Rds nanoparticles drive high and persistent transgene expression RDS 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). As 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. Figure 1 Injection of NMP nanoparticles into P5 rds +/− animals increases Rds mRNA levels. cDNA 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<0.001, **p<0.05. This increase persists through the last time point examined (PI-120).