein expression was confirmed by Western Blot analysis of HEK293 cell lysates. Fundoscopy analysis of C57Bl/6 mice up to 2 months after subretinal delivery shows signal compatible with EGFP-DsRed expression. Western-blot analysis of retinal lysates supports the production of the full length protein. Our results suggest that AAV DNA cargo capacity can be increased up to 12kb in the retina by triple AAV vectors. This bodes well for gene therapy of retinal diseases which require a high DNA cargo capacity. View Large Image | Download PowerPoint Slide