Part:BBa_K404163

pCMV_Z-EGFR-1907_Middle-Linker_[AAV2]-VP23 (ViralBrick-587KO-His-Tag)

pCMV_ZEGFR:1907_MiddleLinker_AAV2-VP23(ViralBrick-587KO-His-Tag)
BioBrick Nr.	BBa_K404163
RFC standard	RFC 10
Requirement	pSB1C3
Source
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

Usage in Biology:

Specific targeting of tumor cells and efficient purification were, 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. The EGF receptor is overexpressed in many 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.

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.

One targeting peptide used by the Freiburg Bioware iGEM team 2010 was the Z_EGFR: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 recombinant production in bacteria (Nord et al. 1997).

The Z_EGFR:1907 Affibody was engineered to specifically bind the EGF receptor with an affinity of a K_D = 2.8 nM (Friedman et al. 2008). Because of 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 Z_EGFR: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).

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).

For safe administration of therapeutic AAV2 particles to human patients, it is important to consider a convenient way of purifying the virus particles. For this purpose we integrated histidine residues into the virus capsid. The high binding affinity of histidine towards Ni2⁺ ions can be exploited for the purification of these viruses via so IMAC (Immobilized Metal Ion Affinity Chromatography) (Koerber et al. 2007).

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.

The pCMV_ZEGFR:1907_MiddleLinker_[AAV2]-VP23 (ViralBrick-587KO-His-Tag) is composed of the Affibody Z_EGFR: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 driven by the CMV promoter.

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 applications.

For this purpose AAV-293 cells are 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.

Characterization:

Transduction Efficacy by Flow Cytometry

For determination of transduction efficacy flow cytometry analysis was conducted. 250.000 AAV-293 cells were transfected with 1 µg total DNA. Different ratios of modified VP2/3 plasmids in respect to the Rep-VP13(587KO)_p5-TATAless plasmid (10:90, 25:75, 50:50) were co-transfected. 72 hours post transfection viruses were harvested and two different cell lines, HT1080 and A431, were transduced. By encapsulating mVenus coding sequence as gene of interest, the amount of transduced cells could be determined via flow cytometry.

Figure1: Flow cytometry. Test of transduction efficiency with HT1080 and A431 cells by detecting mVenus expression from Z_EGFR:1907_Middlelinker_VP2/3 virus particles (Transfection ratio: 50:50 in respect to Rep/Cap plasmid). A) Gating non transduced cells (control); subcellular debris and cellular aggregates can be distinguished from single cells by size, estimated via forward scatter (FS Lin) and granularity, estimated via side scatter (SS Lin). B) : Non transduced cells plotted against mVenus fluorescence (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R6). C) Gating transduced cells. D) Transduced cells plotted against mVenus fluorescence, R10 comprised transduced, mVenus expressing cells. E) Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus fluorescence.

Figure 2 overviews transduction efficacy of all ratio compositions.

Figure 2: Flow cytometry analysis. Transduced and therefore mVenus positive HT1080 and A431 cells, infected with virus particles consisting of different ratios of modified VP2/3 plasmids in respect to theRep-VP13(587KO)_p5-TATAless plasmid (10:90, 25:75, 50:50).

Transduction of HT1080 cells revealed that all viral particles remained infectious with efficacies up to 69 %, regardless of which ratio of pCMV_ZEGFR:1907_MiddleLinker_[AAV2]-VP23(ViralBrick-587KO-His-Tag) plasmids in respect to the Rep-VP13(587KO)_p5-TATAless plasmid was transfected. In general the amount of mVenus positive HT1080 cells decreased only slightly when harboring more modified VP2 subunits. A431 cells, which over express EGF receptor, were generally transduced with reduced efficacy. In comparison to HT1080 cells infection efficacy increased when inserting 50 % modified VP2 plasmids. This indicates that larger peptides could be inserted into the AAV2 capsids without affecting virus assembly and packaging. Further on insertion of the Affibody into the capsid improved infectivity, clearly indicating that EGFR over expressing cells were successfully targeted.

