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Andrea Maddalena, Pasqualina Colella, Ivana Trapani, Renato Minopoli, Carolina Iodice, Alberto Auricchio; Triple AAV vectors to expand AAV cargo capacity in the retina. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3631.
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© ARVO (1962-2015); The Authors (2016-present)
We have recently shown that dual AAV vectors expand AAV cargo capacity in the retina to around 9kb thus allowing their application to the therapy of inherited blinding conditions like Stargardt disease or Usher syndrome type IB (USHIB). However dual AAV vectors would not be sufficient to transfer larger coding sequences like those of genes mutated in USH type ID (CDH23, 10.5 kb in size) which would require the cargo capacity of triple AAV vectors. Our objective is to test the feasibility of gene transfer to the retina mediated by triple AAV vectors.
We have generated triple AAV vectors encoding either a reporter EGFP-DsRed fusion protein or CDH23 under the transcriptional control of the ubiquitous CMV promoter. Triple AAV2 2 were used to infect HEK293 cells in vitro while triple AAV8 vectors were administered by subretinal injection in C57Bl/6 mice. Direct fluorescence or Western blot analysis were used to evaluate transgene expression.
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. Direct fluorescence analysis of retinal cryosections as well as Western blot analysis of retinal lysates will be used to confirm this.
Our results show that the AAV DNA cargo capacity can be increased in vitro to up to 12kb by triple AAV vectors. This bodes well for further testing this platform in the retina in vivo.
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