Testing the specific IgY antibody-based delivery of an siRNA expression
vector into target cells
T. Kegan Brumagen and Jeffrey P. Thompson, Ph.D.
Department of Biology, York College of Pennsylvania, York, PA 17403
Figure 1. Crystal structure of dimerized extracellular
region of FGFR1 binding ligand FGF1 (purple and blue).
From: Plotnikov, et al. 2000
Experimental Procedure
Results (cont.)
Bright Field
Conversion of
mRNA to
cDNA
Formation of conjugate
antigen using Keyhole
Limpet hemocyanin
(KLH) and peptide
Fluorescence
a
PCR amplification of EGFP siRNA
sequence with a thiol-modified reverse
primer (Figure 7)
Amplification
of cDNA
Verification of FGFR1 IIIc
expression
(Figure 5)
Linkage of PDPH and PCR
product
Bacterial amplification and isolation of
expression vectors
pReceiver-M51
(Figure 9)
pEGFP-C1
Electrophoretic Shift Assay
(Figure 8)
2.010 7
7.010 7
1.010 7
6.510 7
pmCherry-N1
6.010 7
Transfection
of COS-7
cells
(Figure 10)
Western Blot
- EGFP siRNA vector
Test EGFP siRNA
down-regulation
(Figure 11)
IgY Conjugate-Mediated EGFP Down-Regulation Experiment
EGFP siRNA Treated
Pre-Treated
pReceiver-M51
+ pmCherry
pEGFP
+ pmCherry
pReceiver-M51
+ pmCherry
IgY Conjugate Treated
pEGFP
+ pmCherry
pReceiver-M51
+ pmCherry
pEGFP
+ pmCherry
Rxn1
2.010 7
5.010 7
1.010 7
4.010 7
3.010 7
2.010 7
1.510 7
7.010 7
1.010 7
6.510 7
5.010 6
6.010 7
pE
G
FP
pR
-M
51
+ IgY/DNA conjugate
Figure 12. Down-regulation of EGFP expression of targeted cells in a cocultured cell population. Mean fluorescence emission at 535 nm and 620
nm for EGFP (green) and mCherry (red), respectively, of the mixed cell
populations. Error bars represent the 95% confidence interval and n = 10 for
all groups. EGFP fluorescence was not significant (ns) following both
treatments (P>0.05) as determined by an un-paired t-test. (a) EGFP and
mCherry fluorescence before and 48 hours after siRNA vector addition to
each co-cultured sample. (b) EGFP and mCherry fluorescence before and 48
hours after IgY/DNA conjugate addition to each co-cultured sample. (c)
Expected EGFP and mCherry fluorescence before and 48 hours after
IgY/DNA conjugate addition to each co-cultured sample.
+ IgY/DNA conjugate
Conclusions
Results
500bp
6.010 7
0
pE
G
FP
7.510 7
- IgY/DNA conjugate
7.010 7
- IgY/DNA conjugate
2.510 7
pE
G
FP
Antibody-mediated
EGFP down-regulation
(Figure 12b)
Non-specific EGFP
down-regulation
(Figure 12a)
EGFP Fluorescence
Baseline
8.010 7
8.010 7
ns
0
a
3.010
ns
7
+ EGFP siRNA vector
*
3.010 7
c
pR
-M
51
pR
-M
51
pE
G
FP
0
Can IgY recognizing an over-expressed, cell-surface protein
receptor be used to selectively deliver siRNA-expressing vectors
into cancer cells?
Rxn2
62
Annealing Temperatures (ºC):
2000 bp
100 bp
63
64
65
66
67
500 bp
a
Figure 5. Amplification of U87MG cDNA with FGFR1 IIIcspecific primers. Both reactions produced predicted PCR
fragments around 2200 bp.
700 bp
100 bp
700 bp
b
b
a
Figure 7. PCR amplification of EGFP siRNA expressing fragments using a
thiol-modified reverse primer. (a) Temperature gradient PCR to determine
appropriate annealing temperature. (b). Samples run with annealing temperature
at 67 to be purified for conjugation to IgY.
Figure 4. Chemical coupling strategy.
(a) Conceptual diagram of an IgY/DNA
conjugate. (b) 3-[2-Pyridyldithio]propionyl
hydrazide (PDPH) is a heterobifunctional
crosslinker that is thiol reactive (red) and
carbonyl reactive (blue).
100 bp
500 bp
Thiolated Reduced
PCR prod.
PCR prod. PCR prod. +PDPH
IgY/DNA
Conjugate
a
b
1. Verify FGFR1 IIIc expression in a human glioblastoma
multiforme cell line, U87MG.
2. Develop anti-human (h)FGFR1 IIIc IgY chicken antibodies.
3. Link IgY antibodies to siRNA-expressing DNA fragments
(Figure 4).
4. Develop a model for specific anti-hFGFR1 IIIc IgY based siRNA
expression vector delivery into target cells.
7.510
7
ns
4.010 7
b
pR
-M
51
Affinity purification of
specific antibodies
(Figure 6)
8.010 7
ns
ns
pE
G
FP
Mammalian Tissue Culture
3.010 7
a
Crosslink DNA fragments to
antibodies using PDPH
EGFP Emission at 535 nm (RFU)
Isolation of total IgY
from immunized hen’s
eggs
Figure 11. The ability of EGFP siRNA expression vector to down-regulate EGFP.
Bright field and fluorescence microscopy of transfected COS-7 cells seventy-two hours
post-transfection with (a) pEGFP-C1, (b) pEGFP-C1 and EGFP siRNA expression vector.
