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Alun R. Barnard, Tanya Tolmachova, Miguel C. Seabra, Robert E. MacLaren; Assessment of Visual Function by Electroretinography following rAAV2-CHM/REP1 Gene Therapy in a Mouse Model of Choroideremia. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1933.
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Choroideremia is an X-linked disease that leads to degeneration of the choriocapillaris, retinal pigment epithelium and photoreceptors. It is caused by mutations in the CHM/REP1 gene, which encodes Rab escort protein-1 (REP1). Electroretinogram (ERG) recording was used to investigate the safety and efficacy of recombinant adeno-associated virus (rAAV) mediated gene delivery of CHM/REP1 in mice.
To examine if REP1 overexpression causes an alteration in retinal function, subretinal injection of 1 µl of high titer (1E+12 gp/ml) rAAV2-REP1 was performed on wildtype mice (C57BL/6, 1 month old, n=5). In all cases, the contralateral eye received an equivalent subretinal injection of rAAV2-EGFP as an internal control for non-transgene specific effects (REP1 cDNA exchanged for EGFP gene, but otherwise identical AAV2 expression cassette).To explore the potential of CHM/REP1 gene therapy to ameliorate retinal function, Chmnull/WT mice were treated with subretinal injection of 2-3 µl of rAAV2-REP1 at high (1E+12 gp/ml, n=4) and low (1E+11 gp/ml, n=5) doses at 1 month of age. Subretinal injection of an isotonic solution was performed on the contralateral eye of each animal, as an internal control for surgical sham effects. Six months after injection retinal function was assessed by ERG.
In rAAV2-REP1 treated eyes, dark-adapted ERG responses could be recorded across a 7 log unit range of flash intensity. Responses from rAAV2-EGFP injected control eyes were significantly smaller (reduced amplitude of the a- and b-waves). In a related experiment, performed using 1/10th of the viral concentration (1E+11 gp/ml for both rAAV2-REP1 and rAAV2-EGFP), response amplitudes were almost identical in both eyes and very similar to those obtained after high titer AAV2-REP1 injection.In both sham and high dose rAAV2-REP1 treated eyes of Chmnull/WT mice, intensity-dependent, dark-adapted ERG responses could be recorded. At high stimulus intensities, there was a small but significant increase in the amplitude of responses from high dose rAAV2-REP1 treated eyes compared to fellow sham treated eyes. Conversely, quantification of ERGs in low dose rAAV2-REP1 treated Chmnull/WT eyes showed a slight reduction in amplitude of a- and b-waves compared to fellow sham treated eyes overall.
No toxic effects of rAAV2-REP1 on retinal function could be identified, even at the highest dose at which toxic effects could be seen following delivery of a similar construct expressing EGFP. A significantly greater improvement in ERG was seen in the high dose compared to low dose rAAV2-REP1, indicating that 1E+11 to 1E+12 gp/ml represents the pharmacological threshold for dosing in this mouse model.
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