Interdisciplinary Nanoscience Center (iNANO), Department of Molecular Biology and Genetics, Aarhus University, 8000 Aarhus C, Denmark.
Department of Biomedicine, Aarhus University, 8000 Aarhus C, Denmark.
J Control Release. 2016 Jun 28;232:143-51. doi: 10.1016/j.jconrel.2016.04.013. Epub 2016 Apr 12.
Major challenges for the clinical translation of small interfering RNA (siRNA) include overcoming the poor plasma half-life, site-specific delivery and modulation of gene silencing. In this work, we exploit the intrinsic transport properties of human serum albumin to tune the blood circulatory half-life, hepatic accumulation and gene silencing; based on the number of siRNA cholesteryl modifications. We demonstrate by a gel shift assay a strong and specific affinity of recombinant human serum albumin (rHSA) towards cholesteryl-modified siRNA (Kd>1×10(-7)M) dependent on number of modifications. The rHSA/siRNA complex exhibited reduced nuclease degradation and reduced induction of TNF-α production by human peripheral blood mononuclear cells. The increased solubility of heavily cholesteryl modified siRNA in the presence of rHSA facilitated duplex annealing and consequent interaction that allowed in vivo studies using multiple cholesteryl modifications. A structural-activity-based screen of in vitro EGFP-silencing was used to select optimal siRNA designs containing cholesteryl modifications within the sense strand that were used for in vivo studies. We demonstrate plasma half-life extension in NMRI mice from t1/2 12min (naked) to t1/2 45min (single cholesteryl) and t1/2 71min (double cholesteryl) using fluorescent live bioimaging. The biodistribution showed increased accumulation in the liver for the double cholesteryl modified siRNA that correlated with an increase in hepatic Factor VII gene silencing of 28% (rHSA/siRNA) compared to 4% (naked siRNA) 6days post-injection. This work presents a novel albumin-mediated cholesteryl design-based strategy for tuning pharmacokinetics and systemic gene silencing.
小干扰 RNA（siRNA）的临床转化面临的主要挑战包括克服其血浆半衰期短、靶向递送和基因沉默的调控等问题。在这项工作中，我们利用人血清白蛋白的固有转运特性来调节其血液循环半衰期、肝积累和基因沉默，其依据是 siRNA 胆固醇修饰的数量。通过凝胶迁移实验，我们证明了重组人血清白蛋白（rHSA）与人源胆固醇修饰 siRNA（Kd>1×10(-7)M）具有很强的特异性亲和力，其亲和力取决于修饰的数量。rHSA/siRNA 复合物表现出降低的核酸酶降解和减少的人外周血单核细胞产生 TNF-α的诱导。在 rHSA 存在下，大量胆固醇修饰的 siRNA 溶解度增加，促进了双链退火和随后的相互作用，从而可以使用多种胆固醇修饰进行体内研究。基于结构活性的 EGFP 沉默筛选，用于选择含有胆固醇修饰的最佳 siRNA 设计，这些设计的胆固醇修饰位于 sense 链内，用于体内研究。我们在 NMRI 小鼠中证明了血浆半衰期的延长，从无修饰的 12min（t1/2）延长至单修饰的 45min（t1/2）和双修饰的 71min（t1/2），使用荧光活体生物成像。生物分布显示，双胆固醇修饰的 siRNA 在肝脏中的积累增加，与肝因子 VII 基因沉默增加 28%（rHSA/siRNA）相比，6 天注射后裸 siRNA 增加 4%（rHSA/siRNA）。这项工作提出了一种基于人血清白蛋白介导的胆固醇设计的新型策略，用于调节药代动力学和全身基因沉默。
