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Qiutang Li, Congwu He, Yu Tao, Xin Zhang, Alvin Luk, Bin Li; Gene therapy for Leber’s hereditary optic neuropathy with mitochondrial ND4 mutation. Invest. Ophthalmol. Vis. Sci. 2022;63(7):61 – A0034.
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Leber’s hereditary optic neuropathy (LHON) is a rare genetic disease caused by mitochondrial gene mutations, such as MT-ND1, MT-ND4, and MT-ND6. MT-ND4 (G11778A) is the most common mutation causing LHON eye diseases worldwide. It causes sudden progressive vision loss in the affected patients, and there is no approved treatment currently available for LHON. This study aims to develop a gene replacement therapy product, NFS-01 (rAAV2-ND4), for the treatment of LHON associated with MT-ND4 mutation.
A recombinant AAV2-based therapeutic vector (NFS-01) was constructed to express a codon optimized human ND4 protein with a mitochondrial targeting sequence. After NFS-01 transduction, ND4 expression and cellular localization in 293T cells were examined by mitochondria/cytosol fractionation following by western blot. The NFS-01 function was investigated by comparing the productions of oxidative phosphorylated ATP and oxygen consumption rate in LHON patient-derived BLT2 hybrid cells with or without rAAV2-ND4 transduction. Furthermore, NFS-01 expression, tissue distribution, and biosafety were studied in rabbits and macaques after intravitreal (IVT) injection.
ND4 protein expressed from rAAV2-ND4 vector was localized to mitochondria. ATP produced through oxidative phosphorylation in the mutant BLT2 cells was increased significantly after NFS-01 infection, indicating that NFS-01 at least partially restored ATP production in the mutant BLT2 cells. Consistently, rAAV2-ND4 significantly increased spare respiratory capacity of BLT2 cells as showed by Seahorse assay in the galactose medium condition. Furthermore, dose-response expression for ND4 gene was demonstrated in the rabbit retina after IVT injection. And the GLP toxicity studies showed no adverse effect or toxicity following IVT injection of NFS-01 in macaque. As part of the GLP studies, biodistribution of the vector was also performed and showed minimal spread of vector genome to spleen and lymph nodes. Neutralizing antibodies against AAV2 were either undetectable or no increase at 1- and 3-months post-injection.
Our gene therapy product NFS-01 allotropically expresses human ND4 protein in mitochondria and compensates for the loss function of ND4 in LHON derived cells. NFS-01 demonstrates effective retina delivery, expression, and safety post IVT injection in rabbits and macaques.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.
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