Transient RORβ Knockdown by Cell-penetrating siRNA Alleviates Retinal Degeneration Caused by Proteotoxicity

Retinitis pigmentosa (RP) is characterized by rod photoreceptor degeneration-driven apoptosis, which secondarily triggers cone photoreceptor loss and eventual blindness. Preserving rod function remains a primary therapeutic objective. Inspired by prior studies demonstrating that neural retina leucine zipper (Nrl) knockdown conferred resistance to rod mutations and induced in vivo rod-to-cone conversion with therapeutic effects in inherited retinal degeneration (IRD) models, we hypothesized that knockdown of retinoid-related orphan receptor beta (RORβ), an upstream regulator of Nrl, could yield similar or superior outcomes. To test this, we developed a cell-penetrating asymmetric siRNA (cp-asiRNA) targeting RORβ (cp-asiRORB), designed to overcome conventional siRNA limitations, including inefficient cellular delivery, off-target effects, and immune activation. Intravitreal administration of cp-asiRORB in RhoP23H mice—an autosomal dominant RP model harboring the P23H rhodopsin mutation—successfully reduced RORβ expression in rod photoreceptors. Contrary to our initial expectation of observing an in vivo reprogramming effect, RORβ knockdown did not seem to induce rod-to-cone conversion. Instead, transient RORβ suppression effectively mitigated apoptosis by enhancing proteostasis, leading to improved rod survival, reduced degeneration, and preserved visual function. Furthermore, single-cell RNA sequencing of retinas treated with cp-asiRORB in RhoP23H mice revealed significant upregulation of proteasomal subunits in RORβ-reduced rod photoreceptors. Consistent with these findings, in HEK293T cells under proteotoxic stress, RORβ knockdown reduced apoptosis, improved cell viability, and diminished aggresome formation. These results demonstrate that transient RORβ suppression alleviates RP progression by augmenting proteasomal activity to resolve proteotoxicity, offering a novel therapeutic avenue for RP patients. All animal experiments were performed in accordance with the guidelines approved by the Institutional Animal Care and Use Committee (IACUC) of Konkuk University (Approval No. KU24219). The mice were housed under a 12-hour light/dark cycle at a temperature of 22 ± 2°C and humidity of 50 ± 10%, with free access to food and water. Two- and eight-week-old C57BL/6J mice were obtained from DBL (Incheon, Korea), while RhoP23H mice and rd10 mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA) and bred under Specific Pathogen-Free (SPF) conditions at the Konkuk University Laboratory Animal Research Center. Genotyping was performed using PCR with specific primers (primer sequences in Supplementary Table S1). Experiments involving RhoP23H mice utilized heterozygous animals, while those involving rd10 mice used homozygous animals. All experimental groups were randomly assigned. Two-week-old RhoP23H and rd10 mice were carefully administered an intravitreal injection of cp-asiRORβ (1 μg/μL), followed by analysis three weeks later. The same procedure was performed on two- and eight-week-old C57BL/6J mice.