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Zongchao Han, Min Zheng, Rajendra Narayan Mitra; Rhodopsin genomic loci DNA nanoparticles transfer led physiological transgene expressions in retinitis pigmentosa. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2905.
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© ARVO (1962-2015); The Authors (2016-present)
Retinitis pigmentosa caused by genetic mutation often leads to complete blindness. Currently, the treatment is limited for the majority of patients. Gene therapy can benefit greatly by replacing the defective gene. However, most of the treatments use minigenes, such as cDNA, which may not ideally express their gene profiles due to lack of crucial endogenous regulatory elements.
In this study, we constructed DNA nanoparticles (NPs) that contain mouse and human full-length rhodopsin genomic loci, including endogenous promoters, all introns, and flanking regulatory sequences of 15-16 kb genomic rhodopsin DNA inserts (gDNA NPs), respectively. We transduced the NPs to primary retinal cell cultures from rhodopsin knockout (RKO) mice in vitro and to RKO mice eyes in vivo, and compared the effects under different functions to their plasmid rhodopsin cDNA NP counterparts that were driven by the ubiquitous promoters at the same transgene copy numbers.
Our in vitro results show that gDNA NPs-mediated rhodopsin expression were predominantly located around the cell membrane, but cDNA NPs-mediated rhodopsin expressions were restricted in both cytoplasm and cell membrane at 2-day post-transfection, indicating partial endoplasmic reticulum (ER) stress on the cDNA transfection. Our in vivo results demonstrate that genomic DNA vectors resulted in consistent high levels [at ~50% of the wild-type (WT) rhodopsin] long-term physiological transgene expressions, whereas their cDNA counterparts exhibited decreased low level expressions [from ~15% of WT rhodopsin at post-injection 1 month (PI-1m) to ~5% of the WT rhodopsin at PI-3m] with sensitivity to ER stress. In addition, electroretinogram (ERG) data showed that a 50% functional rescue was achieved through genomic loci delivery, compared to that of a 5% functional rescue via using cDNAs at PI-3m after subretinal injection.
This study demonstrates for the first time transducing rhodopsin genomic locus using compacted DNA NPs. Given that all endogenous elements were included in the vectors, our work has significant impact on future gene therapy solutions in terms of physiological transgene expressions.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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