Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector.

BACKGROUND: The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector. METHODS/PRINCIPAL FINDINGS: We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector. CONCLUSIONS/SIGNIFICANCE: This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.

Drug delivery-mediated control of RNA immunostimulation.

RNA interference (RNAi) has generated significant interest as a strategy to suppress viral infection, but in some cases antiviral activity of unmodified short-interfering RNA (siRNA) has been attributed to activation of innate immune responses. We hypothesized that immunostimulation by unmodified siRNA could mediate both RNAi as well as innate immune stimulation depending on the mode of drug delivery. We investigated the potential of immunostimulatory RNAs (isRNAs) to suppress influenza A virus in vivo in the mouse lung. Lipidoid 98N12-5(1) formulated with unmodified siRNA targeting the influenza nucleoprotein gene exhibited antiviral activity. Formulations were optimized to increase antiviral activity, but the antiviral activity of lipidoid-delivered siRNA did not depend on sequence homology to the influenza genome as siRNA directed against unrelated targets also suppressed influenza replication in vivo. This activity was primarily attributed to enhancement of innate immune stimulation by lipidoid-mediated delivery, which indicates increased toll-like receptor (TLR) activation by siRNA. Certain chemical modifications to the siRNA backbone, which block TLR7/8 activation but retain in vitro RNAi activity, prevented siRNA-mediated antiviral activity despite enhanced lipidoid-mediated delivery. Here, we demonstrate that innate immune activation caused by unmodified siRNA can have therapeutically relevant effects, and that these non-RNAi effects can be controlled through chemical modifications and drug delivery.

Genetic strategy to prevent influenza virus infections in animals.

The natural reservoirs of influenza viruses are aquatic birds. After adaptation, avian viruses can acquire the ability to infect humans and cause severe disease. Because domestic poultry serves as a key link between the natural reservoir of influenza viruses and epidemics and pandemics in human populations, an effective measure to control influenza would be to eliminate or reduce influenza virus infection in domestic poultry. The development and distribution of influenza-resistant poultry represents a proactive strategy for controlling the origin of influenza epidemics and pandemics in both poultry and human populations. Recent developments in RNA interference and transgenesis in birds should facilitate the development of influenza-resistant poultry.