Bender A., Bergmann S., Fischer A., Günzle J., Kaufmann B., Klingele K., Mall A., Meyerovich K., Morath V., Oschowitzer A., Schelker M., Schindler P. , Schober T., Wagner H., Weingärtner C., Hagen S., Baumann T., Arndt, K. M. & Kristian M. Müller Correspondence to: iGEM team Freiburg, Department of Biology III, Schänzlestraße 1, 79104 Freiburg: [email protected] Gene delivery using adeno-associated viruses holds great promise for the treatment of acquired and inherited diseases. Taking current knowledge on viral vectors into account, the Freiburg iGEM team generated a recombinant, modularized, BioBrick-compatible AAV “Virus Construction Kit”. Our system comprises parts for modified capsid proteins, targeting modules, tumor-specific promoters, and The viral tropism is altered by N-terminal fusion or by loop replacement of the capsid proteins. Functionality of viruses constructed from our kit was demonstrated by prodrug-induced killing of tumor cells upon viral delivery of a thymidine kinase. Incorporating multiple layers of safety, we provide a general tool to the growing field of personalized medicine and demonstrate its use in tumor prodrug-activating enzymes as well as readily assembled vectors for gene delivery and production of non-replicative virus particles. therapy. Altering Tropism Modularization of the AAV-2 Genome 60 HT1080 expressing HSPG binding motif 50 40 30 20 10 0 Tumor-marker targeting: DARPin & Affibody With HSPG HSPG KO Fig. 1.1: Flow cytometry. Viruses carrying mVenus GOI. Fluorescence of living cells was quantified. Using HSPG knock-out viruses, transduction efficiency of HT1080 cells was decreased from 80 % to 16 %. The “Designed Ankyrin Repeat Protein” (DARPin) E_01 (~14 kDa) specifically binds the EGF receptor. We created viral particles with surface-exposed DARPin E_01 (Steiner et al.). • AAV genome was converted to BioBrick format. Infectious Titer/ Genomic Titer [%] Affibodies (~7 kDa) are derived from the Z-domain scaffold of staphylococcal protein A. We used the ZEGFR:1907 Affibody (Friedman et al.) engineered to bind the EGFR for viral capsid modification. • Thymidine Kinase (TK) which activates Ganciclovir . • Thymidine - Guanylate Kinase (TK-mGMK) catalyzing the second Ganciclovir activation • Cytosine Deaminase (CD), which activates 5-Fluorocytosine (original plasmids kindly provided by M. Black and J. Gebert). 140 successfully and GOI Vector plasmid right ITR • ß-Globin intron for enhanced expression. • Poly-A-tag from human Growth Hormone (hGH). • human Telomerase promoter (hTERT) for tumor specific expression. • CMV promoter in combination with mVenus. pHelper Helper plasmid 60 40 prodrug • HSPG-KO increased EGFR specific targeting (Fig. 1.2). The RGD motif Infectious / Genomic Titer RGD insertion into 587 loop Integrins are transmembrane proteins highly expressed in many tumor cell lines and bind to the RGD motif. Therefore, we inserted the RGD motif in the 587 loop of the viral capsid. Compared to the control cell line, transduction efficiency of HeLa cells infected with virus particles displaying RGD was significantly increased (Fig. 1.3). HT1080 as reference 6E-4 Toxic Left Left Right mGMK-TK ITR ITR ITR Left Left ITR ITR drug Control after 24h CD Right ITR Fig. 3.1: Scheme for prodrug-activation approach 0E+0 Fig. 4.2: 24 h treatment of the HT1080 control cell line with viral particles and 20 µM Ganciclovir had a minor effect. EGFR-overexpressing A431 tumor cells were efficiently killed. These data clearly demonstrate that the optimized vector containing the Affibody ZEGFR:1907 fused to VP2/3(587KO_HisTag) is able to kill cells specifically. • Introduction of unique restriction sites for convenient insertion of functional peptides into two major exposed surface loops on the capsid. • New standard plasmid backbone: pSB1C3_001 SalI BamHI PvuII BamHI • SspI SalI • His-Tag for IMAC purification • Biotin Acceptor Protein (BAP) for purification, Control: HSPG-KO Fig. 1.3: qPCR. Genomic titer after