GSK Center for Optical Molecular Imaging and University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Beckman Institute for Advanced Science and Technology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
Nucleic Acid Ther. 2022 Jun;32(3):163-176. doi: 10.1089/nat.2021.0059. Epub 2021 Nov 19.
Antisense oligonucleotides (ASOs), a novel paradigm in modern therapeutics, modulate cellular gene expression by binding to complementary messenger RNA (mRNA) sequences. While advances in ASO medicinal chemistry have greatly improved the efficiency of cellular uptake, selective uptake by specific cell types has been difficult to achieve. For more efficient and selective uptake, ASOs are often conjugated with molecules with high binding affinity for transmembrane receptors. Triantennary N-acetyl-galactosamine conjugated phosphorothioate ASOs (GalNAc-PS-ASOs) were developed to enhance targeted ASO delivery into liver through the hepatocyte-specific asialoglycoprotein receptor (ASGR). We assessed the kinetics of uptake and subsequent intracellular distribution of AlexaFluor 488 (AF488)-labeled PS-ASOs and GalNAc-PS-ASOs in J774A.1 mouse macrophages and primary mouse or rat hepatocytes using simultaneous coherent anti-Stokes Raman scattering (CARS) and two-photon fluorescence (2PF) imaging. The CARS modality captured the dynamic lipid distributions and overall morphology of the cells; two-photon fluorescence (2PF) measured the time- and dose-dependent localization of ASOs delivered by a modified treatment of suspension cells. Our results show that in macrophages, the uptake rate of PS-ASOs did not significantly differ from that of GalNAc-PS-ASOs. However, in hepatocytes, GalNAc-PS-ASOs exhibited a peripheral uptake distribution compared to a polar uptake distribution observed in macrophages. The peripheral distribution correlated with a significantly larger amount of internalized GalNAc-PS-ASOs compared to the PS-ASOs. This work demonstrates the relevance of multimodal imaging for elucidating the uptake mechanism, accumulation, and fate of different ASOs in liver cells that can be used further in complex models and liver tissues to evaluate ASO distribution and activity.
反义寡核苷酸（ASO）是现代治疗学的一种新范例，通过与互补信使 RNA（mRNA）序列结合来调节细胞基因表达。尽管 ASO 药物化学的进步极大地提高了细胞摄取效率，但特定细胞类型的选择性摄取仍难以实现。为了更有效地摄取和选择性摄取，ASO 通常与对跨膜受体具有高结合亲和力的分子结合。三触角 N-乙酰半乳糖胺缀合的硫代磷酸酯 ASO（GalNAc-PS-ASO）被开发出来，通过肝细胞特异性的去唾液酸糖蛋白受体（ASGR）增强靶向 ASO 递送到肝脏中的效率。我们使用同时相干反斯托克斯拉曼散射（CARS）和双光子荧光（2PF）成像来评估 AlexaFluor 488（AF488）标记的 PS-ASO 和 GalNAc-PS-ASO 在 J774A.1 小鼠巨噬细胞和原代小鼠或大鼠肝细胞中的摄取动力学和随后的细胞内分布。CARS 模式捕获了细胞的动态脂质分布和整体形态；双光子荧光（2PF）测量了通过悬浮细胞的改良处理递送到细胞内的 ASO 的时间和剂量依赖性定位。我们的结果表明，在巨噬细胞中，PS-ASO 的摄取速率与 GalNAc-PS-ASO 没有显著差异。然而，在肝细胞中，与在巨噬细胞中观察到的极性摄取分布相比，GalNAc-PS-ASO 表现出外周摄取分布。这种外周分布与内化的 GalNAc-PS-ASO 明显多于 PS-ASO 相关。这项工作证明了多模态成像对于阐明不同 ASO 在肝细胞中的摄取机制、积累和命运的相关性，可进一步用于复杂模型和肝组织中评估 ASO 的分布和活性。
