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Astra Dinculescu, Qiuhong Li, Wen-Tao Deng, Seok-Hong Min, Jianwen Liu, Issam McDoom, William W. Hauswirth; A Self-complementary Y733F Capsid Mutant of AAV8 Prevents Photoreceptor Degeneration and Restores Retinal Function in the Rd6 Mouse Model of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2012;53(14):1634.
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© ARVO (1962-2015); The Authors (2016-present)
Therd6 mouse is a natural model of an RPE-based autosomal recessive retinal degeneration caused by a 4bp deletion in a splice donor site in the membrane-type frizzled related protein Mfrp gene. Our goal was to test the effects of gene therapy on the retinal structure and function in rd6 mice using either a tyrosine-capsid mutant of serotype AAV8 (Y733F) or AAV2 quadruple (Y272,444,500,730F) viral vectors.
One microliter (109 total vector genomes) of either scAAV8 (Y733F) or scAAV2 quadruple (Y272,444,500,730F) vector containing the small chicken β-actin (smCBA) promoter driving the wild-type mouse Mfrp cDNA was delivered subretinally to one eye of P14 rd6 mice, while contralateral eyes remained uninjected. Retinal function in treated rd6 mice was assessed by full-field electroretinography (ERG) at 6 weeks post-injection under scotopic (dark-adapted) or photopic conditions. For morphological analysis, treated 2 month-old rd6 and control eyes were processed for paraffin embedding, and stained with hematoxylin and eosin. MFRP expression in treated eyes was evaluated by immunohistochemistry and Western blot analysis.
Treatment employing the self-complementary fast-acting tyrosine-capsid mutant AAV8 (Y733F) vector provided superior rescue effects compared to the scAAV2 quadruple vector, with both a- and b-wave ERG maximum amplitudes restored to levels similar to wild-type. Both AAV vectors led to robust MFRP expression predominantly in its normal location within the RPE apical membrane and the entire length of its microvilli. Western blot analysis showed the presence of an immunoreactive MFRP band in treated eyes only. Moreover, AAV-treated rd6 retinas had a greater number photoreceptor nuclei, and long, well-organized outer segments, in contrast to untreated eyes, in which the outer segments were shortened. The characteristic accumulation of the abnormal phagocytic cells in the subretinal space was also prevented following treatment.
Subretinal delivery of wild-type mouse Mfrp gene at postnatal day P14 as mediated by a tyrosine-capsid mutant scAAV8 (Y733F) vector can prevent retinal degeneration in the rd6 mouse model of retinitis pigmentosa (RP). Moreover, treatment has led to a significant improvement in both rod and cone photoreceptor function, as assessed by electroretinography. This study indicates that the rd6 mouse model can be useful for future proof of concept experiments on RP caused by mutations in the MFRP gene.
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