Final Dissertation - Gene silencing of a receptor-targeted bioconjugate with enhanced siRNA loading capacity
1. UNIVERSITÀ DEGLI STUDI DI PERUGIA
Dipartimento di Scienze Farmaceutiche
Corso di Laurea Magistrale in Farmacia
UNIVERSITY OF VIENNA
Department of Medicinal Chemistry
Gene silencing of a receptor-targeted bioconjugate with
enhanced siRNA loading capacity
Laureanda Relatori
Marta Marinucci Prof. Antimo Gioiello
Dr. Johannes Winkler
Anno Accademico 2014-2015
2. RNAi is a fundamental pathway in eukaryotic cells by which a sequence-specific RNA
molecule, named small interfering RNA (siRNA) is able to target and cleave complementary
mRNA, thus causing selective gene expression inhibition [1]
RNA interference and gene silencing
RNAi is fundamental for:
• Cell growth
• Tissue differentiation
• Heterochromatin formation
• Cell proliferation
siRNAs are double-stranded molecules of RNA with 3’ overhangs at each end that can
“interfere” with proteins translation [2]
1: Elbashir S.M. et al. (2001). Nature 411, 494-498
2: Dornseifer S. et al. (2015). Nucleic Acid Res. 43(22), 10623-10632
3. RNAi pathways and mechanisms
• Molecules of double-stranded RNA (dsRNA) are
cleaved by the enzyme Dicer into siRNA
• siRNA is incorporated into RISC (RNA-induced
silencing complex), that includes Argonaut 2
• Argonaut 2 unreels siRNA and the sense strand is
cleaved
• Activated RISC, with the antisense strand of siRNA,
selectively degrades mRNA [3]
3: Bernstein E. et al. Nature 409, 363-366
4. Hurdles of the therapy are associated with the need of the siRNAs to reach the cytoplasm,
overcoming many obstacles:
• Absorbance barriers
• Endothelial barriers
• Cell membrane barriers
• Degradation by exonucleases
• Endosomal escape
• Elimination by renal filtration
RNAi dysfunction is linked to cardiovascular diseases, neurological disorders and many
types of cancer[4]
RNAi dysfunction and therapeutic potentiality of synthetic siRNA
4: Dorneseifer S. et al. (2015). Nucleic Acid Res 43(22), 10623-10632
Development of synthetic siRNA as chemical tools and therapeutic agents
5. EpCAM
• Polymeric or lipidic vectors to overcome lysosomal degradation
• Membrane fusion events to alter confirmation in lysosomes
• Chemical modifications of siRNAs to avoid nuclease degradation
• Conjugation with small molecules to improve bio-distribution
• Delivery systems to facilitate uptake into target tissues[5]
Strategies to increase siRNAs therapeutic efficacy
Cellular targetTarget cell Carrier Bioactive compound
DARPin siRNATumour cell
5: Kathryn A. et al. (2009). Nature Rev 8, 129-130
6. • Trans-membrane glycoprotein over-expressed in tumours cells
• Tumour suppressor or oncogene depending on the microenvironment[5]
Epithelial cell adhesion molecule (EpCAM): the cellular target
ONCOGENE
- Inhibits molecular adhesion
- Promotes cell mobility,
proliferation and metastasis
formation
TUMOUR SUPPRESSOR
- Cell adhesion molecule
- Prevents the invasion
5: Van der Gun B.T.F et al. (2010). Carcinogenesis 31 1913-1921
7. Designed Ankyrin repeat proteins (DARPins) as carrier
• Class of non-immunoglobulin binding proteins
• Designed via sequence alignments of natural Ankyrin repeat motifs [6]
- High affinity
- High stability
- Small size
- High expression level
