( Mediating RNA i...)Superior Gene Silencing Using Adenoviral Vectors

How Does pSilencer Adeno 1.0-CMV Work?
An oligonucleotide insert encoding the desired siRNA is cloned into the pre-linearized shuttle vector (provided), which includes a modified CMV promoter for efficient expression of the siRNA sequence. The shuttle vector, containing the desired sequence, is then linearized and introduced into HEK 293 packaging cells (not included) along with the linearized adenovirus backbone (Figure 1). The HEK 293 packaging cells contain the EIA and EIB genes which are not present in the introduced vectors and are required to form infectious particles. Inside the HEK 293 cells, the shuttle vector and the adenoviral backbone plasmid recombine to produce an adenoviral genome including the siRNA expression cassette. The siRNA expression cassette is packaged into infectious virus particles that can be harvested for experiments. Harvested adenoviral particles can then be used to infect cells or animals, or stored for future experiments.

Figure 1. Schematic of pSilencer Adeno 1.0 CMV System Recombination and Packaging. The shuttle vector and backbone plasmid are linearized and then transfected into HEK 293 cells. The HEK 293 packaging cells contain the EIA and EIB genes which are not present in the introduced vectors and are required to form infectious particles. Inside the cell, the plasmids recombine forming a functional genome that is replicated and packaged into adenoviral particles.

siRNA from Adenoviral Vectors Reduce Expression from Transgenic Alleles in vivo
To demonstrate that adenoviruses are useful for decreasing gene expression via the RNAi pathway in animals, Dr. Beverly Davidson and colleagues, our collaborators at the University of Iowa, injected recombinant adenoviruses expressing siRNAs targeting green fluorescent protein (GFP) or -glucuronidase (a negative control) into the brains of transgenic GFP-expressing mice (1). Five days after injection, brain sections were analyzed by fluorescence microscopy and Western blot (Figure 2). GFP expression was reduced in the injected brain hemisphere, indicating the utility of the method for reducing gene expression in animals.

Figure 2. Silencing of Transgenic GFP in Mouse Brain with an Adenoviral Vector (1). Adenovirus engineered to express an siRNA targeting GFP (A, top panels) or a negative control siRNA targeting human -glucuronidase (A, bottom panels) were injected into the brain striatal region of transgenic mice expressing GFP. The adenovirus included a dsRed expression cassette to visualize the localization of the virus. (A) Brain sections were analyzed by fluorescence microscopy 5 days after injection. Green: GFP. Red: dsRed. Left panels: GFP detection. Middle panels: dsRed detection. Right panels: merged images. (B) Brain sections were divided into ipsilateral (il) and contralateral (cl) portions and analyzed by Western blot using antibodies to GFP and -actin (control).

Allele-specific Silencing of Dominant Disease Genes
Due to its inherent specificity, a potential application of siRNAs is the treatment of dominantly inherited diseases. One such neurodegenerative disease is Machado-Joseph disease/spino-cerebellar ataxia type 3. In 70% of diseased carriers, alleles contain a G to C mutation. Dr. Davidson's laboratory constructed plasmids fusing GFP to normal (Atx-Q28-GFP) or mutant polyglutamine (polyQ) alleles (Atx-Q166-GFP) containing a CAG expansion in Ataxin-3 (2). Adenoviral vectors that expressed hairpin siRNAs targeting either the wild type or the mutant allele were designed generated from these plasmids and used to infect Cos-7 cells that had previously been transiently co-transfected with Atx-Q28-GFP (wt) and Atx-Q166 (mutant). (Both plasmids were co-transfected into the Cos-7 cells to mimic the heterozygous adenovirus.) The siRNA expressed from AdG10i had perfect complementarity to the wild type allele, and preferentially suppressed expression of the fusion construct containing that allele (Atx-Q28-GFP, Figure 3A). Conversely, AdC10i preferentially suppressed expression of the mutant allele construct (Atx-Q166, Figure 3A). Finally, these two adenoviral vectors were used to infect PC12 cells that had been created to express normal (Q28) or (Q166) mutant ataxin-3. Again, the viruses were able to specifically target the mutant allele (Figure 3B). These results indicate the utility of viruses for delivering siRNAs that target allele-specific disease genes.

Figure 3. Allele-specific Silencing with Adenoviral Vectors Expressing an siRNA (2). Cos-7 cells were co-transfected with the Atx-3-Q28-GFP (wt) and Atx-Q166 (mutant) constructs and then infected (MOI = 50) with adenovirus expressing siRNAs that had been designed such that a single nucleotide polymorphism (SNP) was in the middle of the target sequence. The siRNA produced by Ad-G10i was perfectly complementary to the wild type construct, whereas that of Ad-C10i was complementary to the mutant construct. (A) Green: GFP. Red: immunofluorescence of the polyQ construct detected with 1C2 antibody. Ad-G10i reduced wt ataxin-3 levels 95% but did not affect mutant ataxin-3 levels. Conversely, Ad-C10i reduced mutant ataxin-3 levels 91% but had no effect on WT ataxin-3 levels. (B) PC12 cells expressing WT (left) or mutant ataxin-3 (right) were infected (MOI = 100) with the Ad-G10i or Ad-C10i virus. Western analysis was performed with antibodies to Ataxin-3 and GAPDH (as a control), and the siRNAs produced by the viruses were able to specifically silence the allele to which they were designed.

The Complete Adenoviral Production System
The pSilencer adeno 1.0-CMV System includes everything needed to produce five preparations of recombinant adenovirus, except the siRNA template oligonucleotide and the HEK 293 packaging cells (HEK 293 cells are available from several sources, including ATCC). The kit includes linearized Shuttle Vector 1.0-CMV (20 rxns), Negative Control Shuttle Vector that encodes a scrambled siRNA sequence, a Positive Control Oligonucleotide Insert that encodes an siRNA targeting GAPDH, and the Adenoviral Backbone that includes a LacZ sequence for monitoring transfection efficiency. Also included are reagents for transfecting the HEK 293 cells with the Shuttle and Backbone Vectors, forward and reverse sequencing primers to verify clones, and 5X Annealing Buffer for preparing the siRNA-encoding oligonucleotides for ligation.

The use of these materials is permitted for research purposes only. Any other use requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.

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