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Targeted delivery of nucleic acids to tumours
Manfred Ogris
Pharmaceutical Biotechnology
Department of Phamacy
University of Munich
05-09-2006
DNA Therapeutics
“... DNA acts as therapeutic prodrug which can
be delivered by a variety of technologies.
The body cells read the genetic information
carried by the DNA and translate it into the
actual drug (usually a protein) …. “
Cancer Gene Therapy
Therapeutic concepts:
• Direct killing of tumor cells (e.g. enzyme/prodrug)
• Block cell cycle/induce apoptosis
• Inhibit tumor cell metastasis/migration
• Inhibit/revert tumor neoangiogenesis
• Stimulate anti-tumor immunity (cytokines, vaccines)
Barriers for systemic gene delivery
tight junction fenestration
plasma
proteins
DNA
blood
cells
extracellular
matrix
target cells
Polyplexes: Towards ‘Synthetic Viruses’
Non-specific
interactions:
Non-target cells
Blood
++
++
*
++
++ ++
++
++
DNA
Shielding
Agent
PEG
Targeting
Ligand
Transport
Domain
(Tf, EGF) (melittin)
** **
*
*
**
** * ***
**** ***
*
*
Bioresponsive
Disassembly
Receptor
mediated
uptake into
target cells
*
* * * **
** * ** ** ** *
*
*** ** *****
** *
*
Compacting
Polycation
Endosomal
release
Kursa 2003 ; Ogris 2003; Boeckle 2005; Walker 2005
Cellular Uptake of Synthetic Viruses
*
** * *
*
** * * ** ****
*
*
**** *****
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**** *****
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Endocytosis
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* * * **
*
** * * ** ** *
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***** *****
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*
DNA
siRNA
NUCLEUS
Nuclear
Trafficking
Endosome
*
* * * **
*
** * * ** ** *
*
***** *****
** *
*
*
** * *
*
** * * ** ****
*
*
**** *****
** *
Endosomal
Release
*
Single Particle Microscopy
DNA/PEI Polyplexes
*
Vesicle, virus
+
+* *+**+*
+*
* * ** *+
* ** *+
++
* *+
*
+*+** *+**+
+
*** * **
+
HUH-7 cells / GFP-tagged tubulin
In vivo Luciferase Imaging
+
+* *+**+*
+*
* * ***+
* ** *+
++
* *+
*
+*+** *+**+
*
*** * *+
+
*
PEI /DNA complexes (unshielded, positive charge)
gene expression
in lungs
and tail
(application site)
I. Hildebrandt et al. Gene Ther. 2003
EGF Polyplexes for Systemic Targeting
HUH-7 s.c.
SCID mice
0.8-1 µm
<+4 mV
PEG
EGF
2 days
mouse 1 – pCMVLuc
mouse 2 – control
Wolschek 2002; Prasmickaite, 2003
Cellular Uptake of Synthetic Viruses
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** * *
*
** * * ** ****
*
*
**** *****
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*
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** * * ** ****
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**** *****
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***** *****
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DNA
NUCLEUS
siRNA
pIC
*
* * * **
*
** * * ** ** *
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***** *****
** *
*
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** * *
*
** * * ** ****
*
*
**** *****
** *
Endosomal
Release
*
Endosomal Escape:
Membrane-active Polymers or/and Peptides
