Nanoparticle-driven Rds expression restores retinal function
The 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.
Table 1  Average full-field ERG values at various timepoints.
Nanoparticle  Naked DNA  Changeb  Pb
Averagea±SEM  #c  Averagea±SEM  #c
PI-30
Scotopic-A   CBA-NMP  134.8±13.3  19  92.9±9.4  6  41.9 µV, 45.1%  0.018
IRBP-NMP  146.7±13.7  15  96.0±11.0  9  50.7 µV, 52.8%  0.018
Photopic-B   CBA-NMP  148.1±11.3  19  98.7±15.2  6  49.4 µV, 50.1%  0.035
IRBP-NMP  134.4±13.7  15  92.4±9.7  9  42.0 µV, 45.4%  0.040
PI-60
Scotopic-A   CBA-NMP  107.7±7.8  5  81.8±17.2  4  25.9 µV, 31.7%  0.061
IRBP-NMP  148.4±8.9  10  70.5±18.8  4  77.9 µV, 110.4%  0.011
Photopic-B   CBA-NMP  117.3±19.7  5  131.0±11.3  4  −13.7 µV, −11.5%  0.594
IRBP-NMP  194.8±16.0  10  64.5±20.4  4  130.3 µV, 202.0%  0.0007
PI-120
Scotopic-A   CBA-NMP  123.9±12.9  5  77.1±17.3  6  46.8 µV, 60.7%  0.086
IRBP-NMP  129.4±9.6  5  67.6±16.2  5  61.8 µV, 91.4%  0.011
Photopic-B   CBA-NMP  120.8±14.83  5  108.0±13.48  6  12.8 µV, 11.8%  0.54
IRBP-NMP  185.6±20.6  5  87.9±20.5  5  97.7 µV, 111.1%  0.009
a  Values are mean µV±S.E.M.
b  Comparison between nanoparticle and naked DNA using 2-tailed un-paired Student's t-test as described in methods.
c  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.
In 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<0.01) 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.
Figure 4  Expression of transferred NMP leads to partial functional rescue of the rds +/−phenotype.
A 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<0.05, 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).