Long-term Transgene Expression And Safety Of a rAAV2 Approach Of Gene Therapy For Leber Hereditary Optic Neuropathy

Abstract

Purpose: : Our main goal aimed at providing a protective gene therapy via rAAV2 (recombinant Adeno Virus Associated type 2) specifying the human 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 due, in 95% of cases, to mutations in ND1, ND4 or ND6 genes located in mitochondrial DNA and encoding respiratory chain (RC) complex I subunits. Three questions were addressed: Is rAAV2_ND4 able to prevent RGC degeneration and vision loss in a rat LHON model? Does the administration of rAAV2_ND4 in the vitreous body lead to a sustained transgene expression without arm to rat eyes? Does rAAV2_ND4 meet the criteria of long-duration transgene expression and safety when administrated to non-human primate eyes?

Methods: : Normal rats and rats mimicking LHON (which expressed mutant ND4 for 10 days before the treatment) received an intravitreal injection of rAAV2_ND4. Morphological and functional evaluations of retinas were obtained by: optomotor tests; eye fundus with a Scanning Laser Ophthalmoscope; histochemistry of retinas and optic nerves; qRT-PCR assays and spectrometric assessments of RC activity. For non-human primate experiments, a similar injection was performed. Animals were followed clinically during 3 months with extensive ophthalmic evaluations. After their death, the distribution of vector DNA molecules in ocular and non ocular tissues was evaluated (qPCR) as well as the abundance of ND4 mRNA (qRT-PCR).

Results: : In the LHON rat model, ND4 expression driven by rAAV2 leads to a sustained and significant benefit for RGC integrity, thus protecting visual function. Normal adult rats subjected to intravitreal injection of rAAV2_ND4 and expressing the transgene for up to 8 months did not show any noticeably morphological or functional change in their retinas. Preliminary data collected on the administration in primate eyes showed the absence of eye toxicity and the presence of vector DNA molecules in retinas and ONs. Experiments are in progress to determine the amount of ND4 mRNA in retinas and ONs and to confirm that long-term expression of ND4 is safe for primate eyes.

Conclusions: : We are about gathering a thorough set of data both in our experimental LHON model and in non-human primates that will undoubtedly validate our in vivo rAAV2_ND4 mediated gene therapy.

Keywords: gene transfer/gene therapy • ganglion cells • optic nerve