In vivo efficacy of NRL knockdown with cell-penetrating siRNA in retinal degeneration

In retinal degenerative diseases, including retinitis pigmentosa (RP) and age-related macular degeneration (AMD), loss of photoreceptors at the macula leads to blindness. Among photoreceptors, rod cells are more vulnerable to degenerative stress than cone cells. Following disruption of neural retina leucine zipper (NRL) expression induced by knockout or the CRISPR/Cas9 system, rod cells have been shown to gain increased expression of several cone cell markers and demonstrate improved survival under degenerative pressure, consequently preventing secondary cone cell degeneration. Here, we report an mRNA silencing approach in which a cell-penetrating asymmetric small interfering RNA (cp-asiRNA) targeting NRL (cp-asiNRL) in postmitotic photoreceptors was used to suppress the expression of the NRL, which determines the fate of developing photoreceptors to rod cells. In mouse models of RP and neovascular AMD, intravitreal delivery of cp-asiNRL substantially increased the expression of cone-specific genes among the photoreceptor population and improved rod cell survival. Furthermore, in retinal organoids, cp-asiNRL also increased the production of photoreceptors expressing cone-specific markers. Our data suggest that cp-asiNRL-mediated NRL suppression in rod cells may be a promising strategy for treating retinal degenerative diseases. Overall design: Rho(P23H/+) mice (male and female, one to four months old) were maintained on a C57BL/6J background (Jackson Laboratory). Rho(P23H/+) transgenic mice were purchased from the Jackson Laboratory (stock no. 000664). The P23H Rho mutation in Rho(P23H/+) transgenic mice leads to the misfolding of rhodopsin in rod photoreceptors. This activates the unfolded protein response, ultimately leading to the death of both rod and cone cells approximately 2 weeks after birth. Therefore, the mice were treated by intravitreal injection of cp-asiNRL (0.4 µg/µl) or PBS as a control on day 14 after birth, and the eyes were subsequently analyzed on day 35.