Figure 10. Fluorescence microscopy of transfected COS-7L cells. Seven days after
transfection with (a) pReceiver-M51, (b) pEGFP-C1, and (c) pmCherry-N1.
pR
-M
51
Selective periodate oxidation of antibody
carbohydrate moities to aldehydes
Chicken IgY proteins (Figure 3) could be an ideal carrier for
siRNA plasmids because they are highly specific, able to be
modified using their carbohydrate moieties, easily produced, and
obtained in high levels.
Objectives
c
3 boosters with
KLH/peptide conjugate
Down-regulation of the oncogene, BCL-2, via siRNA, induced
increased apoptosis in GBM cells(2). However, inefficient in vivo
delivery limits the application of siRNAs as a viable therapeutic
option(6).
Figure 3. Chicken IgY with glycosylation sites.
Black hexagons represent sites that are always
glycosylated, whereas, gray hexagons represent
sites that may or may not be glycosylated
From: Suzuki and Lee, 2003
b
Immunization with
KLH/peptide conjugate
Small interfering RNAs (siRNAs) can be used to specifically
down-regulate genes in mammalian cells. These RNAs, which
mimic viral RNA, are recognized by the cell and used to splice all
matching mRNA transcripts (Figure 2).
Figure 2. Pathway of siRNA-mediated gene downregulation. From: http://www5.appliedbiosystems.com/
tools/pathway/pathway.php?pathway=siRNA%20Pathway
b
mCherry Emission at 620 nm (RFU)
•
a
Obtained EGFP siRNA expression
vector as a template for PCR
mCherry Emission at 620 nm (RFU)
•
Identification of unique
peptide sequence on
hFGFR1 IIIc
Splicing of the fibroblast growth factor receptor 1 (FGFR1) αexon results in FGFR1 splice variant IIIc (FGFR1 IIIc) (Figure 1).
FGFR1 IIIc has been shown to be over-expressed in several
different cancer types including GBM(1)(3).
z
IgY/siRNA vector Linkage
Isolation of
mRNA from
U87MG
b
Figure 9. Plasmid map of pReceiever-M51.
Open reading frame (ORF) contains
FGFR1 IIIc and EGFP genes.
mCherry Emission at 620 nm (RFU)
•
IgY Production
pR
-M
51
•
GBM expression of FGFR1 IIIc mRNA
EGFP Emission at 535 nm (RFU)
•
Glioblastoma multiforme (GBM) is a highly aggressive and
invasive form of brain cancer. Several growth factors and growth
factor receptors have been implicated in increased proliferation,
migration, and survival of GBM cells(1).
EGFP Emission at 535 nm (RFU)
•
a
pE
G
FP
Introduction
b
1. U87MG cells express FGFR1 IIIc mRNA.
2. IgY was successfully produced and affinity purified.
• Believed to bind hFGFR1 IIIc, but need more support.
3. IgY/DNA conjugation appears to have been successful.
4. No significant change in EGFP expression levels for all groups
possibly indicates that the EGFP turn-over time is too long for
down-regulation to be detected or treatment concentrations were
too low.
References
1) Allerstorfer, S., Sonvilla, G., Fischer, H., Spiegl-Kreinecker, S., Gauglhofer, C., Setinek, U., Czech, T., Marosi, C., Buchroithner, J., Pichler, J., Silye, R., Mohr, T., Holzmann, K., Grasl-Kraupp, B., Marian, B.,
Grusch, M., Fischer, J., Micksche, M. and Berger, W. 2008. FGF5 as an oncogenic factor in human glioblastoma multiforme: autocrine and paracrine activities. Oncogene 27(30):4180-90.
2) George, J., Naren L. Banik, N. L., and Ray, S. K. 2009. Bcl-2 siRNA augments taxol mediated apoptotic death in human glioblastoma U138MG and U251MG cells. Neurochemical Research 34(1): 66-7
Expected
Shift
3) Karajannis, M. A., Vincent, L., DiRenzo, R., Shmelkov, S. V., Zhang, F., Feldman, E. J., Bohlen, P., Zhu, Z., Sun, H., Kussie, P., and Rafii, S. 2006. Activation of FGFR1b signaling pathway promotes survival,
migration and resistance to chemotherapy in acute myeloid leukemia cells. Leukemia 20(6):979-86.
4) Plotnikov, A. N., Hubbard, S. R., Schlessinger, J., and Mohammadi, M. 2000. Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand receptor specificity. Cell 101(4): 413-24
5) Suzuki, N. and Lee, Y. C. 2003. Site-specific N-glycosylation of chicken serum IgG. Glycobiology 14(3): 275-92.
6) Song, E., Zhu, P., Lee, S. K., Chowdhury, D., Kussman, S., Dykxhoorn, D.M., Feng, Y., Palliser, D., Weiner, D.B., Shankar, P., Marasco, W.A., and Lieberman, J. 2005. Antibody mediated in vivo delivery of small
interfering RNAs via cell-surface receptors. Nature Biotechnology 23(6):709-17.
1 Kb
700 bp
Figure 6. Affinity purified antibody concentrations of selected 1 ml elution
fractions. (a) Fraction #12 and (b) Fraction #13 contained highest concentration
of eluted IgY.
Figure 8. Shift assay analyzing conjugate formation. Samples from
each stage of the conjugation process were run on a 0.8% agar gel. (a)
Coomassie blue protein stain of the wells. (b) Gelstar Green DNA stain
of the entire gel.
Acknowledgements
I would like to thank Dr. Thompson for his constant support, advice, and insight throughout the course of this project and beyond. I would like to acknowledge
SEPACS for providing a travel grant. Finally, I would like to extend my regards to the Biological Sciences Department at York College of Pennsylvania.