Part:BBa_K404314

DARPin-E01

[DARPin-E01
BioBrick Nr.	BBa_K404314
RFC standard	RFC 25
Requirement	pSB1C3
Source
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Natural protein ankyrin repeat (AR) molecules are motifs that can be found commonly in proteins (Bork 1993). These motifs mediate protein-protein interactions suggesting that AR proteins can be used for designing new binding molecules. Design of structural scaffolds with consensus regions and randomized positions of interacting residues leads to improved biophysical characteristics of targeting molecules (Binz et al. 2003) (Kohl et al. 2003).

The repetitive nature of the ankyrin proteins allows modifications in their variable and modular binding surface. Therefore, consensus sequences of natural ankyrin proteins have been used to design novel and stable scaffolds for binding proteins.

Designed Ankyrin Repeat Proteins (DARPins) are well expressed, monomeric in solution, thermodynamically stable and have the ability to fold fast. In the publication of (Steiner et al. 2008) screening libraries were created by using the signal recognition particle (SRP) translocation pathway for phage display. The selected DARPin E_01 has very high affinities to the target protein ErbB1 and can be used as a potential targeting molecule for our approach by fusing the DARPin to N-terminal VP proteins. Our designed ankyrin repeat protein consists of three internal binding repeats and the C-and N-terminal capping repeats. Each internal repeat module comprises one beta-turn and two hydrophobic alpha helices. The potential interaction residues are located in the beta-turn and the first alpha helix of the AR-proteins.

Characterization

Specific targeting 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.

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.

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. It is expected that these peptides become located on the virus surface either by transit through the pores or by exposure during capsid assembly.

Additionally it is required to knock down the natural tropism of the virus towards its primary receptor heparan sulfate proteoglycan (HSPG) 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 of AAV2 (Opie et al. 2003)

Infectious Titer by qPCR

We transfected 250.000 AAV-293 cells with 1 µg of total DNA composed of equal amounts of Rep/Cap, pHelper and vector plasmid. VP2 fusion plasmids were co-transfected with two different ratios in respect to Rep/Cap(VP2KO). The resulting AAV2 particles were produced in two versions: With or without HSPG binding affinity knock down (587KO).

Viruses were harvested three days post transfection. The genomic titer was determined via qPCR by amplification of a specific sequence located in the CMV promoter of the vector plasmid (Table 1).

Table 1: Quantitative Real-Time PCR. Determination of genomic titer. Data were corrected for negative control value.

Co-transfected Construct	Ratio	Genomic Titer /1ml Corrected For Negative Control
DARPin_MiddleLinker_VP2/3	25:75	4,36E+08
DARPin_MiddleLinker_VP2/3	50:50	3,93E+08
DARPin_MiddleLinker_VP2/3(587KO)	25:75	1,00E+09
DARPin_MiddleLinker_VP2/3(587KO)	50:50	3,99E+08
Control: Rep/Cap	100%	1,55E+08
Control: Rep/Cap(587KO)	100%	5,39E+08

 

We investigated transduction of different cell lines. For this purpose 100.000 HT1080, HeLa or A431 cells were seeded and transduced with 50 µL virus stock and harvested 24 hours later.  Infectious titers were determined via qPCR and normalized to the genomic titers.

Figure 1 shows infection efficacy of DARPin exposing viruses. Transduction of HT1080 cells was almost not affected as long as binding to HSPG was not knocked down. HeLa cells were also infected less efficient compared to the controls. However, A431 cells which over express EGFR were not infected by the controls. Transduction is rescued by integration of the DARPin into the virus capsid. By additionally knocking down the HSPG binding affinity these cells are transduced 10 times better, reaching wild type capsid HT1080 infection efficacy. These results indicated that specific re-targeting of AAV2 virus particles towards EGFR over expressing tumor cells was achieved by N-terminal fusion of targeting motifs to VP2.

Figure 1: DARPin E01 VP2 Fusion. Infectious titers were determined with or without HSPG knock down for HT1080, HeLa and A431 cells. Control:Rep/Cap plasmid with and without HSPG knock down.