Centyrin ligands for extrahepatic delivery of siRNA

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.

Ligand-mediated delivery of RNAi-based therapeutics for the treatment of oncological diseases

“…Centyrins are small, engineered protein that are based on consensus fibronectin (FN3) domains found in human Tenascin C ( 124 ). Centyrins can be engineered to bind any target antigen with high specificity and affinity similar to that of the antibodies ( 124 ).…”

“…Centyrins are small, engineered protein that are based on consensus fibronectin (FN3) domains found in human Tenascin C ( 124 ). Centyrins can be engineered to bind any target antigen with high specificity and affinity similar to that of the antibodies ( 124 ). For example, an EGFR-binding Centyrin was conjugated to a beta-catenin ( CTNNb1 ) targeting siRNA (EGFR-Cent-CTNNb1) followed by evaluation of its activity in EGFR-expressing cancer cells both in vitro and in vivo ( 124 ).…”

“…Centyrins can be engineered to bind any target antigen with high specificity and affinity similar to that of the antibodies ( 124 ). For example, an EGFR-binding Centyrin was conjugated to a beta-catenin ( CTNNb1 ) targeting siRNA (EGFR-Cent-CTNNb1) followed by evaluation of its activity in EGFR-expressing cancer cells both in vitro and in vivo ( 124 ). Treatment of EGFR-expressing A431 cells with EGFR-Cent-CTNNb1 siRNA conjugates induced a significant downregulation of beta-catenin expression at both mRNA and protein levels.…”

Feasibility of cRGD conjugation at 5′-antisense strand of siRNA by phosphodiester linkage extension

“…15 These conjugation strategies significantly improve the stability, biological half-life, and targeting abilities of siRNA drugs and also provide potential methods for extrahepatic siRNA delivery. 16 Introducing a large steric group at the 5 0 -terminus of the antisense strand via a cleavable linkage based on a phosphodiester bond can control the function of the siRNA, since a bulky moiety blocks the siRNA from loading into a RISC. 17 Jain et al 18 designed light-sensitive modifications (caging groups) of antisense strands at the 5 0 -terminus via a phosphodiester linker to block interaction of the duplex with the cellular machinery responsible for RNAi.…”

“… 15 These conjugation strategies significantly improve the stability, biological half-life, and targeting abilities of siRNA drugs and also provide potential methods for extrahepatic siRNA delivery. 16 …”