Persistence of non-viral vector mediated RPE65 expression: Case for viability as a gene transfer therapy for RPE-based diseases

被引:40
|
作者
Koirala, Adarsha [1 ]
Conley, Shannon M. [1 ]
Makkia, Rasha [1 ]
Liu, Zhao [2 ]
Cooper, Mark J. [3 ]
Sparrow, Janet R. [2 ]
Naash, Muna I. [1 ]
机构
[1] Univ Oklahoma, Dept Cell Biol, Hlth Sci Ctr, Oklahoma City, OK 73104 USA
[2] Columbia Univ, Dept Ophthalmol, New York, NY 10032 USA
[3] Copernicus Therapeut Inc, Cleveland, OH 44106 USA
关键词
RPE65; Non-viral gene therapy; Retinal pigment epithelium; DNA nanoparticle; S/MAR; LEBER CONGENITAL AMAUROSIS; COMPACTED DNA-NANOPARTICLES; PIGMENT EPITHELIAL-CELLS; MOUSE MODEL; TRANSGENE EXPRESSION; RETINITIS-PIGMENTOSA; CONE PHOTORECEPTORS; VISUAL FUNCTIONS; IN-VIVO; DELIVERY;
D O I
10.1016/j.jconrel.2013.08.299
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
Mutations in the retinal pigment epithelium(RPE) gene RPE65 are associated with multiple blinding diseases including Leber's Congenital Amaurosis (LCA). Our goal has been to develop persistent, effective non-viral genetic therapies to treat this condition. Using precisely engineered DNA vectors and high capacity compacted DNA nanoparticles (NP), we previously demonstrated that both plasmid and NP forms of VMD2-hRPE65-S/MAR improved the disease phenotypes in an rpe65(-/-) model of LCA up to 6 months post-injection (PI), however the duration of this treatment efficacy was not established. Here, we test the ability of these vectors to sustain gene expression and phenotypic improvement for the life of the animal. NPs or naked DNA were subretinally injected in rpe65(-/-) mice at postnatal day (P) 16 and evaluated at 15 months PI. Quantitative real-time PCR (qRT-PCR) and immunofluorescence were performed at PI-15 months and demonstrated appreciable expression of transferred RPE65 (levels were 32% of wild-type [WT] for NPs and 44% of WT for naked DNA). No reduction in expression at the message level was observed from PI-6 month data. Spectral electroretinography (ERG) demonstrated significant improvement in cone ERG amplitudes in treated versus uninjected animals. Most importantly, we also observed reduced fundus autofluorescence in the eyes injected with NP and naked DNA compared to uninjected counterparts. Consistent with these observations, biochemical studies showed a reduction in the accumulation of toxic retinyl esters in treated mice, suggesting that the transferred hRPE65 was functional. These critical results indicate that both NP and uncompacted plasmid VMD2-hRPE65-S/MAR can mediate persistent, long-term improvement in an RPE-associated disease phenotype, and suggest that DNA NPs, which are nontoxic and have a large payload capacity, expand the treatment repertoire available for ocular gene therapy. (C) 2013 Elsevier B.V. All rights reserved.
引用
收藏
页码:745 / 752
页数:8
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