Abstract
Purpose: :
Müller cells (RMC) span the entire thickness of the retina. These specialized retinal glial cells show a high resistance to pathological insults and play an active role in the regulation of survival and death of photoreceptors following injury through the release of neurotrophic factors. Therefore, they are well-situated targets for therapeutic intervention. GDNF has been shown to be a potent neuroprotective molecule in slowing retinal degeneration. Previous gene therapy studies targeted epithelia and neurons. The purpose of this study is to achieve photoreceptor neuroprotection via sustained secretion of hGDNF from Müller glia by optimizing gene transfer and expression levels in these cells. We expect that hGDNF gene transfer to retinal glia will enhance the endogenous protective functions of RMC. Additional advantages for targeting hGDNF to RMC are to alleviate potential stress to photoreceptors caused by direct gene transfer. Muller cell transfection and secretion opens the possibility to deliver the viral vector by intravitreal injection, avoiding injury from subretinal injection and retinal detachment.
Methods: :
mGFAP and CD44 glial specific promoters (Greenberg et al. IOVS, 2007) packaged into VSV-G pseudotyped lentivirus vectors aswere used to drive hGDNF expression. 3µl of high titer (109-1010 TU/mL) vectors were injected into the superior subretinal space of S334 rats at p15. The S334 transgenic rat is a model of dominant retinitis pigmentosa expressing a C-terminal truncated form of rhodopsin. We correlated the morphological and functional data with histology and scotopic ERGs. Expression levels of the transgenes will bewere compared by measured by ELISA and QPCR for each promoter and compared.
Results: :
In vivo injection of LV mGFAP.GFP and CD44.GFP vectors generates strong expression in RMC and some RPE cells. At 30 days post-injection, ERG recordings demonstrate a consistent preservation of the a and b-wave amplitudes in LV-GDNF injected eyes compared to LV-GFP. These data suggest that hGDNF expressed by Muller cells can slow photoreceptor degeneration in this animal model.
Keywords: gene transfer/gene therapy • Muller cells • retinal degenerations: cell biology