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H. Petrs-Silva, D. Yasumura, M. Matthes, M. LaVail, W. W. Hauswirth, A. Lewin; Suppression of rds Expression by siRNA and Gene Replacement Strategies for Gene Therapy Using AAV Vector. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5336.
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© ARVO (1962-2015); The Authors (2016-present)
Peripherin/RDS is a photoreceptor-specific transmembrane glycoprotein localized to the rim region of outer segment disks in rods and cones. Over 90 human mutations in RDS have been identified and they result in a wide phenotypic spectrum of retinal dystrophies, related with Retinitis pigmentosa and also with a variety of macular dystrophies. To date, attempts to achieve complete structural and functional rescue in animal models of peripherin/rds-induced retinal degeneration have not been successful hindered by the haploinsufficiency phenotype. Our goal is to develop a gene therapy for RDS mutation. For this purpose, we develop an RNA replacement therapy for rds/peripherin in which levels of endogenous mutant and wild-type mRNA are reduced using a siRNA, and an siRNA-resistant version of the RDS mRNA is supplied simultaneously.
All siRNAs and RDS resistant gene were cloned in AAV vectors under the control of H1 and m-opsin promoter, respectively. Through subretinal injections the siRNA and the hardener gene was delivered to photoreceptor cells of mice retinas in recombinant Adeno-associated virus (rAAV). The retinal phenotype was examined, both structurally (histology) and functionally (electroretinography-ERG) in different time points after rAAV delivery.
Initially 4 siRNA were analyzed in order to determine its effect in RDS reductions in vitro and in vivo. All 4 siRNA were able to decrease RDS expression with different potency. Based on these results, 2 siRNA-resistant version of the RDS gene were constructed and analyzed in C57-black mice for its capacity to replace RDSwt and for its resistance to the respective siRNA action. One month after combination injection of vectors containing different siRNAs and the resistant version of RDS, ERG showed that both siRNA-resistant versions of the RDS were not decreased in the presence of its respective siRNA, while siRNA alone decrease retinal function.
With our results we validate the concept of suppression of RDS and replacement strategies of gene therapy with rAAV vectors containing siRNA directing de down regulation of RDS and its replacement by a harderner version. Further analysis is necessary to determine the therapeutic potential of the siRNA and resistant RDS harderner construct in vivo models of RDS/peripherin mutation.
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