Fig 2: Efficient reduction of PTEN after intravitreal injection in adult rats(A) PTEN levels after the intravitreal injection of 6, 30, or 60 µg of BA-434-007 or 60 µg of NTC or BA-434-001. Samples from individual animals (n = 4/group) were run on Western blots and PTEN normalized to GAPDH. (p < 0.05 Dunnett’s multiple comparisons test).(B) Low magnification view of an optic nerve crushed 4 mm from the optic nerve head and treated with BA-434-007. B1 and B2 show higher magnification view of the boxed regions. Bar = 100 µM.(C) For comparison, an animal injected with NTC is shown at the same magnification as B1.(D) The number of regenerating axons counted in bins corresponding to distances of 100–200 µm, 200–500 µm, 500–1000 µm, and 1000–2000 µm past the crush. (Significance was determined by Sidak multiple comparison p < 0.05).(E) The longest regenerating axon was measured in three to five sections per animal. Treatment with BA-434-007 significantly increased regenerating axon length relative to NTC. (two-way ANOVA drug dose: F(1, 172) = 41.95, p < 0.0001; bin distance: F(3, 172) = 25.72, p < 0.0001; Sidak multiple comparison p < 0.05).

Fig 3: BA-434 inhibits PTEN protein expression in primary neurons(A) Uptake of sdRNA by rat primary neurons treated with BA-434-Cy3 and visualized at 1 h, 1 day, and 3 days. Left panel: immunostaining with ß3 tubulin to show all neurites. Right panel: the same field showing Cy3-positive cells with the boxed region shown at higher magnification in the panel on the far right. Red arrowheads point to double-labeled cell bodies, yellow arrowheads highlight the neurites. Scale bar = 100 µm.(B) PTEN mRNA expression 1 day (n = 2), 3 days (n = 3) and 4 days (n = 4) after the administration of BA-434 (1 µM) to rat cortical neurons compared to the NTC control (F(3, 10) = 75.68, p < 0.0001; * indicate p’s < 0.05 by Sidak multiple comparisons test). Shown are means of triplicate samples ±SEM from independent experiments.(C) Hippocampal neurons, plated in custom microfluidic chambers where axons are isolated from cell soma, show positive Cy3 fluorescence in the cell soma (arrowheads), 24 h after the application of BA434-Cy3 to the axon compartment. Left panel shows staining of all processes with ß3 tubulin.(D) The top panel shows the treatment protocol to investigate PTEN expression at 1 day, 4 and 11 days after washing out BA-434 treatment. The middle panel shows representative dose-response Western blots at the three time points. The bottom panel shows the quantitation of the dose-response at 1 day, 4 days, and 11 days after washout. Plotted are means +SEM of three experiments. two-way ANOVA time: F(2, 30) = 5.46 p = 0.0095; dose: F(4, 30) = 27.88 p < 0.0001; * indicate p < 0.05 by Dunnett’s multiple comparison test to NTC within each time.

Fig 4: PTEN protein expression and RGC regeneration tested before the optimization of the sdRNA(A) Staining for PTEN expression in retinal whole-mounts 4 days after the injection of BA-434 shows a clear reduction in PTEN levels on the nasal side of the optic disc, correlating with the injection site. Images at slightly higher magnification taken at the retinal periphery show the reduction of PTEN expression in RGC soma on the nasal side (the injected side) versus the temporal side.(B) CTB labeled regenerating axons 14 days after either NTC treatment (top) or BA-434 treatment (bottom). CTB accumulates immediately proximal to the injury site. Bar = 100 µm.

Fig 5: PTEN protein expression and RGC regeneration tested before the optimization of the sdRNA(A) Schematic representation of the injury and delivery paradigm to study RGC axon regeneration after adult rat optic nerve injury.(B) Western blot in pooled retinal extracts shows a reduction in PTEN protein out to 14 days after the intravitreal injection of 60 µg of sdRNA per eye. GAPDH was used as a control for loading.(C) PTEN and neurofilament staining of a central radial section of a control adult rat retina containing the optic nerve head demonstrates PTEN expression in both RGCs and in photoreceptors.(D) PTEN and neurofilament staining of retinal whole-mounts. PTEN is clearly detected in both RGC soma and axons close to the optic disc. Axonal PTEN staining is not evident at the periphery of the retina while RGC soma staining is still apparent.