title: Periodic-shRNA molecules are capable of gene silencing, cytotoxicity and innate immune activation in cancer cells.
abstract: Large dsRNA molecules can cause potent cytotoxic and immunostimulatory effects through the activation of pattern recognition receptors; however, synthetic versions of these molecules are mostly limited to simple sequences like poly-I:C and poly-A:U. Here we show that large RNA molecules generated by rolling circle transcription fold into periodic-shRNA (p-shRNA) structures and cause potent cytotoxicity and gene silencing when delivered to cancer cells. We determined structural requirements for the dumbbell templates used to synthesize p-shRNA, and showed that these molecules likely adopt a co-transcriptionally folded structure. The cytotoxicity of p-shRNA was robustly observed across four different cancer cell lines using two different delivery systems. Despite having a considerably different folded structure than conventional dsRNA, the cytotoxicity of p-shRNA was either equal to or substantially greater than that of poly-I:C depending on the delivery vehicle. Furthermore, p-shRNA caused greater NF-κB activation in SKOV3 cells compared to poly-I:C, indicating that it is a powerful activator of innate immunity. The tuneable sequence and combined gene silencing, immunostimulatory and cytotoxic capacity of p-shRNA make it an attractive platform for cancer immunotherapy.
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Kevin E
authors.initials: KE
authors.lastname: Shopsowitz
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Connie
authors.initials: C
authors.lastname: Wu
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Gina
authors.initials: G
authors.lastname: Liu
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Erik C
authors.initials: EC
authors.lastname: Dreaden
authors.affiliation: Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Institute for Soldier Nanotechnologies, Massachusetts Institute of Technology, Cambridge, MA 02139, USA hammond@mit.edu.
authors.forename: Paula T
authors.initials: PT
authors.lastname: Hammond
mesh_terms: D000970:Antineoplastic Agents / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D001483:Base Sequence; D053148:Caspase 3 / Q000235:genetics / Q000276:immunology; D053179:Caspase 7 / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000276:immunology; D006801:Humans; D007113:Immunity, Innate; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000096:biosynthesis / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011070:Poly I-C / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000276:immunology / Q000494:pharmacology*; D014158:Transcription, Genetic
chemical_list: D000970:Antineoplastic Agents; D016328:NF-kappa B; D034741:RNA, Small Interfering; D049452:Green Fluorescent Proteins; D008156:Luciferases; D053148:Caspase 3; D053179:Caspase 7; D011070:Poly I-C