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R.A. Bush, Y. Zeng, D. Raz–Prag, S. Kjellstrom, M. Santos–Muffley, P.A. Sieving; Adenovirus–Associated Virus (AAV) Mediated Delivery of Lens Epithelium Derived Growth Factor (LEDGF) Preserves Photoreceptor Function in RCS Rats . Invest. Ophthalmol. Vis. Sci. 2005;46(13):187.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Intraocular injection of LEDGF protects photoreceptors in two animal models of retinal degeneration, light damaged and Royal College of Surgeons (RCS) rats. Protection of cells in vivo and in vitro by LEDGF was associated with increases in small heat shock proteins. This study investigated the protective effects of AAV mediated delivery of LEDGF in RCS rats and the possible role of HSP25 and αß–Crystallin. Methods: Rat LEDGF cDNA was cloned by RT–PCR. A silent mutation was introduced into the ORF to allow viral and endogenous LEDGF to be distinguished. Rat LEDGF was inserted into the vector of pZac2.1 to make Cis AAV with LEDGF driven by a CMV promoter. Recombinant AAV–LEDGF was generated by triple transfection. Twenty RCS rats, at 3 to 7 weeks of age, received subretinal injections of AAV–LEDGF and the fellow eyes received PBS. Five to 22 weeks after injection, Ganzfeld dark– and light–adapted electroretinograms (ERG) were recorded from threshold to 0.6 log cd–s/m2. The criterion for increased preservation of function was a >100% higher amplitude ERG response in the AAV–LEDGF injected eye relative to the contralateral PBS injected eye at the highest flash intensity in dark adapted conditions. RT–PCR and restriction fragment length polymorphism (RFLP) method was performed on all retinas following the final ERG to verify viral LEDGF expression. Real–time RT–PCR was utilized to quantify mRNA of LEDGF, Hsp25 and αß–Crystallin and western blotting was used to evaluate the protein expression of Hsp25 and αß–Crystallin in 8 rats. Results: Out of 16 AAV–LEDGF injected eyes, in which viral LEDGF mRNA was evident, twelve had distinctly high levels and all but one of these showed better preserved retinal function compared to control eyes. Four eyes with low levels of viral LEDGF mRNA and one with a relatively high level did not show functional rescue. One rat followed for 22 weeks still had better preserved retinal function in the AAV–injected eye at 4 months after injection. Quantitative RT–PCR showed that expression of total LEDGF mRNA was 1.6 to 3–fold higher in AAV–LEDGF injected eyes than in control eyes, and was well correlated with functional rescue. However, neither the RNA or protein levels of Hsp25 and αß–Crystallin were associated with ERG differences. Conclusions: AAV–mediated expression of LEDGF produced photoreceptor protection in RCS rats. However, there was no evidence that Hsp25 or αß–Crystallin were directly involved in preserving photoreceptor function.
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