AAV-mediated inhibition of miR-181a/b as a gene-independent therapeutic tool for LHON

Abstract

Purpose : Leber Hereditary Optic Neuropathy (LHON) is the most common mitochondrial disease mainly affecting Retinal Ganglion Cells (RGCs). It shows a high genetic heterogeneity that makes the development of effective treatments particularly difficult. Gene/mutation-independent approaches acting downstream of the genetic defect are thus hugely desirable. We hypothesize that the inhibition of the microRNAs 181a and b (miR-181a/b) involved in mitochondrial functions may be protective in a model of LHON, i.e. the Ndufs4KO mouse.

Methods : At postnatal day 2 (P2), Ndufs4KO mice were injected with Adeno Associate Virus (AAV) carrying tandemly repeated miR-181a/b antisense sequences (miR-181a/b sponge) or with AAV-GFP as control. Treated mice (N=8 mice/condition) were analyzed by Optokinetic response (OKR) test at P35 and Electroretinogram (ERG) at P50. Eyes were then examined by the following histological and molecular analyses (N=3 eyes/condition/experiment): a) immunofluorescence (IF) to assess the number of RGCs, b) western blot (WB) to evaluate the effect of the treatment on mitochondria, c) electron microscopy (EM) to determine the number and morphology of mitochondria. Finally, to assess the safety of our strategy, 2 groups of 5 WT mice were injected with AAV-miR-181a/b sponge or AAV-GFP and analyzed as described above. Two-tailed Student's t-test was used for statistical analysis.

Results : Ndufs4KO mice injected with miR-181a/b sponge showed an evident amelioration of the visual ability. Indeed, both OKR and ERG resulted significantly increased in miR-181a/b sponge-treated mice compared to controls (p=0.005 and ≤0.0006, respectively). Moreover, IF revealed a higher number of RGCs in miR-181a/b sponge-injected eyes with respect to controls (p=0.03), indicating a preservation of RGCs. Interestingly, WB also showed increased mitochondrial proteins, and EM disclosed an increased number of mitochondria and a clear amelioration of the mitochondrial structure, indicating a beneficial effect of miR-181a/b sponge on mitochondrial phenotypes. Notably, retinas from WT mice injected with AAV-miR-181a/b sponge do not show functional or histological abnormalities 1-year post-injections.

Conclusions : Overall, our data demonstrate that AAV-mediated inhibition of miR-181a/b is safe and protective in a LHON mouse model, unveiling a significant therapeutic effect of the sponge therapy and the possibility of translating it in clinics.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.