transfection and infectious titer after transduction were determined by quantitative real-time PCR. rep VP3 CMV VP1up NLS Motif VP2-3 Motif VP2-3 Linker rep 587 motif motif Fig. 6.1: ViralBrick construction Left: Assembly strategy for loop insertions Right: Capsid protein structure cap Purification and Quantification • Virus life cycle was described by ordinary differential equations (ODE). • One model was created for virus production and one for infection. • Time lapse recordings taken with fluorescence microscopy were used to determine viral protein concentration over time. • The model was fitted to data points applying least squares method in logarithmic parameter space. • For virus infection receptor binding was considered. • Depending on modification degree, internalization and transport to the nucleus takes place. • The optimal modification degree was calculated using the model predictions. His-tag Load FT Wash DMEM supernatant DMEM pellet Serum-free supernatant Serum-free pellet 0.30 Antibody-binding motif (Z34C) Figure 8.2 Plates were coated with IgG antibody Cetuximab that can be bound by the Z34C motif. Sandwich ELISA reveal the integration of the Z34C motif in the 587 integration site. Absence of any viral particles in case of 453 insertion might be due to conformational changes or steric effects, disrupting stable VP integration into the viral capsid. 0.25 Protein of interest [1/ml] Absorption at 405nm Virus Infection Efficiency 0.20 0.15 0.10 0.05 0.00 -0.05 4 5 6 7 8 9 10 30 10 0 48.5 97 485 970 Ganciclovir [µM] 7AAD positive cells Annexin-V positive cells A: Gating non-transduced cells (control). B: Non transduced, stained cells plotted against 7AAD Log and AnnV-2 Log C: Gating transduced cells D: Transduced, stained cells plotted against 7AAD Log and AnnV-2 Log Biosafety Figure 8.1 Viral particles displaying His-tags in their loops were purified by immobilized metal ion affinity chromatography (IMAC). Sample and fractions were analyzed by ELISA. Eluted fractions from cells grown in FCS-supplemented medium show high signals, while the signal in serum-free medium is either not present or less intense. - elutions - 40 Fig. 4.4: Testing transduction efficiency and the effect of GCV. Cells were transduced with viruses containing GMK_TK as GOI and treated with 475 µM ganciclovir. 7AAD and Annexin-V staining allows detection of dead and apoptotic cells. Fig. 5.1: Schematic depiction of the N-terminal fusion to VP2. Modeling the AAV Life Cycle 50 453 pSB1C3_001 453 60 20 tropism knock-out in the 587 loop cap (VP1-2-3) CMV D RGD Integrin Binding Motif for targeting HSPG VP2 0E+0 70 Z34C Antibody Binding Motif for targeting 587 Effect on transduced HT1080 cells by different Ganciclovir concentrations 80 C targeting, labeling PvuII BamHI PvuII 90 ViralBricks for multiple applications: • VP1 Control living cells after three days Fig. 4.3: The activated prodrug 5-fluorocyotosine (5-FC) can diffuse through the host cell membrane, widening its toxic effect onto neighbouring cells. This so-called Bystander Effect was tested by mixing transduced HT1080 cells with non-transduced cells and incubating them with 5-FC. After three days, the amount of living cells was calculated via Trypan blue staining. B ViralBricks – A New Standard for Loop Insertions cap (VP1-2-3) 2E-4 mixed with CD-transduced cells Fig. 3.2: Transduced HT1080 cells expressing mVenus SspI rep 4E+6 seeded HT1080 cells VP1 Insertion • Fusion of peptides to the N-terminus of VP2-3 BioBrick • Additional fusion of the unique N-terminal region of VP1 (VP1up BioBrick), providing the essential phospholipase A2 domain – together with a nuclear localization signal (NLS BioBrick) HeLa overexpressing integrin 587_RGD_HSPG-KO Target cells after 24 h 6E+6 mixed with CD-transduced cells and 5-FC • 4E-4 Bystander effect in HT1080 cells: three days with 5-FC Target cells after 0 h 8E+6 A Fig. 1.2: qPCR. Genomic titer after transfection and infectious titer after transduction were determined by quantitative real-time PCR. VP2-3 2E+6 replacing 100 % of VP2 by start codon mutation • Titration and control of the amount of the VP2-targetingsubunit that become incorporated into the virus capsid. 0 Linker Fig. 4.1: All-in-one construct with targeting motif, purification tag and knock-out of the natural tropism. Enzyme Non-toxic VP2 Fusion • Fusion of targeting peptides to the N-terminus of virus protein 2 (VP2-3 BioBrick) according to RFC25 • Expression of the resulting fusion protein in trans to the other structural and regulatory elements of the virus, 80 transduced EGFR-overexpressing A431 cells. In the US, National Institutes of Health (NIH) guidelines classify AAV-2 Vector Systems as Risk Group 1. “… adeno- associated virus (AAV) types 1 through 4; and recombinant AAV constructs, in which the transgene does not encode either a potentially tumorigenic gene product or a toxin molecule and are produced in the absence of a helper virus.” In Germany, viral vectors based on the AAV serotypes 2, 3 and 5 are classified as Biosafety Level 1 as long as the following conditions are fulfilled: 1. Viral particles do not contain AAV-derived sequences other than the ITRs. 2. Viral particles do not contain nucleotide sequences with risk potential. Conclusions, Team, Sponsors and References Conclusions Freiburg iGEM Bioware 2010 • Complete BioBrick-compatible AAV-2 genome modularization • Specific cell targeting • Successful tumor killing • New standards: pSB1C3_001 and ViralBricks • Host lab never worked with AAV-2, Affibodies or DARPins • Fun in the lab, great collaboration with Strasbourg 11 Fraction 453-Z34C 0% Modification degree [%] 587KO-Z34C 100% Biotinylation acceptor peptide (BAP) mVenus Intensity Data 1:2 mVenus Intensity Intensity Time Lapse by Fluorescence Microscopy Control after 0 h 100 specifically • HeLa control cell line showed lower transduction efficiencies. Affibody Additionally, we provide BioBricks for eukaryotic gene expression: We designed two strategies for integration of larger peptides into the virus capsid according to the following steps. Fused peptides are surface-exposed through the capsid pore. 120 0 Key achievements: • Specific cell-killing • Bystander Effect demonstrated N-Terminal Fusions A431 overexpressing EGFR 0 Virus Production and Infection cap (VP1-2-3) cap codes for three proteins VP1, VP2, and VP3. Packaging is defined by the inverted terminal repeats (ITRs). HeLa as reference 20 particles right ITR VP2 fusion: Affibody vs. DARPin 160 viral rep and a Vector Plasmid according to existing systems. • Helper plasmid was taken from existing systems. • We demonstrated better performance than left existing systems. rep cap (VP1-2-3) ITR • 12 site directed mutations were introduced in RepCap plasmid RepCap to facilitate BioBrick compatibility and loop Fig. 2.1: AAV has a single stranded genome with Rep genes for replication, CAP genes for capsid production. insertion. 180 • Modified left ITR • AAV genome was distributed to a RepCap plasmid 70 His6 The ‘vector plasmid’ carries the GOI. We provide as GOI: Amount of living cells Abolishment of the natural viral tropism is essential for specific tumor cell targeting. Therefore we knocked-out the natural tropism towards heparan sulfate proteoglycan (HSPG) (Fig. 1.1). 80 Killing Tumor Cells Dead cells [%] Natural tropism (HSPG) knock-out Gated mVenus positive cells [%] Transduction of HT1080 with viral particles missing HSPG binding motif Gene of Interest (GOI) 1:4 1:8 1:16 1:32 1:64 coated with 200 ng/well Cetuximab Time [min] 1:128 1:250 6 1:512 1:2 1:2 References control Figure 8.3 The aim is to biotinylate viral particles exposing BAP in loop insertions. We showed by ELISA that introduction of the BAP motif was successful, therefore serial dilution of the biotinylated viral vectors was tested. Fig. 9.1: Each layer increases the level of safety for therapeutic application. 1) Ardiani A. & Black ME. (2009). 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