Loh Pei She, Patzel Volker
Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597, Singapore.
Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Block MD4, Level 5, 5 Science Drive 2, Singapore 117597, Singapore.
Pharmaceutics. 2023 Jan 21;15(2):370. doi: 10.3390/pharmaceutics15020370.
Covalently closed dumbbell-shaped DNA delivery vectors comprising the double-stranded gene(s) of interest and single-stranded hairpin loops on both ends represent a safe, stable and efficacious alternative to viral and other non-viral DNA-based vector systems. As opposed to plasmids and DNA minicircles, dumbbells can be conjugated via the loops with helper functions for targeted delivery or imaging. Here, we investigated the non-covalent linkage of tri-antennary N-acetylgalactosamine (GalNAc3) or a homodimer of a CD137/4-1BB-binding aptamer (aptCD137-2) to extended dumbbell vector loops via complementary oligonucleotides for targeted delivery into hepatocytes or nasopharyngeal cancer cells. Enlarging the dumbbell loop size from 4 to 71 nucleotides for conjugation did not impair gene expression. GalNAc3 and aptCD137-2 residues were successfully attached to the extended dumbbell loop via complementary oligonucleotides. DNA and RNA oligonucleotide-based dumbbell-GalNAc3 conjugates were taken up from the cell culture medium by hepatoblastoma-derived human tissue culture cells (HepG2) with comparable efficiency. RNA oligonucleotide-linked conjugates triggered slightly higher levels of gene expression, presumably due to the RNaseH-mediated linker cleavage, the release of the dumbbell from the GalNAc3 residue and more efficient nuclear targeting of the unconjugated dumbbell DNA. The RNaseH-triggered RNA linker cleavage was confirmed in vitro. Finally, we featured dumbbell vectors expressing liver cancer cell-specific RNA -splicing-based suicide RNAs with GalNAc3 residues. Dumbbells conjugated with two GalNAc3 residues triggered significant levels of cell death when added to the cell culture medium. Dumbbell vector conjugates can be explored for targeted delivery and gene therapeutic applications.
共价闭合的哑铃状DNA递送载体，其包含感兴趣的双链基因以及两端的单链发夹环，是病毒和其他基于DNA的非病毒载体系统的一种安全、稳定且有效的替代方案。与质粒和DNA微环不同，哑铃状载体可以通过环与辅助功能基团进行偶联，用于靶向递送或成像。在此，我们研究了通过互补寡核苷酸将三触角N-乙酰半乳糖胺（GalNAc3）或CD137/4-1BB结合适体的同二聚体（适体CD137-2）非共价连接到扩展的哑铃状载体环上，以靶向递送至肝细胞或鼻咽癌细胞。将用于偶联的哑铃状环大小从4个核苷酸扩大到71个核苷酸并不影响基因表达。GalNAc3和适体CD137-2残基通过互补寡核苷酸成功连接到扩展的哑铃状环上。基于DNA和RNA寡核苷酸的哑铃-GalNAc3偶联物被源自肝母细胞瘤的人组织培养细胞（HepG2）从细胞培养基中摄取，摄取效率相当。RNA寡核苷酸连接的偶联物引发的基因表达水平略高，可能是由于RNaseH介导的连接子切割、哑铃状载体从GalNAc3残基上释放以及未偶联的哑铃状DNA更有效地靶向细胞核。在体外证实了RNaseH触发的RNA连接子切割。最后，我们展示了带有GalNAc3残基、表达基于肝癌细胞特异性RNA剪接的自杀性RNA的哑铃状载体。当添加到细胞培养基中时，与两个GalNAc3残基偶联的哑铃状载体引发了显著水平的细胞死亡。哑铃状载体偶联物可用于靶向递送和基因治疗应用的探索。
