The in vivo analysis should enlighten our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression. The following constructs have been subcloned separately into the AAV context to accomplish those tasks:
positive control,
off-targeting construct,
synthetic tuning construct and
on-targeting construct.
All but one virus were packaged by the AAV rep and cap gene with Adenovirus 5 (Ad5) as a helper plasmid. Accordingly, one virus construct was packaged into a shuffled cap gene from our homology based capsid shuffling attempt.
The positive control (see sidebar, fig. 1) consisted of the SV40 promoter driving a firefly luciferase (luc2) gene, thereby leading to an unspecific expression of the luciferase protein in all mice tissues. In addition to packaging this construct into a wild type AAV virus, the positive control was also packaged as a transgene into our shuffled capsid which after random selection was already able to positively transduce Huh7 and HepG2 cellsin vitro.
The off-targeting construct (see sidebar, fig. 2) was composed of an SV40 promoter driving a firefly luciferase (luc2) gene with binding sites against miR-122 behind it. In order to achieve the highest expression in all mice cells but the liver cells - a single perfect binding site of miR-122 was used for in vivo study.
The synthetic tuning construct (see sidebar, fig. 3) consisted of two viruses injected at the same time in the mice. The one virus packaged the expression construct of shRNA haat driven by the H1 promoter ("tuning" construct, see sidebar, fig.3). The second virus packaged the following transgene: SV40 promoter driving luc2 with shRNA haat binding site behind it ("tuned" construct, see sidebar, fig. 4). In order to ensure a synthetic tuning effect, a perfect binding site and one with a bulge that was introduced at position 9-12 were used for in vivo experiments, respectively. Those two binding sites should lead to a significant knockdown in the first case and a slight repression of luciferase expression in the latter as compared to the positive control.
The on-targeting construct consisted of two independent viruses which were co-infected into mice, as well. One of these viruses packaged the Tet Repressor (TetR) driven by an SV40 promoter ("repressor" construct, see sidebar, fig. 5). The expression of TetR is under the control of miR-122 as four binding sites of this miRNA were cloned into the 3’UTR of the gene. The second virus was composed of an SV40 promoter driving the Tet operator (TetO2) which monitors the expression of luc2 ("operator" construct, see sidebar, fig. 6). With this setup, luc2 expression should be inhibited by the TetR in all mice tissues except for liver cells, where TetR is down-regulated by miRNA 122.
mice injection
In order to assess tissue tropism of the The TVroute was chosen so as to assess AAV serotype tissue tropism;
the luciferase transgene was used for visualizing the relative vector
distribution in all the animals in a real-time manner
bioluminescence imaging
Discussion
Methods
contructs
production of recombinant virus
The viruses were produced in HEK 293-T cells and purified on an iodixanol gradient according to the virus production protocol.
The mouse experiments were conducted in accordance with the animal facility of the german cancer research institute of Heidelberg. Female NMRI mice were purchased from....??. At 8-10 weeks of age, the animals were injected in the tail vein (TV), with ~ 1x1011 particles of AAV-SV40-luciferase in 200µl of 1x phosphate-buffered saline. The mice are transferred to a holding device which restrains the mouse while allowing access to the tail vein. The tails were warmed before the injections and injections were carried out using 27 gauge needles. All the mice recoverd from the injection quickly without loss of mobility or interruption of grooming activity [http://2010.igem.org/Team:Heidelberg/Project/Mouse_Infection#References (Zincarelli et al., 2008)].
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