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.2010 Apr;24(4):1178-91.
doi: 10.1096/fj.09-139147. Epub 2009 Dec 1.

Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa

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Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa

Xue Cai et al. FASEB J.2010 Apr.

Abstract

The purpose of the present study was to test the therapeutic efficiency and safety of compacted-DNA nanoparticle-mediated gene delivery into the subretinal space of a juvenile mouse model of retinitis pigmentosa. Nanoparticles containing the mouse opsin promoter and wild-type mouse Rds gene were injected subretinally into mice carrying a haploinsufficiency mutation in the retinal degeneration slow (rds(+ or -)) gene at postnatal day (P)5 and 22. Control mice were either injected with saline, injected with uncompacted naked plasmid DNA carrying the Rds gene, or remained untreated. Rds mRNA levels peaked at postinjection day 2 to 7 (PI-2 to PI-7) for P5 injections, stabilized at levels 2-fold higher than in uninjected controls for both P5 and P22 injections, and remained elevated at the latest time point examined (PI-120). Rod function (measured by electroretinography) showed modest but statistically significant improvement compared with controls after both P5 and P22 injections. Cone function in nanoparticle-injected eyes reached wild-type levels for both ages of injections, indicating full prevention of cone degeneration. Ultrastructural examination at PI-120 revealed significant improvement in outer segment structures in P5 nanoparticle-injected eyes, while P22 injection had a modest structural improvement. There was no evidence of macrophage activation or induction of IL-6 or TNF-alpha mRNA in P5 or P22 nanoparticle-dosed eyes at either PI-2 or PI-30. Thus, compacted-DNA nanoparticles can efficiently and safely drive gene expression in both mitotic and postmitotic photoreceptors and retard degeneration in this model. These findings, using a clinically relevant treatment paradigm, illustrate the potential for application of nanoparticle-based gene replacement therapy for treatment of human retinal degenerations.-Cai, X., Conley, S. M., Nash, Z., Fliesler, S. J., Cooper, M. J., Naash, M. I. Gene delivery to mitotic and postmitotic photoreceptors via compacted DNA nanoparticles results in improved phenotype in a mouse model of retinitis pigmentosa.

