Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides

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• 作者：Kariem Ezzat ; Yoshitsugu Aoki ; Taeyoung Koo ; Graham McClorey ; Leif Benner ; Anna Coenen-Stass ; Liz O鈥橠onovan ; Taavi Lehto ; Antonio Garcia-Guerra ; Joel Nordin ; Amer F. Saleh ; Mark Behlke ; John Morris ; Aurelie Goyenvalle ; Branislav Dugovic ; Christian Leumann ; Siamon Gordon ; Michael J. Gait ; Samir El鈭扐ndaloussi ; Matthew JA Wood
• 刊名：Nano Letters
• 出版年：2015
• 出版时间：July 8, 2015
• 年：2015
• 卷：15
• 期：7
• 页码：4364-4373
• 全文大小：444K
• ISSN：1530-6992

文摘

Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2鈥睴methyl phosphorothioate (2鈥睴Me), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2鈥睴Me as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2鈥睴Me. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles.