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M. C. Debrinsky, IV, S. Ellouze, S. Augustin, A. Bouaita, H. Cwerman-Thilbault, M. Simonutti, M. Paques, P. Rustin, J.-A. Sahel; A Faithful Model of Optic Neuropathy Created by an Induced Nd4 Gene Heteroplasmy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3137.
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© ARVO (1962-2015); The Authors (2016-present)
Our main goal aimed at providing a protective gene therapy via rAAV2 (recombinant Adeno Virus Associated type 2) specifying the ND4 gene to Leber Hereditary Optic Neuropathy (LHON) patients. LHON is characterized by selective death of retinal ganglion cells (RGC) and optic nerve (ON) atrophy resulting in central vision loss. ~95% of LHON patients harbor mutations in ND1, ND4 or ND6 genes located in mitochondrial DNA and encoding respiratory chain (RC) complex I subunits.The lack of a reliable animal model required for understanding physiopathology and evaluating therapies impedes any progress for treating LHON.
For creating a faithful LHON model, rat retinal ganglion cells (RGC) were transduced with the human mutant version of ND4 carried by a rAAV2 vector. Protection against RGC and ON degeneration in these rats was searched by transduction with rAAV2 vector carrying human wild-type ND4. For this, eyes were subjected to electroporation with mutant ND4 three weeks after rAAV2 administration, for overcoming the delay in rAAV-driven gene expression. For transgene expression we used optimized allotopic expression which consists on forcing mRNA sorting to the mitochondrial surface. Extensive evaluations were performed to determine whether rats recapitulated LHON hallmarks and protection can be achieved safely. Rats were subjected to optomotor test for visual acuity evaluation. Scanning Laser Ophthalmoscope and Optic Coherence Tomography visualizations allowed estimating nerve fiber density, blood vessel organization and retinal thickness. Studies were completed by RC function assessments and histochemistry.
We confirmed that our model recapitulated LHON hallmarks: (i) RC complex I defect in ONs; (ii) significant reduction of nerve fibers; (iii) reactive gliosis and microglial cell activation. These changes were permanent and led to irreversible decline of visual function. Remarkably, the effect of mutant ND4 was counteracted by wild-type ND4 expression, thus, protecting mitochondrial function, RGC integrity, and visual behavior in a long-lasting fashion.
Our strategy was successful for creating a valuable model of LHON, which will allow deciphering physiopathology and become the recipient for testing novel therapies. Next step toward our goal of clinical trials for preventing blindness in LHON patients will be to assess the long-term safety of rAAV-ND4 gene therapy with respect to retinal morphology and function in non-human primates.
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