Centyrin ligands for extrahepatic delivery of siRNA.

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.

Selection of high-affinity Centyrin FN3 domains from a simple library diversified at a combination of strand and loop positions.

Alternative scaffold molecules represent a class of proteins important to the study of protein design and mechanisms of protein-protein interactions, as well as for the development of therapeutic proteins. Here, we describe the generation of a library built upon the framework of a consensus FN3 domain sequence resulting in binding proteins we call Centyrins. This new library employs diversified positions within the C-strand, CD-loop, F-strand and FG-loop of the FN3 domain. CIS display was used to select high-affinity Centyrin variants against three targets; c-MET, murine IL-17A and rat TNFα and scanning mutagenesis studies were used to define the positions of the library most important for target binding. Contributions from both the strand and loop positions were noted, although the pattern was different for each molecule. In addition, an affinity maturation scheme is described that resulted in a significant improvement in the affinity of one selected Centyrin variant. Together, this work provides important data contributing to our understanding of potential FN3 binding interfaces and a new tool for generating high-affinity scaffold molecules.