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bioRxiv · 10.1101/2022.06.15.496353

A Mechanistic Study on the Cellular Uptake, Intracellular Trafficking, and Antisense Gene Regulation of Bottlebrush Polymer-Conjugated Oligonucleotides

Zhang, L.·Wang, Y.·Chen, P.·Wang, D.·Zhang, Z.·Ruimeng, W.·Kang, X.·Fang, Y.·Lu, H.·Cai, J.·Ren, M.·Dong, S.·Zhang, K.
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Abstract

We have developed a non-cationic transfection vector in the form of a bottlebrush polymer-antisense oligonucleotide (ASO) conjugate. 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 lacks a mechanistic understanding regarding 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 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 functions as a steric blocker. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/496353v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@56e08eorg.highwire.dtl.DTLVardef@1b09510org.highwire.dtl.DTLVardef@a785corg.highwire.dtl.DTLVardef@1641874_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Zhang, L., Wang, Y., Chen, P., Wang, D., Zhang, Z., Ruimeng, W., Kang, X., Fang, Y., Lu, H., Cai, J., Ren, M., Dong, S., Zhang, K.. 2022-06-17. A Mechanistic Study on the Cellular Uptake, Intracellular Trafficking, and Antisense Gene Regulation of Bottlebrush Polymer-Conjugated Oligonucleotides. https://doi.org/10.1101/2022.06.15.496353

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