Abstract
Purpose :
Glia-based gene therapies hold promise to alleviate or cure many retinal degenerative disorders (retinitis pigmentosa, macular degeneration...). Adeno-associated virus (AAV)-mediated transduction of Müller glia remained difficult until the development of two variants, 7m8 and ShH10. Both variants can efficiently transduce Müller glia from the vitreous with different tropism profiles, but specificity remains a challenge. In this study, we show that selective glial expression requires specific promoters to restrict off-target expression. We evaluated the tropism of 7m8 and ShH10 for glia with either a ubiquitous promoter CAG, or glial promoters GLAST and gfaABC1D driving eGFP expression in wildtype and rd10 mice.
Methods :
pAAV.scCAG.eGFP, pAAV.scgfaABC1D.eGFP and pAAV.GLAST.eGFP plasmids were packaged into 7m8 and ShH10 capids. Viruses were injected intravitreally in P60 WT C57BL/6J and rd10 mice. Fundus imaging confirmed high transgene expression 4 weeks post-injection. Retinas were flatmounted/cross-sectioned to analyze cell tropism. Retinas were stained using GFAP and GS antibodies as markers for astrocytes/glia. Retinas were also dissociated to isolate eGFP+ cells and RNA extracted for gene expression analysis. Transcription factor binding sites (TFBS) in the gfaABC1D and GLAST promoters were identified and analyzed to understand cell specificity.
Results :
ShH10 showed a higher tropism for Müller glia than 7m8 but expression was more concentrated along the vasculature. 7m8 was able to reach Müller glia pan-retinally. The gfaABC1D (688bp) promoter drove strong and selective expression in Müller glia, compared to the GLAST promoter (2.2kb), which showed ganglion off-target expression. The TFBS analysis confirmed this finding, with a higher number of glia-specific transcription factor binding motifs found in the gfABC1D promoter. Changes in Müller glia during degeneration did not affect the tropism pattern of the different vectors tested, but the efficiency of transduction was lower in the degenerated retina.
Conclusions :
Selectively targeting glia is critical to more precisely “dissect” the role of glia in the healthy and diseased retina. It represents an attractive approach for novel therapies (e.g. dedifferentiation of glia into photoreceptors) requiring no off-target expression. Our study shows that gfaABC1D promoter, in combination with 7m8 leads to strong and selective Müller glia expression from the vitreous.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.