This Small Business Innovation Research (SBIR) Phase I project will demonstrate targeted delivery of small interfering RNA (siRNA) to HER2 overexpressing tumor cells using a unique Shiga-like toxin 1A (SLT1A) scaffold. Molecular Templates has developed a SLT1A library of variants containing a unique 12 amino acid insert, allowing each SLT1A variant unique binding characteristics. SLT1A toxin intrinsically facilitates endocytosis and intracellular routing into the cytosol. This proposed work will demonstrate targeted intracellular delivery of siRNA to HER2 overexpressing breast cancer cells using proprietary HER2-specific SLT1A variants. This work will enable the development of a platform technology that can selectively bind to diseased cells, force cellular uptake of the siRNA-toxin construct, and ensure proper intracellular routing of the siRNA into the cytosol for maximal therapeutic benefit.
The broader/commercial impacts of this research will be to provide a novel drug delivery technology that could lead to new therapeutics for a variety of human diseases. As siRNA therapy is being pursued for various genetic, viral, and cancerous diseases, technologies to selectively deliver siRNA to diseased tissue are needed. Current innovations in siRNA delivery are focused on selective binding to diseased cells using nanoparticles conjugated to antibodies, aptamers, or peptides. However, these ligand-targeted approaches often poorly facilitate endocytosis, endosomal escape, and/or cytosolic delivery of siRNA. Thus, the intrinsic characteristics of our SLT1A variant library to accomplish proper intracellular delivery of siRNA along with selective binding to diseased cells of interest would help overcome a significant problem in delivery of siRNA therapeutics.
Molecular Templates has developed novel Engineered Toxin Bodies (ETBs) derived from the ribosome-inhibiting protein Shiga-like toxin 1. These ETBs contain the catalytically active SLT-1A subunit (SLT1A) which inhibits protein synthesis through depurination of adenine residue 4324 of 28S rRNA and induces apoptotic cell death. Further, ETBs contain an engineered binding domain which selectively binds a disease-associated antigen in order to specifically deliver the cytotoxic SLT1A payload only to diseased cells. For this Phase I work, Molecular Templates developed an HER2-specific ETB capable of selectively killing HER2-overexpressing (HER2+) cancer cells. We aimed to demonstrated delivery of short interfering RNA (siRNA) to HER2+ breast cancer cells using this ETB. Through genetic engineering we were able to attenuate the ETB catalytic activity and provided for site-specific conjugation of a siRNA molecule to the ETB. Through optimization of conjugation and purification methods we achieved purified ETB-siRNA conjugate species. This conjugate was applied to HER2+ breast cancer cells and provided a mild inhibition of the siRNA target mRNA expression, but no inhibition was observed by protein western blot. We also observed by immunofluorescence that the ETB was able to efficiently deliver a siRNA payload to HER2+ cells which was predominantly localized to endosomes. These results indicate that the ETB was able to deliver siRNA molecules selectively to HER2+ cells, but likely due to poor endosomal escape unable to efficiently suppress target gene expression.
