Terapia gênica neuroprotetora e regenerativa para a neurodegeneração glaucomatosa baseada na superexpressão do miR-17

Abstract

Glaucoma, a multifactorial neurodegenerative disorder, is characterized by progressive degeneration of the axons that make up the optic nerve and subsequent loss of retinal ganglion cells (RGCs). It is currently the leading cause of irreversible blindness worldwide, with more than 60 million people affected. Current treatments focus on lowering intraocular pressure. Despite treatment, in a given group of patients, the disease continues to progress. Given this, it is imperative to develop new therapeutic strategies, focused on protecting the RGCs from death, their axons from degeneration and simultaneously being able to promote the regeneration of injured axons. Therefore, this work tested the effectiveness of neuroprotective and regenerative gene therapy for glaucoma, based on the forced expression of miR-17. Recombinant adeno-associated virus (rAAV) vectors overexpressing miR-17 (rAAV.miR-17) and expressing the mCherry protein (rAAV.CTRL) were used to transduce cortical neurons in vitro and RGCs in vivo of Wistar rats. The rAAV vectors showed high transduction efficiency both in vitro and in vivo. The culture was subjected to a scratch lesion and evaluation of neuritic regeneration was performed. The rAAV.miR-17 was able to significantly increase neurite regeneration in vitro. For the in vivo analysis, RGCs were transduced with rAAV vectors, retrogradely traced by FluoroGold and subsequently an optic nerve crush was performed. Neuroprotection of RGCs was observed by overexpression of miR-17, with a significant increase in RGCs survival 28 days after the lesion in the three regions of the retina quantified. Immunostaining for neurofilament, GFAP and Iba1 was performd in optic nerve cryosections. Axonal staining by antibody against neurofilament and by the expression of mCherry revealed axonal protection in the optic nerve in the proximal and distal regions to the lesion. There was no change in astrocytes and microglia in the optic nerve by GFAP and Iba1 satining, respectively. Bioinformatics analysis using the miRWalk and miRTarBase platforms allowed the identification of predicted and validated miR-17 targets, respectively. Functional analysis of gene ontology of the predicted target genes of miR-17 showed that miR-17 has several predicted target genes involved with regeneration, degeneration and cell death. In conclusion, the present study demonstrated the neuroprotective and regenerative effect of the gene therapy based in the overexpression of miR-17 in models of neurodegeneration.