- Rapidly selected from a library to bind desired targets
6: Winkler J. et al. (2009). Mol. Cancer Ther. 8(9)
8. Produce, analyse and evaluate gene silencing effect for multiplexed DARPin-conjugate of
dT18 oligonucleotide in EpCAM positive (MDA-MB-468) and negative (HEK293) cell lines
AIMS OF THE WORK
Ec4-C-dT18 3’ Sulfo-SMCC Ec4-C-dT18 3’ SPDP
dT18
spacer linker DARPin
dT18
spacer linker DARPin
9. Produce, analyse and evaluate gene silencing effect for multiplexed DARPin-conjugate of
dT18 oligonucleotide in EpCAM positive (MDA-MB-468) and negative (HEK293) cell lines
AIMS OF THE WORK
1
• Solid phase synthesis, analysis and purification of the dT18
oligonucleotide
2
• Oligonucleotide conjugation with Sulfo-SMCC or SPDP for
the linkage with 4-arm spacer
3
• Binding of the complex oligonucleotide-linker to DARPin
4
• In vitro activity evaluation by Dual-Luciferase Reporter Assay
15. Binding of the complex oligonucleotide-linker to DARPin
4
1
2
3
• Removal of non reacted dT18 by nickel-NTA
Ni-NTA resin Ec4-C-dT18 3’Sulfo-SMCC
+
Ni-Ec4-C-dT18 3’Sulfo-SMCC
• Purification with complementary-biotinylated DNA• Purity monitoring with SDS-PAGE
3 2 1
1: Protein-Ladder
2: Ec4-Cys
3: Conjugate
16. • Removal of non reacted dT18 by nickel-NTA
• Purification with complementary-biotinylated DNA
• Final purification with SDS-PAGE
dT18 3’ SPDP Ec4-C-dT18 3’ SPDP
Nucleophilic substitution
Binding of the complex oligonucleotide-linker to DARPin
4
1
2
3
25°, overnight, GuHCl
17. EpCAM positive
cell
EpCAM negative
cell
+ pEGFP-Gluc (Firefly luciferase)
pCMV-GLuc (Renilla luciferase)
and Lipofectamine
+ anti-Luc siRNA
and Lipofectamine
luminescence attenuationhigh luminescence
4
1
2
3
In vitro activity evaluation
high luminescence luminescence attenuation
18. DUAL-LUCIFERASE REPORTER ASSAY
• Firefly luciferase is the experimental reporter
• Renilla luciferase is the internal control
In vitro activity evaluation
4
1
2
3
19. Luciferase assay in MDA-MB-468 cells
0.3 μl LF/pmol siRNA guarantees
efficient downregulation at 10 and 1
pmol siRNA
The reduction of siRNA/well
concentration is associated with a
reduced down-regulation
Conditions:
• siRNA dilutions: 10 pmol/well, 1 pmol/well, 0.1 pmol/well, 0.01 pmol/well
• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA, 0.03 μl/pmol siRNA,
0.003 μl/pmol siRNA
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
*
AIMS: establish the minimum amount of LF that guarantees efficient transfection and the siRNAs
concentration to allow down-regulation.
20. The gene silencing effect is smaller
than in MDA-MB-468 cells
Even if the higher amount of
LF/pmol siRNA is used, the extent
of down regulation is lower
Luciferase assay in HeLa cells LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
Conditions:
• siRNA dilutions: 10 pmol/well, 1 pmol/well, 0.1 pmol/well, 0.01 pmol/well
• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA, 0.03 μl/pmol siRNA,
0.003 μl/pmol siRNA
*
21. siRNA
siRNA
Luciferase assay
siRNA
4-arm spacer
DARPinEpCAM
siRNA
In order to increment siRNA cellular uptake a 4-arm spacer was prepared and used:
AIM: produce a delivery system in order to increment cellular uptake and endosomal release using the
minimum amount of LF and increasing the siRNAs quantities.
