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Merck

Centyrin ligands for extrahepatic delivery of siRNA.

Molecular therapy : the journal of the American Society of Gene Therapy (2021-02-19)
Donna Klein, Shalom Goldberg, Christopher S Theile, Richard Dambra, Kathleen Haskell, Elise Kuhar, Tricia Lin, Rubina Parmar, Muthiah Manoharan, Mark Richter, Meizhen Wu, Jeannine Mendrola Zarazowski, Vasant Jadhav, Martin A Maier, Laura Sepp-Lorenzino, Karyn O'Neil, Vadim Dudkin
초록

RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.

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Sigma-Aldrich
Anti-CTNNB1 antibody produced in rabbit, Prestige Antibodies® Powered by Atlas Antibodies, affinity isolated antibody, buffered aqueous glycerol solution