Transient RORß 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 (RORß), an upstream regulator of Nrl, could yield similar or superior outcomes. To test this, we developed a cell-penetrating asymmetric siRNA (cp-asiRNA) targeting RORß (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 RORß expression in rod photoreceptors. Contrary to our initial expectation of observing an in vivo reprogramming effect, RORß knockdown did not seem to induce rod-to-cone conversion. Instead, transient RORß 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 RORß-reduced rod photoreceptors. Consistent with these findings, in HEK293T cells under proteotoxic stress, RORß knockdown reduced apoptosis, improved cell viability, and diminished aggresome formation. These results demonstrate that transient RORß suppression alleviates RP progression by augmenting proteasomal activity to resolve proteotoxicity, offering a novel therapeutic avenue for RP patients. Overall design: 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-asiRORß (1 µg/µL), followed by analysis three weeks later. The same procedure was performed on two- and eight-week-old C57BL/6J mice.

色素性视网膜炎（Retinitis Pigmentosa，RP）以视杆光感受器变性驱动的细胞凋亡为特征，该过程会继发性触发视锥光感受器丢失，最终导致失明。保留视杆功能仍是当前主要的治疗目标。受既往研究启发——该研究显示，神经视网膜亮氨酸拉链（Neural Retina Leucine Zipper，Nrl）敲低可对抗视杆细胞突变，并在遗传性视网膜变性（Inherited Retinal Degeneration，IRD）模型中诱导体内视杆向视锥转化，且具有治疗效果——我们提出假设：作为Nrl上游调控因子的类视黄醇相关孤儿受体β（Retinoid-Related Orphan Receptor Beta，RORβ），其敲低可产生相似或更优的治疗效果。
为验证这一假设，我们开发了一款靶向RORβ的细胞穿透型不对称小干扰RNA（Cell-Penetrating Asymmetric Small Interfering RNA，cp-asiRNA，以下简称cp-asiRORB），旨在克服传统小干扰RNA的局限性，包括细胞递送效率低下、脱靶效应以及免疫激活。
在携带P23H视紫红质突变的常染色体显性RP模型RhoP23H小鼠中实施玻璃体内给药后，cp-asiRORB成功降低了视杆光感受器内的RORβ表达水平。与我们最初预期的体内重编程效应相反，RORβ敲低似乎并未诱导视杆向视锥转化。相反，暂时性RORβ抑制通过增强蛋白质稳态有效减轻了细胞凋亡，进而改善了视杆细胞存活、减轻了视网膜变性并保留了视觉功能。
此外，对RhoP23H小鼠经cp-asiRORB处理的视网膜进行单细胞RNA测序分析发现，在RORβ表达降低的视杆光感受器中，蛋白酶体亚基的表达显著上调。与上述发现一致，在处于蛋白毒性应激状态的HEK293T细胞中，RORβ敲低可减少细胞凋亡、提升细胞活力并减少聚集体形成。上述结果表明，暂时性RORβ抑制可通过增强蛋白酶体活性以缓解蛋白毒性，从而延缓RP的疾病进展，为RP患者提供了全新的治疗思路。
整体实验设计：所有动物实验均按照建国大学（Konkuk University）机构动物护理与使用委员会（Institutional Animal Care and Use Committee，IACUC）批准的指南进行（批准编号：KU24219）。小鼠饲养于12小时光/暗循环环境中，温度控制为22±2℃，湿度为50±10%，自由摄食饮水。2周龄和8周龄的C57BL/6J小鼠购自韩国仁川的DBL公司；RhoP23H小鼠与rd10小鼠则购自美国缅因州巴尔港的杰克逊实验室（Jackson Laboratory），并在建国大学实验动物研究中心的无特定病原体（Specific Pathogen-Free，SPF）环境中繁育。采用聚合酶链式反应（Polymerase Chain Reaction，PCR）与特异性引物进行基因分型（引物序列见补充表S1）。针对RhoP23H小鼠的实验使用杂合子动物，而针对rd10小鼠的实验则使用纯合子动物。所有实验组均采用随机分组。对2周龄的RhoP23H小鼠与rd10小鼠小心实施玻璃体内注射cp-asiRORB（浓度为1 µg/µL），并于3周后进行分析。同样的操作流程也应用于2周龄和8周龄的C57BL/6J小鼠。