22. Luciferase assay in MDA-MB-468 cells siRNA
Sulfo
SMCC
SPDP
4-arm spacer increases the down-regulation of luciferase gene when compared with
the siRNA monomer
The trend of the down-regulation is likely to be concentration dependent
The higher amount of LF/pmol siRNA is associated with a better transfection
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
Conditions:
• siRNA dilutions: 40 pmol/well, 20 pmol/well, 4 pmol/well
• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA
*
0.075 μl/pmol siRNA0.3 μl/pmol siRNA
*
23. Higher is the concentration of 4-arm spacer, greater is the down regulation
The higher amount of LF/pmol siRNA is associated with better transfection
Luciferase assay in HeLa cells siRNA
SPDP
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
Conditions:
• siRNA dilutions: 40 pmol/well, 20 pmol/well, 4 pmol/well
• LF amounts: 0.3 μl/pmol siRNA, 0.075 μl/pmol siRNA
0.3 μl/pmol siRNA 0.075 μl/pmol siRNA
24. 1: 4-arm spacer+3x siRNA+ conjugate+LF
2: 4-arm spacer+3x siRNA+ LF
3: 4-arm spacer+3x siRNA+conjugate
4: 4-arm spacer+3x siRNA
5: siRNA (positive control) + LF
4-arm spacer seems to be more effective
in gene silencing than siRNA as monomer
The full assembly resulted in a slight
target down regulation
4-arm spacer with conjugate and without
LF did not produce significant down
regulation confirming the role of LF in
transfection
Luciferase assay in MDA-MB-468 cells
Conditions:
• 4-arm spacer dilutions: 10 pmol/well, 1 pmol/well
• LF amounts: 0.001 μl/pmol siRNA
• 4-arm spacer was used alone or with Sulfo-SMCC with or without LF
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
MDA-MB-468
25. • dT18 oligonucleotide was efficaciously synthesized
• DNA oligonucleotides dT sequences were successfully attached to an EpCAM-specific
DARPin
• Analytical data clearly showed successful removal of unreacted components and high
substance purity
• The use of conjugate attached to a 4-arm spacer seems to increase the cargo-uptake
per receptor internalization though no significant gene silencing effect was found
• The oligonucleotide complexation can be a useful strategy to produce stronger effects
than standard siRNA-transfection
CONCLUSIONS…
• More efficient methods for endosomal escape need to be investigated
• Use of different modified oligonucleotides will be explored
…and FUTURE PROSPECTIVES
27. The use of conjugates is not associated
with higher down regulation if compared
with 4-arm spacer without conjugate
LF is essential for transfection
Luciferase assay in MDA-MB-468 cells
Conditions:
• 4-arm spacer dilutions: 333 pmol/well, 167 pmol/well,33 pmol/well
• No LF amounts was used
• 4-arm spacer was used alone or with Sulfo-SMCC or with SPDP
MDA-MB-468
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
28. The use of conjugate is not associated
with higher down regulation if compared
with 4-arm spacer alone
LF is necessary for knock-down
Luciferase assay in MDA-MB-468 cells
Conditions:
• 4-arm spacer dilutions: 10 pmol/well, 1 pmol/well
• LF amounts: 0.001 μl/pmol siRNA
• 4-arm spacer was used alone or with Sulfo-SMCC with or without LF
MDA-MB-468
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
29. The use of conjugate is not associated
with higher down regulation if
compared with 4-arm spacer alone
LF is necessary for knock-down
Luciferase assay in MD-MB-468 cells
Conditions:
• 4-arm spacer dilutions: 10 pmol/well, 1 pmol/well
• LF amounts: 0.005 μl/pmol siRNA
• 4-arm spacer was used alone or with Sulfo-SMCC with or without LF
MDA-MB-468
LF
4-arm
spacer
EpCAM
+cells
siRNA
Sulfo
SMCC
SPDP
![RNAi is a fundamental pathway in eukaryotic cells by which a sequence-specific RNA
molecule, named small interfering RNA (siRNA) is able to target and cleave complementary
mRNA, thus causing selective gene expression inhibition [1]
RNA interference and gene silencing
RNAi is fundamental for:
• Cell growth
• Tissue differentiation
• Heterochromatin formation
• Cell proliferation
siRNAs are double-stranded molecules of RNA with 3’ overhangs at each end that can
“interfere” with proteins translation [2]
1: Elbashir S.M. et al. (2001). Nature 411, 494-498
2: Dornseifer S. et al. (2015). Nucleic Acid Res. 43(22), 10623-10632](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)