PMC:2669177 / 6288-25169 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/2669177","sourcedb":"PMC","sourceid":"2669177","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2669177","text":"Results\n\nRds nanoparticles drive high and persistent transgene expression\nRDS expression and localization to the distal connecting cilium in the mouse rod photoreceptor cell begin around postnatal day 5 (P5) [26], [32] (i.e., before OS formation), a time that precedes the onset of retinal degeneration in the rds model. Hence, we selected P5 as the physiologically appropriate developmental stage for therapeutic intervention. Two vectors were generated, each expressing the full-length cDNA of normal mouse peripherin/rds (NMP), one under the control of the ubiquitously expressed chicken beta-actin promoter (CBA), and the other employing the well characterized photoreceptor-specific promoter for the human interphotoreceptor retinoid-binding protein (IRBP) [33]. Acetate compacted nanoparticles containing the vectors (Figure S1) or controls were injected subretinally into rds+/− mice at P5 and followed for up to four months. The controls chosen for this study were saline (vehicle) and uncompacted plasmid DNA (called “naked DNA”) carrying the same therapeutic transgene (CBA-NMP or IRBP-NMP).\nAs shown in Figure 1, injection of both CBA-NMP and IRBP-NMP nanoparticles resulted in significantly elevated expression of Rds message, as measured by qRT-PCR. At post-injection day 2 (PI-2), mRNA levels in CBA-NMP and IRBP-NMP nanoparticle- injected eyes were at least three- to four-fold higher than the saline or naked DNA-injected eyes (Figure 1). Eyes injected with IRBP-NMP maintained elevated expression until PI-14, then stabilized at levels two- to three-fold higher than controls, while eyes injected with CBA-NMP stabilized at similar levels at PI-7. Neither saline nor naked DNA produced a significant alteration in Rds mRNA levels (Figure 1A), compared to uninjected eyes. Elevated mRNA levels were maintained for up to four months (PI-120), the longest time point examined.\nFigure 1 Injection of NMP nanoparticles into P5 rds +/− animals increases Rds mRNA levels.\ncDNA from eyes injected with saline, naked DNA (A) or nanoparticle DNA (B) at PI-2 through PI-120 was prepared and analyzed by qRT-PCR to determine relative Rds mRNA levels. Because Rds primers amplify from the NMP (nanoparticle) and the WT (endogenous) allele but not from the Rds mutant allele, expression values are reported as fold change from the uninjected contralateral control eye. Values shown are averages±S.D. (N = 3–6 mice per group). (A) Injection of saline or naked DNA does not alter Rds message levels at any time point. (B) Conversely, injection of both CBA-NMP and IRBP-NMP compacted DNA nanoparticles leads to a significant, two- to four-fold increase in total Rds message level compared to the naked DNA injected eyes (*p\u003c0.001, **p\u003c0.05. This increase persists through the last time point examined (PI-120).\n\nCompacted DNA nanoparticles efficiently transfer RDS to all photoreceptor cells\nWe next examined the identity of the cells that took up the exogenously delivered NMP cDNA and the efficiency of gene product expression within the retina over time, using immunohistochemistry. The entire eye was cut and every sixth section was collected and assessed, enabling us to examine gene expression in multiple regions throughout the retina. Due to an epitopic modification in the NMP carboxyl terminus (P341Q), which is not present in wildtype RDS and does not result in retinal disease or vision loss [24], the transferred Rds gene product can be detected selectively even on a normal RDS background using a monoclonal antibody (mAB 3B6) that recognizes the P341Q epitope. [For demonstration of the selectivity of mAB 3B6 for transferred RDS (NMP) as opposed to native (endogenous RDS), see Figure S2 and our previous publications [24], [34].] The endogenous mouse RDS protein (and to a much lesser extent transgenic NMP protein) is labeled with the RDS-CT antibody [35]. Although normal OS development has not yet begun at P7 (PI-2) [36], Figure 2 (top row) shows expression of both transferred (Figure 2A and 2B) and native RDS (Figure 2C) protein in the tip of the photoreceptors. By PI-7 (P12), distinct outer and inner nuclear layers are apparent and NMP/RDS staining in the tips of nascent OSs is visible as a thin immunopositive layer adjacent to the photoreceptor nuclei. NMP distribution in the OSs persisted through the latest time point examined (PI-30). NMP also co-localized with native RDS and was usually limited to the OS layer (Figure 2); no NMP was detected in eyes