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X. Cai, M. Cooper, M. Naash; Effective Gene Transfer for Leber Congenital Amaurosis With Compacted Dna Nanoparticle. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1737.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in the RRE65 gene cause Leber congenital amaurosis (LCA) in humans. Successful gene therapy for RPE65 deficiency has been achieved with viral delivery. Here we test the efficacy of non-viral compacted DNA nanoparticles expressing human RPE65 (hRPE65) to rescue the phenotype in the rpe65-/- mice.
Plasmids encoding hRPE65 cDNA driven by CMV, chicken β-actin (CBA), or the RPE-specific vitelliform macular dystrophy (VMD2) promoters were generated and compacted into rod-like nanoparticles with a 30-mer lysine peptide conjugated to 10 kDa polyethylene glycol (CK30PEG10k). Nanoparticles or naked DNA were delivered to the subretinal space of the right eyes of postnatal (P) 5 (1 µg) or P30 (4 µg) mice while the left eyes served as uninjected controls. Mice injected with saline were also used as controls. Gene expression was analyzed by qRT-PCR at 2, 7 and 30 days post-injection (PI), and transgene distribution and expression patterns were analyzed by immunohistochemistry (IHC). Functional improvement was assessed by ERG at PI-30, 60 and PI-120.
For three constructs injected at P5 and P30, mRNA expression in the nanoparticle injected eyes was over 8-fold higher than naked DNA injected eyes at PI-2 and gradually declined at later timepoints although several fold higher than controls. At the protein level, P5 and adult injections of CMV- and VMD2-hRPE65 nanoparticles resulted in expression of hRPE65 in RPE and photoreceptors that was sustained for up to PI-30, the latest time point tested. Both saline and naked DNA injected eyes showed no trace of RPE65 at the message and protein levels. Ganglion cell expression was only observed with the CMV-RPE65 nanoparticles. Strong and uniform expression in the RPE layer was seen at PI-30 with CMV- and VMD2-hRPE65 particles injected in adults. Retinal function was slightly improved in adult injection with all nanoparticles when compared to controls. Full expression profiles and ERG results from the P5 injection cohort are pending.
Compacted DNA nanoparticles efficiently deliver therapeutic genes to RPE cells in young and adult mouse model of RPE65-LCA. Our results demonstrate the clinical potential of nano-technology based gene therapy for providing a safe, highly efficient and sustained therapeutic delivery of genes to the RPE cells.
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