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NS Dejneka, F-QQ Liang, AM Maguire, J Bennett; AAV-Mediated Delivery of Rhodopsin to the Rhodopsin Knockout Mouse (rho-/-) . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4600.
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Purpose:Rhodopsin knockout mice (rho-/-) lack outer segments, and photoreceptor degeneration is nearly complete by post-natal day 90 [Lem et al. (1999) PNAS, 96: 736-741.]. This study aimed to deliver rhodopsin to these animals via an adeno-associated virus 2 (AAV2), and assess potential rescue effects. Methods:An AAV2, expressing the murine rhodopsin cDNA in tandem with the marker enhanced green fluorescent protein was prepared. Rho-/- mice (post-natal days 3-9) subsequently received the virus subretinally (single eye) and were maintained for 12 weeks. Contralateral eyes served as untreated controls. Five, 8 and 12 weeks post-injection, eyes were enucleated, fixed, cryoprotected and assessed histologically. Samples were analyzed for rhodopsin (primary antibody, rho4D2) and EGFP expression, as well as for evidence of photoreceptor rescue, using fluorescent microscopy. Results:Five weeks post-injection, EGFP expression localized to photoreceptor cell bodies and inner segments. Immunohistochemistry revealed strong rhodopsin labeling localized to the tips of the inner segments. A similar expression pattern was also evident at 8 weeks post-treatment. At this time there also appeared to be local rescue of photoreceptors surrounding the site of injection; however, there was no evidence for outer segment regeneration, and the rescue effect was not maintained. By 12 weeks of age treated eyes degenerated to that of controls, and rhodopsin and EGFP expression was no longer evident. Conclusion:AAV2-mediated delivery of rhodopsin to rho-/- mice does not appear to rescue the rho-/- phenotype. Rescue may be dependent on the time of delivery (i.e. onset of rhodopsin expression should mimic that of the normal endogenous state) as well as the rate of onset of AAV-mediated transgene expression. Studies are underway to deliver different AAV serotypes and to deliver the transgene in utero,in order to maximize potential rescue effects.
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