Endosomolytic polymers: e.g. polyethylenimine (PEI)
proton sponge effect, discovered by J.P. Behr (Boussif et al. 1995)
Endosomolytic peptides:
D-melittin, N-terminal
(as alternative or in combination)
Source
peptide
optimal pH
Influenza virus
HA-2
acidic
INF6: GLFG AIEG FIEN GWEG WEGn IDGW WYGG CG
Melittin-SH: CIGA VLKV LTTG LPAL ISWI KRKR QQ
Bee venom
Melittin
Plank 1992 ; Ogris 2001; Boeckle 2005
neutral
PEI
EGFR-targeted Poly IC:
Intracellular Distribution
+Mel
+Mel
Fluorescein labeled polyIC
-Mel
-Mel
A. Shir, M. Ogris, E. Wagner, A. Levitzki PLOS Medicine 2006
pH-Specific Endosomal PEG Deshielding
extra -
intra- cellular
Bioreversible hydrazone linker
DNA
N
++++
N
Endosomal
acidification
++++
+
+
+
+
pH responsive linker
- stable at pH 7, labile at pH 5.5
G. Walker et al, Mol. Ther. 2005
C
H
++++
PC-PEG
N
H
PEG polyplex deshielding
pH 5: less than 1 hr / 37°C
pH 7: no deshielding in 6 hrs /37°C
Ligand plus pH-reversible PEG Shield
** *
*
* DNA
*
*
*
**
* * *
*
Tf
log RLU
+7
+6
TfR-targeting
+5
K562
+4
Non
Stable Reversibly
shielded shielded shielded
or EGF
log RLU
+8
+7
+6
+5
G. Walker et al, Mol. Ther. 2005
EGFR-targeting
Renca-EGFR
pH-reversible PEG Shield: in vivo
*
EGF
1E+7
log RLU/ organ
** *
**
* DNA
*
**
* *
* *
EGFR-targeted reversibly shielded small polyplexes
mediate highest gene expression in sc HUH7 tumors
1E+6
1E+5
1E+4
1E+3
spleen
kidney
non shielded
G. Walker et al, Mol. Ther. 2005
liver
heart
stable shielded
lung
tumor
reversibly shielded
Therapeutic Applications ....
Cancer gene therapy: GDEPT
CPA: Chemotherapy vs. GDEPT
P450/CPA Gene Therapy
• Human Hepatoma in
SCID mice
1600
control
1400
CPA
1000
CYP/CPA
800
bGal/CPA
tumorvolume
1200
600
400
200
0
0
5
10
15
20
days after tumor setting
25
Synthetic RNA: a therapeutic approach
EGF Receptor-Targeted Synthetic DoubleStranded RNA Eliminates Glioblastoma, Breast
Cancer, and Adenocarcinoma Tumors in Mice
A. Shir, M. Ogris, E. Wagner, A. Levitzki
PLoS Medicine, 2006
dsRNA Complex Formation
Targeting domain: EGF
(select desired interaction)
++
++
Compacting domain: PEI
++
dsRNA
Shielding domain: PEG
(block undesired interactions)
Endosomal domain: Melittin
(intracellular release)
dsRNA
/ PEI
Targeted Poly (I• C) Eliminates
Glioblastoma Tumors From Mouse Brains
20 days tumors
10 days tumors
No treatment
No treatment
conjugate control
pIC/PEI-PEG-EGF+Mel
100
Survival
of mice (%)
0
0
20
40
60
80
100
120
140
160
180
200
Key Issues for Cancer Gene Therapy
Improve targeting
- Overcome systemic barriers
- Target tumor cells/tumor endothelium
Enhance efficiency
- intracellular release by membrane-active peptides
- optimize into bioresponsive systems (e.g. PEG deshielding)
Choice of therapeutic concept
- Combined effects with chemotherapeutics
- Induction of immune responses
Arkadi Zintchenko, postdoc
Greg Walker, postdoc
Ernst Wagner
Sabine Boeckle
Julia Fahrmeir
Carolin Fella
Katharina v.
Gersdorff
Julia Klöckner
Veronika Knorr
Nicole Tietze
Collaborators:
LMU
Christoph Bräuchle
Karla de Bruin
Ralf Bausinger
Jerusalem U
Alexei Shir
Alexander Levitzki
EC FP6 ‘GIANT‘
Sanders Stiftung
DFG SFB486 ‘NanoMan‘
Dr. Mildred Scheel-Stiftung