UC San Diego

The discovery of RNAi and the subsequent demonstration that synthetic short interfering RNA (siRNA) could silence all mRNA expression in a sequence dependent manner offered tremendous potential as a therapeutic to treat all genetic disease. However, siRNA is both too large (>14,000 Da) and too charged (40+ phosphates) to passively diffuse across the cell membrane, requiring a targeting domain to deliver the siRNA therapeutic. Conjugation of siRNA to tris-N-acetylgalactosamine (GalNAc) targeting liver asialoglycoprotein receptor (ASGPR) revolutionized RNAi therapeutics and the problem of hepatic delivery can now be considered solved. However, siRNA therapeutics have not seen the same success in extra-hepatic tissues as the biology of these tissues has proven more difficult for siRNA targeting and delivery.

To address the issue of siRNA delivery, our laboratory developed short interfering ribonucleic neutrals (siRNN) whose charged phosphodiester backbone has been neutralized by bioreversible phosphotriester groups. The first generation siRNN utilized a t-butyl-s-acyl-2-thioethyl (tBu-SATE) phosphotriester group that allowed enhanced in vivo GalNAc-siRNA delivery. To adapt this technology for extra-hepatic delivery, we modified the tBu-SATE phosphotriester to allow site-specific conjugation through copper catalyzed Click chemistry. Conjugation of mannose to siRNNs effectively delivered siRNN to CD206+ macrophages and elicited a robust RNAi response in a model of tumor-associated macrophages (TAMs). Unfortunately, additional ligand/receptor pairs like mannose/CD206 and GalNAc/ASGPR that are tissue specific, highly expressed, and rapidly internalize are extremely limited. An alternative to small ligands is the use of antibodies. Traditional antibody targeted therapies often rely on conjugations that result in a poorly defined, heterogeneous mixtures of antibody-drug conjugates. To conjugate siRNNs to antibodies in a quantitative manner, we developed a site-specific enzymatic conjugation strategy. Unfortunately, the resulting antibody RNA conjugates (ARC) failed to deliver the siRNN into the cytoplasm, likely due to endosomal entrapment. To avoid entrapment, endosomal escape domains (EED) were incorporated into the ARC through a hydrazone conjugation phosphotriester to form an ARC-EED. Taken together, this work describes the development of novel, well defined, site-specific, multifunctional, and multivalent siRNN conjugates capable of extra-hepatic targeting and endosomal escape.