A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides

Lei Zhang; Yuyan Wang; Peiru Chen; Dali Wang; Tingyu Sun; Zheyu Zhang; Ruimeng Wang; Xi Kang; Yang Fang; Hao Lu; Jiansong Cai; Mengqi Ren; Sijia S. Dong; Ke Zhang

doi:10.1039/D2CB00149G

A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides†

Lei Zhang,‡^ab Yuyan Wang,‡^b Peiru Chen,^b Dali Wang,^b Tingyu Sun,^b Zheyu Zhang,^b Ruimeng Wang,^b Xi Kang,

^b Yang Fang,^b Hao Lu,^b Jiansong Cai,^b Mengqi Ren,^b Sijia S. Dong

*^b and Ke Zhang

*^bc

Author affiliations

* Corresponding authors

^a Chemicobiology and Functional Materials Institute, School of Chemistry and Chemical Engineering, Nanjing University of Science and Technology, Nanjing 210094, P. R. China

^b Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115, USA
E-mail: s.dong@northeastern.edu, k.zhang@northeastern.edu

^c Departments of Chemical Engineering and Bioengineering, Northeastern University, Boston, Massachusetts 02115, USA

Abstract

We have developed a non-cationic transfection vector in the form of bottlebrush polymer-antisense oligonucleotide (ASO) conjugates. Termed pacDNA (polymer-assisted compaction of DNA), these agents show improved biopharmaceutical characteristics and antisense potency in vivo while suppressing non-antisense side effects. Nonetheless, there still is a lack of the mechanistic understanding of the cellular uptake, subcellular trafficking, and gene knockdown with pacDNA. Here, we show that the pacDNA enters human non-small cell lung cancer cells (NCI-H358) predominantly by scavenger receptor-mediated endocytosis and macropinocytosis and trafficks via the endolysosomal pathway within the cell. The pacDNA significantly reduces a target gene expression (KRAS) in the protein level but not in the mRNA level, despite that the transfection of certain free ASOs causes ribonuclease H1 (RNase H)-dependent degradation of KRAS mRNA. In addition, the antisense activity of pacDNA is independent of ASO chemical modification, suggesting that the pacDNA always functions as a steric blocker.

RSC Chemical Biology

A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides†

Abstract

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A mechanistic study on the cellular uptake, intracellular trafficking, and antisense gene regulation of bottlebrush polymer-conjugated oligonucleotides

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