RNAi is an evolutionarily conserved process in metazoans by which double-stranded RNA directs sequence-specific degradation of mRNA. Studies have shown that RNAi can be triggered by the introduction of synthetic 21-nt RNA duplexes [7
], often referred to as “short interfering RNA” (siRNA), or by the expression of RNA duplexes in a hairpin structure [8
], referred to as “short hairpin RNA” (shRNA), which can be processed into siRNA by cellular RNA endonucleases. RNAi has been shown to be effective in interfering with viruses such as HIV, hepatitis B virus, respiratory syncytial virus, poliovirus, rhinovirus, severe acute respiratory syndrome—associated coronavirus, and dengue virus in cell culture and, in a few cases, in animals.
Directly relevant to influenza control, studies have shown that siRNA that is specific for conserved regions of influenza virus genes potently inhibit replication of a broad spectrum of influenza viruses in cell lines, chicken embryos, and mice [9
]. Stable expression of influenza-specific shRNA via a lentiviral vector in a cell line renders the cells refractory to influenza virus infection [10
]. Similarly, introduction of the same lentiviral vector into the mouse lung results in significant inhibition of virus production in vivo. Together, these findings suggest the possibility of developing influenza-resistant poultry by transgenic expression of influenza-specific shRNA.
Lentivirus has been the vector of choice for stable expression of shRNA in cells and animals [11
]. Recently, lentivirus-mediated transgenesis has been shown to be very efficient in birds. More than 70% of founder birds contain the vector sequences, and 4%–45% of founder birds give rise to germline transmission [12
]. In contrast, transgenesis in chickens by direct DNA injection or infection with oncoretroviral vectors is much less efficient, often requiring the screening of thousands of birds to obtain a single germline transmission [14
]. Furthermore, unlike transgene expression from oncoretroviral vectors, which is often silenced by epigenetic modifications during early ontogeny, transgene expression from integrated lentiviral vectors has been found to be stable for 4 generations (B.B.S. and C.L., unpublished data).
A critical consideration in developing influenza-resistant poultry is the prevention of the emergence of resistant viruses. One approach is to use siRNA targeting the conserved regions of influenza virus genes, because these regions either do not change or change much less frequently than do other regions, probably owing to structural and/or functional constraints. Targeting the highly conserved region potentially allows the siRNA to remain effective despite antigenic drift and shift. It also has the potential to reduce the emergence of viable resistant variants. Another approach is to simultaneously express multiple shRNAs. The mutation rate of influenza virus is estimated to be 1.5 × 10-5
mutations/nucleotide/infection cycle [15
]. If 4 shRNAs are expressed simultaneously, the probability of the emergence of a resistant virus can be reduced to 1 resistant virus/3 × 1021
virions. This probability can be assessed by use of the following example. The United States produced ~9 billion broiler chickens in 2006. For a resistant virus to arise, each chicken would have to produce 3 × 1011
infectious virions. Under the assumption that 1 infected cell produces 3 × 103
new infectious virions, 108
cells/chicken would have to be infected. Thus, lentiviral vectors expressing ≥4 shRNAs specific for the conserved regions of the influenza virus genome should be used for transgenic poultry production.