Infectious Titer by qPCR

We transfected 250.000 AAV-293 cells with 1 µg total DNA amount. Modified VP2/3 fusion plasmids were co-transfected to the Rep/Cap(VP2KO) plasmid in a 50:50 ratio. 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 the genomic titer. Data were corrected for negative control value.

Co-transfected Construct	Ratio	Genomic Titer /1ml Corrected For Negative Control
Modified VP2/3 plasmid	50:50	5,44E+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 (Fig. 3).

Figure 3: Infectious titers were determined for HT1080, HeLa and A431 cells. Control:Rep/Cap plasmid with and without HSPG knock down.

Figure 3 clearly demonstrates that HSPG affinity knocked down viruses have, in comparison to unmodified viruses, only slightly infectious properties towards HT1080 and HeLa cells. Both controls were nearly were not able to transduce A431 cells. In contrast infectivity was rescued by integrating the Affibody Z_EGFR:1907 into the virus capsids. These data emphasizes the functionality of the VP2 fusion construct for specifically targeting tumor cells for therapeutic or imaging applications.

Killing cells: Time-Lapse

HT1080 and A431 cells were transduced with Z_EGFR:1907_MiddleLinker_[AAV2]-VP23(ViralBrick-587KO-His-Tag) viruses packaged with the guanylate kinase fused to the thymidine kinase coding sequence (GMK-TK). A time series of pictures was started directly after adding 20 µM ganciclovir (Fig. 4).

Figure 4 a: Time-lapse. HT1080 (control) cells transduced with GMK-TK packaged viruses and treated with 20 µM ganciclovir A) 0 hours, B) 7 hours, C) 15 hours and D) 23 hours post transduction.

Figure 4 b: Time-lapse. A431 cells transduced with GMK-TK packaged viruses and treated with 20 µM ganciclovir A) 0 hours, B) 7 hours, C) 15 hours and D) 23 hours post transduction.

Virus and ganciclovir treatment only slightly affects the morphology of HT1080 cells. After 23 hours of incubation in 20 µM ganciclovir the cells had almost the same appearance as at time point zero (Fig. 4 a). In comparison to that A431 epidermoid carcinoma cells were efficiently killed after transduction and ganciclovir add-on: After 23 hours nearly all cells were lysed (Fig. 4 b).

These results clearly demonstrate that we were able to specifically target EGFR over-expressing tumor cells via capsid integrated Affibody and that these transduced cells were efficiently killed by expressing the GMK-TK which converted prodrug ganciclovir into its cell-toxic monophosphate.

Validating Integration of modified Viral Proteins into the Virus Capsid: ELISA

Transfection of AAV-293 producer cells was performed in five 10 cm petri dishes with 3.6x10^6 cells, resulting in a confluency of about 70-80% according to the standard protocol either with cells grown in GIBCO® FreeStyle™ 293 Expression Medium (Invitrogen, protein- and animal-origin free) or in DMEM supplemented with 10% FCS (PAA). Cells were spun down at 200 x g for five minutes and the samples were divided into the pellet and the supernatant fractions. Physical cell lysis was performed by four cycles of freeze and thaw for all four samples. 48 h post transduction the cell lysate / supernatant fractions were incubated with 800 µl of His-Affinity Gel (kindly provided by Zymo Research, USA) at 4 °C for 18 hours with 200 rpm constant agitation. The beads were then collected in 5 ml gravity-flow columns and washed five times with one column volume of PBS each. The His-affinity gel was subsequently washed with PBS, 25 mM Imidazole to remove unspecifically bound proteins. Elution was performed in an second step with PBS, 500 mM Imidazole to elute the His-tagged viral vectors. The genomic titer of the purified viral vectors was detected via q-PCR. In an ELISA, viral vectors were captured employing the monoclonal antibody A20 (kindly provided by PD Dr. J. Kleinschmidt, DKFZ, Heidelberg) that exclusively recognizes assembled AAV capsids. His-Tags present in assembled viral capsids were subsequently detected with an HRP-tagged secondary anti-His-Tag antibody (1:2000 diluted, A7058, Sigma). HRP presence was detected using the peroxidase substrate ABTS. Generation of blue-green color (absorption at 405 nm) was measured in a Tecan Sunrise plate reader. Sample data were blanked with the average of the non-template controls (NTC).

