Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2,, DK-2100, Copenhagen Ø, Denmark.
Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences,, University of Copenhagen, Jagtvej 162, DK-2100, Copenhagen Ø, Denmark.
Pharm Res. 2019 Jan 9;36(3):37. doi: 10.1007/s11095-018-2566-3.
Antisense oligonucleotides (ASOs) are promising therapeutics for specific modulation of cellular RNA function. However, ASO efficacy is compromised by inefficient intracellular delivery. Lipid-polymer hybrid nanoparticles (LPNs) are attractive mediators of intracellular ASO delivery due to favorable colloidal stability and sustained release properties.
LPNs composed of cationic lipidoid 5 (L) and poly(DL-lactic-co-glycolic acid) were studied for delivery of an ASO mediating splice correction of a luciferase gene transcript (Luc-ASO). Specific purposes were: (i) to increase the mechanistic understanding of factors determining the loading of ASO in LPNs, and (ii) to optimise the LPNs and customise them for Luc-ASO delivery in HeLa pLuc/705 cells containing an aberrant luciferase gene by using a quality-by-design approach. Critical formulation variables were linked to critical quality attributes (CQAs) using risk assessment and design of experiments, followed by delineation of an optimal operating space (OOS).
A series of CQAs were identified based on the quality target product profile. The L content and L:Luc-ASO ratio (w/w) were determined as critical formulation variables, which were optimised systematically. The optimised Luc-ASO-loaded LPNs, defined from the OOS, displayed high loading and mediated splice correction at well-tolerated, lower doses as compared to those required for reference L-based lipoplexes, L-modified stable nucleic acid lipid nanoparticles or LPNs modified with dioleoyltrimethylammonium propane (conventional cationic lipid).
The optimal Luc-ASO-loaded LPNs represent a robust formulation that mediates efficient intracellular delivery of Luc-ASO. This opens new avenues for further development of LPNs as a broadly applicable technology platform for delivering nucleic acid cargos intracellularly.
反义寡核苷酸（ASO）是一种有前途的治疗方法，可特异性调节细胞 RNA 功能。然而，由于细胞内递送效率低下，ASO 的疗效受到影响。由于具有良好的胶体稳定性和持续释放特性，脂质-聚合物杂化纳米颗粒（LPN）是细胞内 ASO 递送的有吸引力的介质。
研究了由阳离子脂质体 5（L）和聚（DL-乳酸-共-乙醇酸）组成的 LPN 对介导荧光素酶基因转录本（Luc-ASO）剪接校正的 ASO 的递送。具体目的是：（i）增加决定 ASO 在 LPN 中加载的因素的机制理解，（ii）通过质量源于设计方法，优化 LPN 并针对含有异常荧光素酶基因的 HeLa pLuc/705 细胞中的 Luc-ASO 递送对其进行定制。使用风险评估和实验设计将关键制剂变量与关键质量属性（CQA）相关联，然后划定最佳操作空间（OOS）。
根据质量目标产品概况确定了一系列 CQA。L 含量和 L:Luc-ASO 比（w/w）被确定为关键制剂变量，并进行了系统优化。从 OOS 中定义的优化的 Luc-ASO 负载的 LPN 显示出高负载，并介导剪接校正，所需剂量低于参考 L 为基础的脂质体、L 修饰的稳定核酸脂质纳米颗粒或用二油酰基三甲基丙胺（常规阳离子脂质）修饰的 LPN。
最佳的 Luc-ASO 负载的 LPN 代表一种稳健的制剂，可介导 Luc-ASO 的有效细胞内递送。这为进一步开发 LPN 作为一种广泛适用于细胞内递送核酸载体的通用技术平台开辟了新途径。
