Part:BBa_K404156

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

[https://parts.igem.org/Part:BBa_K404156 pCMV_Z-EGFR-1907_Middle-Linker_(AAV2)-VP23(ViralBrick-587KO-Empty)
BioBrick Nr.	BBa_K404156
RFC standard	RFC 10
Requirement	pSB1C3
Source
Submitted by	[http://2010.igem.org/Team:Freiburg_Bioware FreiGEM 2010]

This part is used for cotranfection with parts containing VP1up (BBa_K404164-BBa_K404166)

Affibody Z-EGFR-1907

(BBa_K404302)

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). Affibodies can be used for example for tumor targeting (Wikman et al., 2004) and diagnostic imaging applications (Orlova et al., 2006; Orlova et al., 2007). 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). The EGF receptor is overexpressed in certain types of tumors, e.g. in breast (Walker & Dearing, 1999), lung (Hirsch et al., 2003) and bladder (Colquhoun & Mellon, 2002) carcinomas, and is therefore a suitable target for cancer imaging or therapeutic applications. Because of their good tumor uptake, and their 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 was chosen for therapeutic applications by the Freiburg iGEM Team 2010 (Friedman et al., 2008; Göstring et al., 2010).

CMV

CMV promoter is derived from human Cytomegalovirus, which belongs to Herpesvirus group. All family members share the ability to remain in latent stage in the human body. CMV is located upstream of immediate-early gene. However, CMV promoter is an example of widely used promoters and is present in mammalian expression vectors. The advantage of CMV is the high-level constitutive expression in mostly all human tissues [Fitzsimons et al., 2002].

Middle Linker ( Gly-Gly-Ser-Gly)x2

(BBa_K243005)

This part is a linker, it can be used to connect two parts and add additional space between them. That can be necessary to avoid interactions between these parts.

Capsid

(BBa_K404006)

The AAV capsid consists of 60 capsid protein subunits. The three cap proteins VP1, VP2, and VP3 are encoded in an overlapping reading frame. Arranged in a stoichiometric ratio of 1:1:10, they form an icosahedral symmetry. The mRNA encoding for the cap proteins is transcribed from p40 and alternative spliced to minor and major products. Alternative splicing and translation initiation of VP2 at a nonconventional ACG initiation codon promote the expression of VP1, VP2 and VP3. The VP proteins share a common C terminus and stop codon, but begin with a different start codon. The N-terminus of VP1 plays an important role in infection and contains a motif highly homologous to a phospholipase A2 (PLA2) domain and nuclear localization signals (BR)(+). VP2 contrains basic regions, too.

ViralBrick 587-KO empty

The primary receptor of AAV-2 is the heparan sulfate proteoglycan (HSPG) receptor (Perabo et al. 2006). Its 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. Opie et al. have shown that two point mutations (R585A and R588A) are sufficient to eliminate the heparin binding affinity in AAV2. (Opie et al. 2003). This ViralBrick has been created to introduce this knockout into other constructs. The biobricks with containing this knockout are annotated with „HSPG-ko“.

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 VP2 fusion construct in respect to the Rep/Cap(587KO_VP2KO) plasmid were co-transfected. 72 hours post transfection viruses were harvested and two different cell lines, HT1080 and A431, were transduced with 1 mL virus stock. By encapsulating mVenus coding sequence, the amount of transduced cells could be determined via flow cytometry.

Figure 1: 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.

HT1080

A431

Figure 2: Flow cytometry analysis. Transduced and therefore mVenus positive HT1080 and A431 cells, infected with virus particles consisting of different ratios of VP2 fusion construct in respect to Rep/Cap(587KO_VP2KO) plasmid.

Transduction of HT1080 cells revealed that all viral particles regardless of which motifs inserted into the capsids remained infectious with efficacies up to 69 %. In general the amount of mVenus positive cells decreased only slightly when harboring more modified VP2 subunits. This indicates that larger peptides could be inserted into the AAV2 capsids without affecting virus assembly and packaging. A431 cells, which over express EGF receptor, were generally transduced with reduced efficacy.

This indicated that VP1 tolerated larger peptides inserted downstream of its unique N-terminal region and that this modification still allowed virus assembly and packaging.

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 – with or without HSPG binding knock down – were co-transfected in respect to Rep/Cap(VP2KO). 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
Affibody_MiddleLinker_VP2/3	25:75	2,20E+08
Affibody_MiddleLinker_VP2/3	50:50	2,39E+08
Affibody_MiddleLinker_VP2/3(587KO)	25:75	1,17E+09
Affibody_MiddleLinker_VP2/3(587KO)	50:50	5,44E+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 3 shows infection efficacy: 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 overexpress EGFR were not infected by the controls. Transduction is rescued by integration of the Affibody into the virus capsid. These results indicated that specific targeting of AAV2 virus particles towards EGFR over expressing tumor cells was achieved by N-terminal fusion of targeting motifs to VP2.

qPCR

Figure 3: Affibody Z_EGFR:1907 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.

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 2721
Illegal SapI site found at 1632

References

Mellon. 2002. Epidermal growth factor receptor and bladder cancer.Postgraduate medical journal78, 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 biology376, no. 5: 1388-402. doi:10.1016/j.jmb.2007.12.060. http://www.ncbi.nlm.nih.gov/pubmed/18207161.
Fitzsimons, H.L., Bland, R.J. & During, M.J., 2002. Promoters and regulatory elements that improve adeno-associated virus transgene expression in the brain. Methods San Diego Calif, 28(2), pp.227-236. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12413421.
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.
Orlova, Anna, Vladimir Tolmachev, Rikard Pehrson, Malin Lindborg, Thuy Tran, Mattias Sandström, Fredrik Y Nilsson, Anders Wennborg, Lars Abrahmsén, and Joachim Feldwisch. 2007. Synthetic affibody molecules: a novel class of affinity ligands for molecular imaging of HER2-expressing malignant tumors. Cancer research 67, no. 5 (March): 2178-86. doi:10.1158/0008-5472.CAN-06-2887. http://www.ncbi.nlm.nih.gov/pubmed/17332348.
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 treatment53, 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.