Document 2523

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). Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo. Cancer Gene Ther. 17:86.
2) Warrington KH Jr et al., (2004). Adeno-associated virus type 2 VP2 capsid protein is nonessential and can tolerate large peptide insertions at its N terminus. J Virol. 78:6595.
3) Friedman et al., (2008). Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule. J Mol Biol. 376:1388.
4) Boucas J et al., (2009). Engineering adeno-associated virus serotype 2-based targeting vectors using a new insertion site-position 453-and single point mutations. J Gene Med. 11:1103.
5) Girod A et al., (1999)Genetic capsid modifications allow efficient re-targeting of adeno-associated virus type 2. Nat Med. 5:1438.
6) Göstring, L. et al., (2010) Quantification of internalization of EGFR-binding Affibody molecules: Methodological aspects. Int J Oncol. 36:757.
7) Jing, X.J. et al., (2001) Inhibition of adenovirus cytotoxicity, replication, and E2a gene expression by adeno-associated virus. Virology 291:140.
8) Levy, H.C. et al., (2009) Heparin binding induces conformational changes in Adeno-associated virus serotype 2. J Struct Biol. 165:146.
9) Opie, S., Jr, K.W. & Agbandje-, M., (2003) Identification of amino acid residues in the capsid proteins of adeno-associated virus type 2 that contribute to heparan sulfate proteoglycan binding. J Virol.
77:6995.
10) Steiner, D., Forrer, P. & Plückthun, A., (2008) Efficient selection of DARPins with sub-nanomolar affinities using SRP phage display. J Mol Biol. 382:1211.
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