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Figures

Figure 1.
Figure 1.
Injection of MOP-NMP nanoparticles into P5 and P22rds+/− animals increasesRds mRNA levels. cDNA samples from eyes injected with saline, naked DNA, or nanoparticle DNA at P5 (A) or P22 (B) and from PI-2 through PI-120 were prepared and analyzed by qRT-PCR to determine relativeRds mRNA levels. BecauseRds primers amplify both native (endogenous) and transgenic (exogenous)Rds genes, expression values are reported as fold change relative to the uninjected contralateral eye (control). Values are averages ±se (n=3–6 mice/group). Injection of nanoparticles at P5 (A) or P22 (B) led to statistically significant increases inRds mRNA levels. Naked DNA and saline injection did not lead to any increase compared with the contralateral uninjected control eye. There was no significant difference between mRNA levels from eyes injected with nanoparticles at P5 and P22. *P < 0.01.
Figure 2.
Figure 2.
Transgenic (nanoparticle-dependent) NMP colocalizes with endogenous RDS. Frozen retinal sections from eyes collected at multiple ages (PI-2 to PI-30) were immunostained for NMP (mAB 3B6, green) and total RDS (RDS-CT, red) with a nuclear counterstain (DAPI, blue). Transferred RDS from eyes injected with nanoparticles at P5 (A) and P22 (B) is detected beginning at PI-2. Expression remains strong through the latest time point analyzed (PI-30) and colocalizes with native RDS. Expression is limited to the OSs or nascent OSs and is not detected in any other retinal cell types, subcellular compartments, or layers.n = 3–5 mice/group. INL, inner nuclear layer. Scale bars = 20 μm.
Figure 3.
Figure 3.
MOP-NMP nanoparticle injection drives gene expression in cone photoreceptors. Double immunolabeling for transferred RDS (3B6, green) and cone OSs (S-opsin, red) with nuclear counterstain (DAPI, blue) was performed at PI-30 from eyes injected at P5 (A) or P22 (B). Images are single planes from a spinning disk confocal image stack. Representative cones from nanoparticle-injected animals are shown for each treatment. Cones in eyes injected with nanoparticles express transgenic NMP. Naked DNA-injected eyes express no transgenic NMP. Controls (right) are from saline-injected (A) and uninjected (B) animals.n = 3–5 animals/group. ROS, segment; COS, cone outer segment. Scale bar = 5 μm.
Figure 4.
Figure 4.
MOP-NMP nanoparticle injection does not elicit an acute cytokine response. qRT-PCR for TNF-α and IL-6 was completed at PI-2 and PI-30 on cDNA prepared from whole eyes for animals injected at P5 or P22. No significant differences were observed between TNF-α (A) or IL-6 (B) levels in either injected sample groups (MOP-NMP nanoparticle, naked DNA, or saline treatment) or uninjected animals. Significant cytokine expression was detected in identically prepared positive control samples from theB. cereus endophthalmitis model.
Figure 5.
Figure 5.
MOP-NMP nanoparticle injection does not elicit an acute macrophage response.A) Immunolabeling with the macrophage/microglia marker F4/80 was performed at PI-2 and PI-30 in eyes injected with nanoparticles or naked DNA at P5 or P22. No labeling was detected.B) Positive control image is from an experimental endophthalmitis model to confirm antibody recognition (arrows indicate infiltrating macrophages). Negative controls include saline-injected and uninjected sections, and sections from nanoparticle-injected eyes on which normal rat IgG was used instead of the F4/80 primary antibody. GCL, ganglion cell layer. Scale bar = 20 μm.
Figure 6.
Figure 6.
Transferred NMP leads to increased expression of photoreceptor-specific proteins in therds+/− retina.A–C) Message levels of photoreceptor proteins at PI-30 were analyzed by qRT-PCR for both P5 and P22 injected mice. MOP-NMP nanoparticle injections (but not naked DNA or saline) lead to increases inRom-1 (A), rhodopsin (B), and cone S-opsin (C) message after P5 and P22 injection.n = 3 animals/group.D,E) Protein levels at PI-30 after nanoparticle, naked DNA, or saline P5 and P22 injections were examined. Representative SDS-PAGE/Western blots from individual retinas are shown (n=5–6/group). MOP-NMP nanoparticle injections at P5 and P22 increase RDS and ROM-1 (20 μg/lane;D), and rhodopsin (RHO; 10 μg/lane;E). Actin is shown as a loading control.
Figure 7.
Figure 7.
Expression of transferred NMP leads to partial functional rescue of therds+/− phenotype.A) Representative scotopic and photopic traces from naked DNA-injected (gray trace) and nanoparticle-injected eyes (black trace) at PI-30.B) Scotopic a-wave (top) and b-wave (middle) amplitudes from eyes injected with MOP-NMP nanoparticles are elevated compared with naked DNA injected eyes after P5 and P22 injection but do not reach levels seen in age-matched WT animals. Photopic b-wave (bottom) amplitudes from eyes injected with MOP-NMP nanoparticles are elevated compared with naked DNA-injected eyes after P5 and P22 injection and meet or exceed levels seen in age-matched WT animals. Amplitudes are means ±se (N values in Table 1). *P < 0.05,§P < 0.05vs. nanoparticle treatment.
Figure 8.
Figure 8.
Transferred NMP leads to structural rescue of therds+/− phenotype. Light micrographs (top rows) and electron micrographs (bottom rows,n=3–5/group) from the temporal side ofrds+/− eyes were examined. After P5 (A) or P22 (B) injection, moderate ultrastructural rescue is detected in the OSs of nanoparticle-injected eyes (arrows) at PI-30; significant ultrastructural improvement in OSs of nanoparticle-injected eyes is apparent by PI-120. OS discs are properly aligned and flattened, and improved OSs do not exhibit the swirl-like structures typical of therds+/−. IS, inner segment layer; RPE, retinal pigment epithelium. Scale bars = 10 μm.
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