CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, Telangana 500007, India.
CSIR-Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, Telangana 500007, India.
J Control Release. 2016 Apr 28;228:120-131. doi: 10.1016/j.jconrel.2016.03.007. Epub 2016 Mar 4.
RNA interference represents a novel therapeutic approach to modulate several neurodegenerative disease-related genes. However, exogenous delivery of siRNA restricts their transport into different tissues and specifically into the brain mainly due to its large size and the presence of the blood-brain barrier (BBB). To overcome these challenges, we developed here a strategy wherein a peptide known to target specific gangliosides was fused to a double-stranded RNA binding protein to deliver siRNA to the brain parenchyma. The designed fusion protein designated as TARBP-BTP consists of a double-stranded RNA-binding domain (dsRBD) of human Trans Activation response element (TAR) RNA Binding Protein (TARBP2) fused to a brain targeting peptide that binds to monosialoganglioside GM1. Conformation-specific binding of TARBP2 domain to siRNA led to the formation of homogenous serum-stable complex with targeting potential. Further, uptake of the complex in Neuro-2a, IMR32 and HepG2 cells analyzed by confocal microscopy and fluorescence activated cell sorting, revealed selective requirement of GM1 for entry. Remarkably, systemic delivery of the fluorescently labeled complex (TARBP-BTP:siRNA) in ΑβPP-PS1 mouse model of Alzheimer's disease (AD) led to distinctive localization in the cerebral hemisphere. Further, the delivery of siRNA mediated by TARBP-BTP led to significant knockdown of BACE1 in the brain, in both ΑβPP-PS1 mice and wild type C57BL/6. The study establishes the growing importance of fusion proteins in delivering therapeutic siRNA to brain tissues.
RNA 干扰代表了一种调节多种神经退行性疾病相关基因的新型治疗方法。然而，由于 siRNA 体积较大且存在血脑屏障 (BBB)，其外源性传递限制了其在不同组织中的运输，特别是进入大脑。为了克服这些挑战，我们开发了一种策略，即将靶向特定神经节苷脂的肽与双链 RNA 结合蛋白融合，将 siRNA 递送至脑实质。设计的融合蛋白命名为 TARBP-BTP，由人类反式激活反应元件 (TAR) RNA 结合蛋白 (TARBP2) 的双链 RNA 结合域 (dsRBD) 与靶向结合单唾液酸神经节苷脂 GM1 的脑靶向肽融合而成。TARBP2 结构域与 siRNA 的特异性结合导致具有靶向潜力的同质性血清稳定复合物的形成。进一步通过共聚焦显微镜和荧光激活细胞分选分析，发现神经母细胞瘤 2a 细胞、IMR32 细胞和 HepG2 细胞对该复合物的摄取，揭示了 GM1 对进入的选择性要求。值得注意的是，在阿尔茨海默病（AD）的 ΑβPP-PS1 小鼠模型中全身性递送荧光标记的复合物（TARBP-BTP：siRNA）导致其在大脑半脑中有独特的定位。此外，TARBP-BTP 介导的 siRNA 传递导致 ΑβPP-PS1 小鼠和野生型 C57BL/6 小鼠大脑中的 BACE1 显著敲低。该研究确立了融合蛋白在将治疗性 siRNA 递送至脑组织方面的重要性日益增加。