injected with saline (Figure 2C and Figure S3) or naked DNA (not shown). Occasionally, NMP expression was detected in RPE cells after nanoparticle injection (Figure 2). Expression in the RPE was highly variable; while many animals had some RPE expression, others did not. RPE expression was much more common in eyes injected with CBA-NMP nanoparticles, consistent with its role as a ubiquitous promoter. On the other hand, IRBP-NMP staining in the RPE was almost always limited to PI-2. NMP detection with mAB 3B6 in the OSs was heterogeneous, with stronger signal in areas closer to the injection sites. However, we estimate that a majority of photoreceptors expressed the product of the transferred gene (based on a qualitative assessment of mAB 3B6 immunostaining in successive retinal sections). The choice of promoter did not have any apparent effect on cellular distribution within the photoreceptor: both CBA-NMP and IRBP-NMP nanoparticles exhibited similar distribution patterns at all time points examined.\nFigure 2 Transferred NMP co-localizes with endogenous RDS.\nFrozen 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 CBA-NMP (A) and IRBP-NMP (B) nanoparticles is detected at PI-2. Expression remains strong through the latest time point analyzed (PI-30) and co-localizes 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. (C) No NMP is detected in saline-injected control eyes, but native RDS is detected beginning at PI-2 (P7), consistent with normal ocular development. Scale bars, 20 µm. N = 3–5 mice per group. Abbreviations: RPE, retinal pigment epithelium; OS, outer segment layer; ONL, outer nuclear layer; INL, inner nuclear layer.\n\nRds nanoparticles improve expression levels of key visual transduction proteins\nOur next step was to determine whether nanoparticle-driven expression of NMP results in rescue of the rds +/− disease phenotype. To measure biochemical rescue, we assayed the levels of several photoreceptor-specific proteins known to be decreased by RDS deficiency. Figure 3 (panels A and D) shows that expression levels of the RDS binding partner ROM-1 were increased, both in terms of message (by qRT-PCR) and protein (by Western blot analysis), compared to uninjected controls, at PI-30. Consistent with the mRNA data presented in Figure 1, expression of RDS protein was also increased in NMP nanoparticle-injected eyes. Expression of rhodopsin (the rod visual pigment) is necessary for phototransduction and proper photoreceptor maintenance, and is significantly decreased in the rds +/− retina [37]. We show that injection of NMP nanoparticles led to increased rhodopsin message (Figure 3B) and protein (Figure 3E) levels. We also observed a similar increase in the message level of short-wavelength cone opsin (S-opsin, Figure 3C) after nanoparticle injection, although no alteration in S-opsin protein level was detected (Figure 3F), likely due to lack of cone degeneration at this age.\nFigure 3 Transferred NMP leads to increased expression of photoreceptor-specific proteins in the rds +/− retina.\n(A–C) cDNA was collected at PI-30, and message levels of photoreceptor genes were analyzed by qRT-PCR. (A) CBA-NMP nanoparticle injection leads to a modest increase in Rom-1 message levels, while IRBP-NMP nanoparticle injection increases expression four- to five-fold over levels in uninjected control eyes. (B,C) CBA-NMP and IRBP-NMP nanoparticle injections lead to increases in rod (B) and cone (C) opsins. (A–C); N = 3 animals per group. (D–F) Protein levels at PI-30 after nanoparticle injection were examined. Representative SDS-PAGE/Western blots from individual retinas are shown (N = 5–6 animals per group). (D) CBA-NMP and IRBP-NMP nanoparticle injections increase RDS and ROM-1 protein levels (protein load: 20 µg per lane). (E) Increases in rhodopsin protein (RHO) are detected after injection of both CBA-NMP and IRBP-NMP nanoparticles (protein load: 10 µg per lane). (F) No change in S-opsin (S-ops) protein level is detected after nanoparticle injection (protein load: 50 µg per lane). (G) Double immunolabeling for transferred RDS (mAB 3B6, green) and cone OSs (S-opsin, red) with nuclear counterstain (DAPI, blue) was performed on frozen sections from PI-30 eyes. Representative cones from two different animals are shown for each treatment. Cones in eyes injected with CBA-NMP or IRBP-NMP nanoparticles express transferred NMP (top and middle rows). Saline injected eyes express no transferred NMP (bottom row). Scale bar, 5 µm; N = 3–5 animals per treatment group. Abbreviations: OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer * = p\u003c0.05. Since the photoreceptor population in the mouse retina consists of 95–97% rods [38], [39], the results presented in Figure 2 are consistent with the conclusion that the two types of nanoparticles drove gene expression in rods and that their products were delivered with fidelity to the OS. However, it was not clear from those data whether transferred RDS protein was expressed in cones. Therefore, double labeling for NMP and S-opsin was performed on PI-30 eyes. Two representative cones from each nanoparticle-injected and control eye are shown in Figure 3G (single, 0.5-µm slices of spinning disk confocal image stacks). Most cones from nanoparticle-injected eyes expressed NMP when consective sections were evaluated from the same eye. S-opsin immunopositive cone cells that lacked NMP expression were mainly located in areas far from the injection site. No NMP-positive cells were detected in saline-injected eyes (Figure 3G, bottom row).\n\nNanoparticle-driven Rds expression restores retinal function\nThe rds +/− mouse adRP model exhibits reduced electroretinogram (ERG) responses indicative of early-onset slow rod degeneration followed by late-onset slow cone degeneration [26], [40]. In order to assess functional rescue of this phenotype after treatment, full-field ERGs were obtained from nanoparticle-injected and control mice. Initial ERGs were obtained and analyzed at PI-30 (see Table 1). Average scotopic a-wave amplitudes, indicative of rod function, were increased with statistical significance after injection of either CBA-NMP or IRBP-NMP nanoparticles, compared to amplitudes from eyes injected with naked DNA or saline. In order to confirm that the naked DNA had no adverse effect, a subset of animals was injected with saline only. Scotopic a-wave amplitudes for saline injected animals were not significantly different from those injected with either CBA-NMP or IRBP-NMP naked DNA (p = 0.2634). Interestingly, nanoparticles led to an improvement in cone function. The magnitude of rescue varied considerably with both nanoparticles, most likely due to variations in particle uptake and/or relative activity of CBA vs. IRBP promoters in rods and cones. Several nanoparticle-injected animals exhibited significantly greater-than-average rescue; 6/15 (IRBP-NMP) and 5/19 (CBA-NMP) treated animals had 90% increase in scotopic a-wave amplitudes, compared to naked DNA-injected controls. Similarly, 4/15 (IRBP-NMP) and 6/19 (CBA-NMP) animals had at least a 70% increase in cone ERG amplitudes.\nTable 1 Average full-field ERG values at various timepoints.\nNanoparticle Naked DNA Changeb Pb\nAveragea±SEM #c Averagea±SEM #c\nPI-30\nScotopic-A CBA-NMP 134.8±13.3 19 92.9±9.4 6 41.9 µV, 45.1% 0.018\nIRBP-NMP 146.7±13.7 15 96.0±11.0 9 50.7 µV, 52.8% 0.018\nPhotopic-B CBA-NMP 148.1±11.3 19 98.7±15.2 6 49.4 µV, 50.1% 0.035\nIRBP-NMP 134.4±13.7 15 92.4±9.7 9 42.0 µV, 45.4% 0.040\nPI-60\nScotopic-A CBA-NMP 107.7±7.8 5 81.8±17.2 4 25.9 µV, 31.7% 0.061\nIRBP-NMP 148.4±8.9 10 70.5±18.8 4 77.9 µV, 110.4% 0.011\nPhotopic-B CBA-NMP 117.3±19.7 5 131.0±11.3 4 −13.7 µV, −11.5% 0.594\nIRBP-NMP 194.8±16.0 10 64.5±20.4 4 130.3 µV, 202.0% 0.0007\nPI-120\nScotopic-A CBA-NMP 123.9±12.9 5 77.1±17.3 6 46.8 µV, 60.7% 0.086\nIRBP-NMP 129.4±9.6 5 67.6±16.2 5 61.8 µV, 91.4% 0.011\nPhotopic-B CBA-NMP 120.8±14.83 5 108.0±13.48 6 12.8 µV, 11.8% 0.54\nIRBP-NMP 185.6±20.6 5 87.9±20.5 5 97.7 µV, 111.1% 0.009\na Values are mean µV±S.E.M.\nb Comparison between nanoparticle and naked DNA using 2-tailed un-paired Student's t-test as described in methods.\nc Number of animals tested. Although WT eyes can completely recover from P5 subretinal injections, we observed that ERG amplitudes from saline- and naked DNA-injected eyes in the rds+/− tended to be lower than in uninjected eyes (data not shown). These data, in combination with our earlier work on adult rds +/− mice [41], suggest that the rds +/− eye is more fragile than the normal eye and that subretinal injections per se in the mutant may cause adverse effects on visual function which would need to be overcome by any treatment. This idea is further supported by the wide variation in nanoparticle-mediated functional rescue, and highlights the need to assess rescue in every treated animal.