In Vivo Fermentation Production of RNA Interference Agents

Novel in vivo approaches for manufacturing RNA agents are warranted to complement conventional methods such as in vitro transcription and chemical synthesis. Recently, we have identified and utilized unique tRNA-fused pre-miRNA carriers to establish a robust RNA molecular bioengineering technology that allows one to consistently and efficiently produce target, bioengineered or biologic RNA molecules bearing small interfering RNA (siRNA) payloads (BioRNA/siRNA). Here, we describe a streamlined method with a detailed protocol for the rational design, molecular cloning, heterologous overexpression, and chromatography purification of large quantity (multi-milligrams from 0.25 L of bacterial culture) and high quality (> 98% homogeneity and <5 endotoxin unit/μg RNA) of target BioRNA/siRNA molecules that are ready for basic research as well as experimental therapy.