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Helene Cwerman-Thibault, Sebastien Augustin, Christophe Lechauve, Jessica Ayache, Manuel Simonutti, Jose-Alain Sahel, Marisol Corral-Debrinski; Design of a clinical study of gene therapy for Leber Hereditary Optic Neuropathy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1913.
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Since six years, we are involved in the development of a gene therapy to prevent retinal ganglion cell (RGC) loss and optic atrophy. Our aim is to succeed in the design of a clinical trial for Leber Hereditary Optic Neuropathy (LHON) due to the ND4 (NADH dehydrogenase 4) substitution G11778A, affecting 70% of patients. ND4 encodes a subunit of respiratory chain complex I; the mutation leads to complex I deficiency. In this study we determined: (1) the kinetics of transgene expression in rat RGCs after intravitreal administration of AAV2-ND4 and the subcellular localization of the protein produced by the vector. (2) Since the inner limiting membrane (ILM) could be a barrier for successful RGC transduction in patients, we assessed 3 different routes of AAV administration in non-human primate eyes.
Adult rats received intravitreal (IVT) injection of AAV2_ND4. RNA was isolated from whole retinas to determine ND4 mRNA abundance at different post-injection times. Mitochondrial crude extractions were examined by western blot and Blue Native gels to assess if the human ND4 was efficiently translocated into the organelle and may be detected in assembled complex I. For non-human primates, 3 administration routes were compared using an AAV2_GFP vector: (1) single IV injection; (2) injection after vitrectomy; (3) injection after vitrectomy and ILM peeling. One month later, vector DNA level was evaluated in ocular and non ocular tissues as well as GFP mRNA abundance; retinal structure was also studied by histology and immunolabelling.
Human ND4 mRNA was detected in rat retinas as early as 4 weeks after vector administration; its level remains stable up to 8 months later. Preliminary data indicated that ND4 protein localized to mitochondria. In non-human primates, we detected both GFP mRNA and protein in RGCs. The safest route for AAV2 administration was the single IV injection since no abnormalities were evidenced in any part of the eye. By contrast vitrectomy and ILM peeling induced inflammatory cells in the anterior segment of the eye.
We are about gathering a thorough set of data both in rat and in non-human primates that will undoubtedly validate our AAV2_ND4 gene therapy. Moreover, we prove both the safety and the efficiency of IV administration for RGC transduction in non-human primates thus preparing the clinical trial for LHON.
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