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| | ===Usage and Biology=== | | ===Usage and Biology=== |
| − | Specific targeting and purification of tumor cells was, besides producing recombinant virus particles for therapeutical applications, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. <br/>
| + | [[Media:Freiburg10 FACSDataA431FavouritePart.png]] |
| − | For development of targeting strategies against EGF receptor (EGFR) over-expressing cancer cells, exhaustive literature search for engineering the surface of the Adeno-associated virus 2 (AAV2) was performed. The EGF receptor is overexpressed in certain types of tumors, e.g. in breast (Walker and Dearing 1999), lung (Hirsch et al. 2003) and bladder (Colquhoun and Mellon 2002) carcinomas, and is therefore a suitable target for cancer imaging or therapeutic applications. <br/>
| + | [[Media:Freiburg10 FACSDataFavouritePart.png]] |
| − | Besides insertion of targeting motifs into the viral protein 1 (VP1) open reading frame (ORF), we designed a method for fusing larger motifs to the N-terminus of VP2. We expected that that peptides fused to the N-terminus of VP2 become located on the virus surface either by transit through the channels or by exposure during capsid assembly.
| + | [[Media:Freiburg10 FACSDataHT1080FavouritePart.png]] |
| − | One targeting peptide used by the Freiburg iGEM team_Bioware was the ZEGFR:1907 Affibody. Affibodies are small (6 kDa), soluble high-affinity proteins. They are derived from the IgG-binding B domain of the Staphylococcal protein A, which was engineered to specifically bind to certain peptides or proteins. This so-called Z domain consists of an antiparallel three-helix bundle and is advantageous due to its proteolytic and thermodynamic stability, its good folding properties and the ease of production via recombinant bacteria (Nord et al. 1997). <br/>
| + | [[Media:Freiburg10 qPCRFavouritePart.png]] |
| − | The ZEGFR:1907 Affibody was engineered to specifically bind the EGF receptor with an affinity determined to be KD = 2.8 nM (Friedman et al. 2008). Because of its good tumor uptake, and its property to become internalized into the target cells with an efficiency of 19 – 24% within one hour – compared to 45% of the natural ligand EGF - the ZEGFR:1907 Affibody (Z-EGFR-1907, BBa_K404302) was designed according to the Freiburg RFC25 standard for fusing it to the N-terminus of VP2/3 (Göstring et al. 2010; Friedman et al. 2008).<br/>
| + | [[Media:Freiburg10 TimeLapseA431.png]] |
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| + | [[Media:Freiburg10 TimeLapseHT1080.png]] |
| − | Additionally the natural tropism of the virus towards its primary receptor heparan sulfate proteoglycan (HSPG) needed to be knocked down in order to prevent infection of healthy cells (Perabo et al. 2006). The binding motif consists of five amino-acids located on the capsid surface: R484/R487, K532, R585/587. (Trepel et al. 2009). The positively charged arginine residues interact with the HSPGs' negatively charged acid residues. Two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity in AAV2 (Opie et al. 2003). <br/>
| + | [[Media:Freiburg10 ELISA.PNG]] |
| − | Since the aim behind engineering therapeutic AAV2 vectors is a safe administration to human patients, it is important to consider a convenient way of purifying the virus particles. For this purpose we integrated six histidine residues into virus capsid. The high binding affinity of Histidine towards Ni2+ ions can be exploited for the purification of these viruses via so called „Immobilized Metal Ion Affinity Chromatography“ (Koerber et al. 2007). <br/>
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| − | The ViralBrick-587KO-His-Tag (BBa_K404211) combines the R585A and R588A point mutations with the Histidine-Tag inserted in the capsid loop at amino acid position 587.<br/>
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| − | The pCMV_ZEGFR:1907_MiddleLinker_[AAV2]-VP23 (ViralBrick-587KO-His-Tag) is composed of the Affibody ZEGFR:1907 (Z-EGFR-1907, BBa_K404302) coupled to the N-terminus of AAV2 VP2/3 sequence ([AAV2]-VP23, BBa_K404150) via Middle Linker (Middle Linker ( Gly-Gly-Ser-Gly)x, BBa_K243005). The ViralBrick 587KO-His-Tag (ViralBrick-587KO-His-Tag, BBa_K404211) was inserted into the surface exposed loop at amino acid position 587. The expression of the resulting fusion construct is controlled by the CMV promoter. <br/>
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| − | This composite part was created in order to produce recombinant virus particles which can be easily purified and specifically target EGFR over expressing tumor cells for therapeutic application.
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| − | For this purpose AAV-293 cells need to be co-transfected with this part, [AAV2]-Rep-VP13(ViralBrick-587KO-empty)_p5-TATAless (BBa_K404004), any gene of interest located between the AAV2 left ([AAV2]-left-ITR, BBa_K404100) and the right ([AAV2]-right-ITR, 1BBa_K404100) inverted terminal repeats and the pHelper plasmid.
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