Figure 5: A) Schematic overview of the sandwich ELISA for the detection of His-tagged viral particles. B) ELISA from viral particles produced by AAV-293 cells in DMEM or Free Style medium, divided into cell pellet and cell culture supernatant samples. The particles were purified using Ni-NTA affinity chromatography with Imidazole in PBS as washing and elution agent. C) Absorption measurements from plate shown in B. Undiluted Äkta fractions converted ABTS peroxidase substrate at 405 nm. D) As C, whereas Äkta fractions were 10-fold diluted.

Presence of the His-affinity tag in the viral capsid was detected and the ELISA enabled quantification of the purification procedure efficiency. The absorbance measured for the elution fractions of the 1/10 diluted samples sums up to 2.3 for the DMEM- and 0.5 for the Free Style 293-grown cells, assigning the DMEM-grown cells a five times higher production efficiency. Comparison between the cell pellet and the supernatant fractions revealed that 70 - 80% of the viral particles can be found inside the producer cells.

According to these results, producer cells should be grown in complex media for in vitro and cell culture experiments. Use of serum-free produced viral vectors is recommended for mouse or other animal experiments and possible therapeutical applications where even the presence of traces amounts of fetal calf serum should be avoided. Combination with different purification approaches such as gel filtration chromatography using i.e. Superdex 200 columns (GE Healthcare) enables the production of highly purified viral vectors for several different applications.

References

Colquhoun, a J, and J K Mellon. 2002. Epidermal growth factor receptor and bladder cancer. Postgraduate medical journal 78, no. 924 (October): 584-9. doi:10.1136/pmj.78.924.584. http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1742539&tool=pmcentrez&rendertype=abstract.

Friedman, Mikaela, Anna Orlova, Eva Johansson, Tove L J Eriksson, Ingmarie Höidén-Guthenberg, Vladimir Tolmachev, Fredrik Y Nilsson, and Stefan Ståhl. 2008. Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. Journal of molecular biology 376, no. 5: 1388-402. doi:10.1016/j.jmb.2007.12.060. http://www.ncbi.nlm.nih.gov/pubmed/18207161.

Göstring, Lovisa, Ming Tsuey Chew, Anna Orlova, Ingmarie Höidén-guthenberg, Anders Wennborg, Jörgen Carlsson, and Fredrik Y Frejd. 2010. Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects. International Journal of Oncology 36, no. 4 (March): 757-763. doi:10.3892/ijo_00000551. http://www.spandidos-publications.com/ijo/36/4/757.

Hirsch, Fred R, Marileila Varella-Garcia, Paul a Bunn, Michael V Di Maria, Robert Veve, Roy M Bremmes, Anna E Barón, Chan Zeng, and Wilbur a Franklin. 2003. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 21, no. 20 (October): 3798-807. doi:10.1200/JCO.2003.11.069. http://www.ncbi.nlm.nih.gov/pubmed/12953099.

Nord, K, E Gunneriusson, J Ringdahl, S Ståhl, M Uhlén, and P A Nygren. 1997. Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain. Nature biotechnology 15, no. 8 (August): 772-7. doi:10.1038/nbt0897-772. http://www.ncbi.nlm.nih.gov/pubmed/9255793.

Walker, R a, and S J Dearing. 1999. Expression of epidermal growth factor receptor mRNA and protein in primary breast carcinomas. Breast cancer research and treatment 53, no. 2 (January): 167-76. http://www.ncbi.nlm.nih.gov/pubmed/10326794.

Wikman, M, a-C Steffen, E Gunneriusson, V Tolmachev, G P Adams, J Carlsson, and S Ståhl. 2004. Selection and characterization of HER2/neu-binding affibody ligands. Protein engineering, design & selection : PEDS 17, no. 5 (May): 455-62. doi:10.1093/protein/gzh053. http://www.ncbi.nlm.nih.gov/pubmed/15208403.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 2195
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 665
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 2754
Illegal SapI site found at 1632