The importance of virus shuttles for human gene therapy canot be understated. The Adeno-associated virus (AAV) is a non-pathogenic virus that can infect human tissue but does not replicate without a helper virus (like Adenovirus or Herpex simplex virus). Therefore it is ideal for the delivery of transgenes in vivo. AAVs are small (4.8kb genome size), nonenveloped viruses that exist in various natural serotypes with different properties in tropism and transduction speed and efficiency. The most intensively studied AAV serotype is AAV2, which is used in several ongoing clinical trials aiming at muscle tissue or central nervous system diseases.
AAV viruses are easily manipulated, as they do not require any viral genes in cis (that is, on the same construct) genes needed for packaging (cap) and other structures (rep) that usually make up the viral genome can be delivered in trans from a second vector that is co-transfected. The only requirement for a gene to be packaged into an AAV virus is that it has to be flanked by Inverted Terminal Repeats (ITR) that serve as a packaging signal.
For delivery of our transgenes into mice, we chose AAV serotype 8 as a carrier. This serotype, which has been isolated from rhesus monkey, has been shown to infect rapidly and especially target heart and liver. Since we are using the liver-specific miRNA 122 for On- and Off-targeting, this would be our ideal vector.
gene therapy
Adeno-associated viruses
miRNAs
bioluminescence imaging
Results
constructs
In order to enable in vivo analysis of our gene therapy approach using AAV tropism as well as miRNA binding sites as trigger for expression the following constructs have been subcloned into the AAV context: (1) positive control, (2) off-targeting construct, (3) synthetic tuning construct and (4) on-targeting construct. All but of one virus were packaged in the adeno-associated virus (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.
(1) The positive control 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 selection pressure was already able to positively transduce Huh7 and HepG2 cells in cell culture.
(2) The off-targeting construct 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 perfect binding site of miR-122 was used for in vivo study.
(3) The synthetic tuning construct 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. The second virus packaged the following transgene: SV40 promoter driving luc2 with binding sites for shRNA haat behind it. 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 knockdown in the first case and a repression of luciferase expression in the latter in comparison to the positive control.
(4) 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. 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. With this setup, luc2 expression should be inhibited by the TetR in all mice tissues except for liver cells, where TetR is downregulated 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 on HEK 293-T cells and purified on an iodixanol gradient according to 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 ~1x10^11 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 usind 27 gauge needles. All the mice recoverd from the injection quickly without loss of mobility or interruption of grooming activity.
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