Epitope-tagged recombinant AAV vectors for expressing neurturin and its receptor in retinal cells

Purpose: Neurturin (NTN) is a potent neuronal survival factor in the centra l and peripheral nervous systems. We previously described altered expressio n of mRNAs for NTN and one of its receptor components, GFR alpha -2 in dege nerative retinas of rd/rd mice. Towards assessing the potential for transfe r of these genes to counteract retinal degeneration, we examined recombinan t adeno-associated virus (rAAV) constructs for expression of NTN and GFR al pha -2 transgenes in retinal cells in vitro and for the effect of transgene expression on retinal function following intraocular delivery in rd/rd mic e. Methods: The rAAV constructs incorporated epitope tags to facilitate discri mination between transgenic and endogenous expression. Expression of murine NTN was driven by a CMV promoter and a partial murine opsin promoter was u sed to drive expression of human GFR alpha -2. rAAV preparations were used to infect mouse retinal cell cultures and for intraocular injection of pred egenerative rd/rd mice. Endogenous and transgene expression was analyzed by immunofluorescence. Photoreceptor function in treated mice was assessed by electroretinography. Results: Both vectors delivered and expressed their transgenes in vitro and in vivo. Differential targeting was achieved in vivo through the use of al ternative promoters. Under the conditions examined, no functional rescue of rd photoreceptors was observed. Conclusions: Therapeutic treatment of the rd model of retinal degeneration does not appear to be effected by simple modulation of the expression of NT N or GFR alpha -2, and may therefore depend on additional synergistic facto rs. Our AAV constructs will facilitate the development of combinatorial app roaches to the treatment of central and peripheral neurodegenerations.