Centyrin ligands for extrahepatic delivery of siRNA
Donna Klein1, Shalom Goldberg1, Christopher S Theile2
1Janssen Pharmaceuticals, Spring House, PA, USA.
Abstract:
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.
Insights
New Centyrin-siRNA conjugates offer targeted gene silencing for cancer therapy. This approach overcomes delivery challenges, enabling potent knockdown of oncogenes and suppressed tumor growth, paving the way for novel anti-cancer drugs.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- RNA interference (RNAi) holds promise for treating diseases by silencing target genes.
- Development of RNAi therapeutics is hindered by inefficient delivery and intracellular barriers.
- Targeted delivery and overcoming intracellular barriers are crucial for effective RNAi cancer therapy.
Purpose of the Study:
- To develop a novel drug delivery system combining Centyrins and small interfering RNAs (siRNAs) for targeted gene silencing in cancer cells.
- To evaluate the efficacy of Centyrin-siRNA conjugates in achieving specific gene knockdown and suppressing tumor growth.
- To demonstrate the generalizability and potential of this approach for developing new anti-cancer drugs.
Main Methods:
- Site-specific conjugation of Centyrins (targeting plasma membrane receptors) to chemically modified siRNAs.
- In vitro cell culture and in vivo mouse models (tumor xenografts) were used to assess delivery and gene silencing.
- Testing of Centyrin-siRNA conjugates targeting various receptors (EGFR, BCMA, PSMA, EpCAM) and genes (CTNNb1, CD68, KLKb1, SSB1).
- Evaluation of dual-target silencing using single Centyrins conjugated to two different siRNAs.
- Assessment of tumor cell growth suppression by EpCAM Centyrin CTNNb1 siRNA conjugates in colorectal cancer models.
Main Results:
- An EGFR Centyrin-CTNNb1 siRNA conjugate achieved potent and specific target gene knockdown in EGFR-positive tumor cells in vitro and in vivo.
- The Centyrin-siRNA conjugate approach demonstrated generalizability across multiple receptor-gene targets.
- Simultaneous silencing of two different genes was achieved using a single Centyrin fused to two siRNAs.
- An EpCAM Centyrin-CTNNb1 siRNA conjugate suppressed tumor cell growth in a relevant colorectal cancer model.
Conclusions:
- Centyrin-siRNA conjugates represent a promising platform for targeted delivery of RNAi therapeutics to cancer cells.
- This approach effectively overcomes key barriers to RNAi drug development, including delivery and intracellular barriers.
- Centyrin-siRNA conjugates have the potential to be developed into a new class of potent anti-cancer drugs for selective oncogene silencing.
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