Research Project
7646776
DESCRIPTION (provided by applicant): Nano-delivery of siRNA and RNA-based splice-site switching oligoribonucleotides (SSO) is an important new approach with chemotherapeutic potential against cancer. Here we will focus on manufacturing RNA-bound nanoparticles (RNPs). For this purpose gold, manganese or zinc-based core nanoparticles (NPs) will be prepared and RNA attached via delivery-enhancing materials (protamine or PAMAM dendrimer). In addition to a delivery advantage, this approach will greatly enhance RNA's stability against chemical and enzymatic degradation. Stabilization of the RNA by the RNPs will be confirmed by electrophoretic and liquid chromatography analysis after exposure to accelerated chemical or enzymatic degradation assays. The RNPs will be further characterized by electron microscopy, UV, fluorescence and dynamic laser light scatter spectroscopy. Functional nuclear delivery of the RNPs will be evaluated in the Hela 705 splice-site switching assay by delivering a bound RNA oligonucleotide which overlaps the aberrant splice site, corrects splicing and switches-on Luciferase. Ultimately, as proof-of-principle to the important anti-cancer potential of this approach, biological activity of RNPs containing siRNA or SSO targeted against B-Raf-an important model cancer target will be investigated. Keywords: SSO=splice-site switching oligonucleotide, RNP=RN nanoparticle PUBLIC HEALTH RELEVANCE: This project focuses on nano-manufacturing nanoparticles of gold, manganese and zinc with RNA bound to them (RNPs). In preliminary experiments we have formed such RNPs attaching RNA via delivery enhancing materials (protamine or alternatively PAMAM dendrimer). Given these data and our previous experience with these nanomaterials in combination with DNA, here we will show the RNPs will provide the RNA resistance to chemical and enzymatic degradation and substantial delivery advantage. Finally as proof-of- principle for the chemotherapeutic potential of our approach here we will demonstrate that RNPs can deliver siRNA or splice-site switching oligomers against B-Raf an important model cancer target. Thus this application addresses a very important barrier for the success of RNA- derived chemotherapy, namely how to combine RNA with nanomaterials so as to retain structure-function.
