LHON Gene Therapy Clinical Trial: Serial Pretreatment Evaluations and ND4 Expression in the Human Eye

Abstract

Purpose: : To demonstrate AAV mediated ND4 gene expression in the human eye and select LHON subjects with the G11778A mutation in mtDNA for gene therapy

Methods: : Serial PERG, OCT and clinical evaluations of 19 patients and 16 asymptomatic carriers were obtained at baseline, 6 and 12 months. A normal human eye, removed during an orbital exenteration was placed into tissue culture media immediately after removal of the cornea. We injected 120 µl of scAAV containing a nuclear-encoded ND4 linked to the ATPc mitochondrial targeting sequence and a FLAG epitope. Two days later, the retina was stained with an anti-FLAG antibody and examined by confocal microscopy. RGCs expressing the ND4FLAG chimera were counted relative to DAPI labeled cells.

Results: : ETDRS acuity was stable at one year with a mean of 13.00 letters relative to 11.87 at baseline. One patient improved from 4.50 to 35.00 a year later. HVF mean defect (MD) was -25.31 at baseline and -24.66 at one year. Only one patient improved by 7.26 MD. PNF-H (square root) was unchanged at 0.44 ng/ml relative to 0.41 ng/ml at baseline. RNFL was 53 µm at one year and 57 µm at baseline. Two patients had RNFL of 112 µm and 119 µm that dropped to 64 µm and 63 µm respectively at one year. PERG amplitudes of 0.47 µV at one year and 0.49 µV at baseline were 50% of normal. In the patient who recovered vision, PERG improved by 42%. In carriers, baseline acuity was 87.41 and 86.84 at one year. MD was -1.82 at baseline and -1.37 at one year. PNF-H increased from 0.59 ng/ ml to 0.67 ng/ml (p = 0.13). Two carriers had pNF-H > 1 that increased a year later. One carrier developed transient visual loss. Average RNFL was 104 µm at baseline and did not change at one year. Half the carriers had RNFL values greater than 100 µm. PERG dropped from 1.00 µV at baseline to 0.78 µV at one year (p = 0.0013). Decrements in the PERG were seen in both those with normal (p= 0.01) or increased RNFL (p = 0.01). Punctate and perinuclear expression of ND4FLAG surrounding DAPI labeled nuclei was predominantly seen in the RGC layer of the human retina. Counts of ND4FLAG positive cells were 6780 cells/mm². Counts of DAPI labeled RGC nuclei were 8069 /mm². Thus, 84% of human RGCs expressed the AAV delivered ND4 gene.

Conclusions: : While gene therapy with a scAAV-ND4 targets almost all human RGCs, whether restoring mitochondrial dysfunction in patients with stable RNFL loss by AAV injections of wild-type ND4 will improve visual loss remains to be determined.

Keywords: mitochondria • neuro-ophthalmology: optic nerve