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J. Liu, A.M. Timmers, A.S. Lewin, W.W. Hauswirth; In vivo somatic knockdown of PDE RNA in normal mice using AAV–vectored ribozymes is enhanced by addition of a constitutive transport element (CTE) . Invest. Ophthalmol. Vis. Sci. 2004;45(13):4775.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To create an animal model of retinal degeneration, we tested whether a CTE linked to a Ribozyme (Rz) might efficiently guide the Rz to its PDEγ RNA target. CTE has been proposed to bind a helicase that unwinds target mRNAs and facilitates cleavage. Methods: Four Rz and four Rz–CTE constructs targeted to specific sites in the mouse PDEγ gene were designed and packaged into serotype 5 AAV vectors. Among them, pRZ198 and pRZ266 were targeted to accessible sites located in predicted RNA loop regions while pRZ35 and pRZ42 targeted predicted inaccessible sites within stable RNA stem structure. Rz and Rz–CTE activities in vitro were performed against a 430nt RNA target under enzyme–saturating conditions. For subretinally injected and partner control retinas, retinal responses to light were measured by dark adapted ERG amplitudes at 3, 6 and 9 weeks post–treatment. Microarray analysis identified changes in retinal gene expression profiles resulting from rAAV–Rz treatment. Results: To test the efficacy of the Rz & Rz–CTE in vitro, we performed RNA cleavage analyses against a 430nt RNA target. All four Rz and all four RZ–CTE constructs efficiently cleaved the RNA substrate with good kcat and Km values. Therefore, CTE addition did not alter in vitro ribozyme activity. In contrast, after subretinal injection of equal titers of the four AAV–Rz and four AAV–Rz–CTE vectors to the right–eyes of normal C57BL mice and AAV–GFP or AAV–inactive–Rz to the left–eyes, ERG amplitudes showed that Rz coupled–CTEs were more than 20% more active than the corresponding non–CTE Rz for each target site. RNA loop targeted Rz198 and Rz266 remained more active than the RNA stem targeted Rz35, Rz42. Histology and microarray results confirmed this observation. Conclusions: Rz–CTE–mediated in vivo somatic knockdown of the wild type PDEγ gene can create an animal model of retinal degeneration more efficiently than the Rz alone. Our data suggests that the CTE assists ribozymes through RNA transport and not through RNA unwinding. AAV vector–delivered Rz–CTEs may be useful tools as retinal gene inhibitors in many contexts.
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