\nIn order to determine whether functional rescue persisted at later timepoints, animals that demonstrated the hallmarks of rescue at PI-30 were selected for follow-up at PI-60 and PI-120 (Table 1 and Figure 4). Injection of CBA-NMP nanoparticles did not result in long-term functional rescue of rods (Table 1, and Figure 4A, bottom) or cones (Table 1, and Figure 4C, bottom). In striking contrast, ERG amplitudes from IRBP-NMP nanoparticle-injected eyes continued to be elevated at both PI-60 and PI-120 when compared to naked DNA-injected controls (Table 1, and Figure 4B and 4D, bottom). Cone function continued improving between PI-30 and PI-60 (p\u003c0.01) before stabilizing near WT levels at PI-120; based on two-way ANOVA, age was not an interacting factor in any case. In addition to the overall (average) improvement in cone ERG function at PI-120, in 6/10 (PI-60) and 2/5 (PI-120) cases IRBP-NMP nanoparticle injection led to photopic ERG levels that exceeded the mean value for uninjected WT animals (for example, at PI-120, treated subject 1, 245.6 µV vs. age-matched WT average 204.9±24.5 µv, N = 8). This suggests that IRBP-NMP nanoparticle-mediated NMP expression is capable of overcoming damage due to subretinal injection and can slow or rescue the functional degeneration associated with RDS haploinsufficiency.\nFigure 4 Expression of transferred NMP leads to partial functional rescue of the rds +/−phenotype.\nA subset of individual injected animals identified from PI-30 ERG analysis (Table 1) was chosen for follow-up. (A,B) Top: scotopic traces from naked DNA (gray) and nanoparticle (black) injected eyes at PI-30. Bottom: (A) Scotopic a-wave amplitudes from eyes injected with CBA-NMP nanoparticles are elevated at PI-30, but drop almost back to baseline at PI-60 and PI-120. (B) IRBP-NMP nanoparticle-injected animals retain improved rod function (as measured by scotopic a-wave) through PI-120. (C,D) Top: photopic traces from naked DNA (gray) and nanoparticle (black) injected eyes at PI-30. Bottom: (C) Cone function (as measured by photopic b-wave) does not remain substantially improved past PI-30 in eyes injected with CBA-NMP nanoparticles. (D) Photopic b-wave amplitudes in IRBP-NMP nanoparticle-injected animals are improved at PI-30 and continue improving at PI-60 (* = p\u003c0.05, PI-30 vs. PI-60) before stabilizing at the last time point examined (PI-120). Amplitudes are means±standard error (N values are in Table 1).\n\nOS ultrastructure is substantially improved by increased Rds expression\nFinally, we analyzed NMP nanoparticle-mediated structural rescue of photoreceptors in the rds +/− retina, using both light and electron microscopy, at PI-30 and PI-120, in comparison with uninjected controls. Photoreceptors in the rds +/− retina typically exhibit very short OSs with misaligned and whorl-like disc membranes. At PI-30, there was a modest increase in outer nuclear layer (ONL) thickness (Fig 5A, top), and many individual OSs exhibit improved ultrastructure (arrows, Figure 5A, bottom). By PI-120, however, virtually all photoreceptors examined showed noticeable structural improvement (Figure 5B). Consistent with the ERG results (see Figure 4), structural rescue was more pronounced in the IRBP-NMP-injected eyes compared to CBA-NMP-injected eyes at PI-120, but both exhibited OSs with orderly stacks of disc membranes.\nFigure 5 Transferred NMP leads to structural rescue of the rds +/− phenotype.\nLight micrographs (top row) and electron micrographs (bottom row, N = 3–5 animals per group) from rds +/− were examined. (A) At PI-30, moderate ultrastructural rescue is detected in the OSs of nanoparticle injected eyes (arrows). (B) By PI-120 significant ultrastructural improvement in OSs of nanoparticle injected eyes is apparent. OS discs are properly aligned and flattened and OS do not exhibit the swirl-like structures typical of the rds +/−. RPE, retinal pigment epithelium; OS, outer segment layer; IS, inner segment layer; ONL, outer nuclear layer. Scale bar, 10 µm. In order to determine the extent of these structural improvements, we undertook a series of morphometric analyses of the nanoparticle injected-eyes compared to controls. Histological images were collected from each eye at 200 µm, 400 µm, and 600 µm from the optic nerve head (both temporally and nasally) and vertical rows of ONL nuclei and OS thickness were measured. Figure S4 shows results from two representative experimental animals, with the average values (± standard deviation) obtained from uninjected control eyes shaded in gray (accompanying supplemental methods found in Text S1). At PI-30, injected animals showed little or no increase in the number of rows in the ONL (top panels), but a definite increase in OS thickness (bottom panels). At PI-120, there was no morphometric evidence of any histological benefit from the CBA-NMP nanoparticles, while IRBP-NMP nanoparticles led to a slight increase in both the number of rows in the ONL and in OS thickness. The increase in ONL rows at PI-120 is particularly relevant, since it suggests that nanoparticle-mediated increases in RDS may slow photoreceptor cell death in the rds 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