An Optimized Allotopic Expression Approach Leads to Generate a Rat Model of Retinal Ganglion Cell Degeneration and to Subsequently Rescue Cell Death

Abstract

Purpose: : Leber Hereditary Optic Neuropathy (LHON) leads to retinal ganglion cell (RGC) and optic nerve degeneration and remains resistant to any treatment. Our aim is to provide a therapy that will impede blindness of patients affected by LHON. We have optimized the nuclear expression of ATP6, ND1 and ND4 mitochondrial genes, by the addition of cis-acting elements which ensure the transport of their mRNAs to the mitochondrial surface. The optimization of this approach, known as "allotopic expression" led to the rescue of mitochondrial dysfunction in fibroblasts from patients bearing mutations in ATP6, ND1 or ND4 genes. We are currently examining the benefit for mitochondrial function of our vectors in an animal model that we created by the allotopic expression of the human ND4 gene carrying the G11778A mutation, responsible of 60% of LHON cases.

Methods: : We injected intravitreally in 24 eyes the human mutated ND4 gene, combined with mRNA cis-acting elements ensuring the efficient delivery of the polypeptide inside the mitochondria. As controls, we injected also 12 eyes with either the GFP or the wild-type ND4 gene. Gene transduction was obtained by electroporation (ELP). Rats were euthanized 25 days later and the effect of gene transfer was assessed in retinal sections and RGC cultures. We also performed a second ELP in eyes treated with ND4-G11778A during 14 days.

Results: : There was a 24% diminution of RGC number when the mutated version of the gene was expressed for 25 days compared to the wild-type gene (p=0.0004). Interestingly, RGCs which expressed the mutated gene for 25 days were unable to develop neuritic processes in culture indicating a deleterious effect of this expression. Notably, no adverse biological effect for RGC function and viability neither in vivo nor ex vivo were observed when the wild-type ND4 gene was transduced by ELP. Most importantly, the introduction of the wild-type gene in eyes which expressed the ND4-G11778A gene for 14 days, significantly promote RGC survival. Indeed, the number of RGCs in eyes which expressed either the mutated gene alone or the mutated gene for 14 days followed by the expression of the wild-type gene were not significantly different.

Conclusions: : We are in the process of demonstrating the proof-of-principle that our approach results in significant improvements of RGC function in the experimental model of LHON. If we succed, our protocol of gene therapy will permit the development of clinical trials to treat patients suffering for visual impairment due to mitochondrial dysfunction.

Keywords: gene transfer/gene therapy • ganglion cells • mitochondria