Abstract
Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide a possible solution. However, initial reports of antibody–siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody–siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.
Highlights
A groundbreaking discovery in 1998, RNA interference (RNAi) describes the fundamental process in eukaryotes in which double-stranded RNAs induce the cleavage of mRNAs with complementary sequences [1]
antibody–Small interfering RNA (siRNA) conjugates (ARCs) were generated in two primary steps: the amine-tagged siRNA was reacted with a NHS-linker to form a thiol-reactive siRNA-linker adduct, and this adduct was reacted with thiol groups on the THIOMAB to covalently link the siRNA via a thio-ester bond (Figure 1a)
We focused on analysing this negative result using anti-Her2 ARCs because we possessed a deep arsenal of reagents related to Her2, and anti-Her2 delivery of siRNAs had been previously reported [9,11,25]
Summary
A groundbreaking discovery in 1998, RNA interference (RNAi) describes the fundamental process in eukaryotes in which double-stranded (ds) RNAs induce the cleavage of mRNAs with complementary sequences [1]. The main hurdle to realizing the therapeutic promise of RNAi is the safe and effective systemic delivery of siRNA. Clinical trials of siRNA-based drugs employed local delivery, such as direct injection into the vitreous humor of the eye or systemic administration of lipid-based vehicles that primarily deliver to the liver [2]. Subsequent clinical trials have employed systemic methods to deliver siRNAs against cancer targets, and these trials stand as major milestones in the field. The first such trial relied on nanoparticle carriers and reported silencing in non-liver tumors, and a more recent trial, using lipid nanoparticles, reported regression of liver metastases in endometrial cancer patients
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