Genomic DNA nanoparticles rescue rhodopsin‐associated retinitis pigmentosa phenotype

Abstract

Mutations in the rhodopsin gene cause retinal degeneration and clinical phenotypes including retinitis pigmentosa (RP) and congenital stationary night blindness. Effective gene therapies have been difficult to develop, however, because generating precise levels of rhodopsin expression is critical; overexpression causes toxicity, and underexpression would result in incomplete rescue. Current gene delivery strategies routinely use cDNA-based vectors for gene targeting; however, inclusion of noncoding components of genomic DNA (gDNA) such as introns may help promote more endogenous regulation of gene expression. Here we test the hypothesis that inclusion of genomic sequences from the rhodopsin gene can improve the efficacy of rhodopsin gene therapy in the rhodopsin knockout (RKO) mouse model of RP. We utilize our compacted DNA nanoparticles (NPs), which have the ability to transfer larger and more complex genetic constructs, to deliver murine rhodopsin cDNA or gDNA. We show functional and structural improvements in RKO eyes for up to 8 months after NP-mediated gDNA but not cDNA delivery. Importantly, in addition to improvements in rod function, we observe significant preservation of cone function at time points when cones in the RKO model are degenerated. These results suggest that inclusion of native expression elements, such as introns, can significantly enhance gene expression and therapeutic efficacy and may become an essential option in the array of available gene delivery tools.

Let There Be Light: Gene and Cell Therapy for Blindness

“…In general, they allow transfection of cells with larger pieces of DNA and carry lower risk of immune responses associated with viral gene delivery. 33,34 However, lack of long-term gene expression is a major limitation of such vectors. For example, clinical trials using polyethylene glycol-substituted 30-mer lysine peptide-based nanoparticles and lipid-mediated vectors to deliver therapeutic genes to the nasal mucosa of patients with cystic fibrosis reported no detectable gene expression, although vector DNA was detectable for at least 2 weeks.…”

DNA nanoparticles are safe and nontoxic in non-human primate eyes

“…They were as follows: 1) the pscCBA-GFP vector in which GFP reporter gene expression is directed by the chicken beta-actin promoter 19 (CBA, known here as CBA-GFP, 5,770 bp 19 ) originally provided to us by Dr Arun Srivastava, Department of Medicine at the University of Florida; 2) the pEPI-S/MAR vector (backbone originally provided by Dr Isa Stehle/ Hans Lipps, University of Witten/Herdecke) in which GFP expression is driven by the RPE-specific human vitelliform macular dystrophy 2 promoter 2 (VMD2, −585/+38, known here as VMD2-GFP, 20 6,778 bp); and 3) the pEPI-S/ MAR vector in which GFP expression is under the control of the photoreceptor-specific promoter rhodopsin kinase (RK, −385/+86, known here as RK-GFP 8 ). Endotoxin-free (,5 EU/mg) plasmid DNA was generated by Aldevron, L.L.C.…”

“…2,3 We have shown that they efficiently transfect both photoreceptors 3,4 and the RPE, 2,4,5 and can improve the disease phenotype in the rds +/− and rhodopsin-knockout models of retinitis pigmentosa, 3,[6][7][8] the Abca4 −/− model of Stargardt's dystrophy, 9 and the Rpe65 −/− model of Leber's congenital amaurosis.…”

Gentherapie als Behandlungskonzept für erbliche Netzhauterkrankungen

“…Nanopartikel, die an das therapeutische Gen gebunden werden, sind eine weitere Möglichkeit des nicht viralen Gentransfers. Dieser Nukleinsäu rekomplex kann nun durch rezeptorver mittelte Endozytose in die Zelle aufge nommen werden [4]. …”