Design of AAV vectors for delivery of RNAi

Abstract

Adeno-associated viral vectors have emerged as an important tool for human gene therapy, having demonstrated high transduction efficiency in a broad range of target tissues, a good safety profile in animal models and human clinical trials, and prospective long-lasting gene expression. First discovered 20 years ago, RNA interference (RNAi) has become another important tool for human gene therapy, enabling scientists to move on from classical gene transfer to gene silencing approaches, or combinations thereof. In this chapter, we describe a simple step-by-step method that will allow gene silencing novices to design their own artificial miRNAs against a target of their choice, clone these miRNAs into an AAV-based vector, and rapidly screen for highly efficient artificial miRNAs. The described method takes into consideration recent advances in the field including miRNA processing from various cellular miRNA backbones, choice between polymerase II and III promoters, and the potential impact of these factors on toxicity as it relates to off-targeting and to saturation of the endogenous RNAi machinery.