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Targeted delivery of nucleic acids to tumoursTargeted delivery of nucleic acids to tumours
Manfred OgrisManfred Ogris
Pharmaceutical Biotechnology Pharmaceutical Biotechnology Department of PhamacyDepartment of PhamacyUniversity of MunichUniversity of Munich
05-09-200605-09-2006
DNA TherapeuticsDNA Therapeutics
““... ... DNADNA acts as acts as therapeutic prodrugtherapeutic prodrug which which
can be delivered by a variety of technologies. can be delivered by a variety of technologies.
The The body cellsbody cells read the genetic information read the genetic information
carried by the DNA and carried by the DNA and translatetranslate it into the it into the
actual drugactual drug (usually a protein) …. “ (usually a protein) …. “
Cancer Gene TherapyCancer Gene Therapy
Therapeutic concepts:Therapeutic concepts:
• Direct killingDirect killing of tumor cells (e.g. enzyme/prodrug)of tumor cells (e.g. enzyme/prodrug)
• BlockBlock cell cycle cell cycle/induce/induce apoptosisapoptosis
• Inhibit tumor cellInhibit tumor cell metastasis/migrationmetastasis/migration
• Inhibit/revert tumorInhibit/revert tumor neoangiogenesisneoangiogenesis
• Stimulate anti-tumorStimulate anti-tumor immunityimmunity (cytokines, vaccines)(cytokines, vaccines)
Barriers for systemic gene deliveryBarriers for systemic gene delivery
plasma proteins
DNA
fenestration
extracellular matrix
target cells
blood cells
tight junction
DNADNA
Polyplexes: Towards ‘Synthetic Viruses’Polyplexes: Towards ‘Synthetic Viruses’
Kursa 2003 ; Ogris 2003; Boeckle 2005; WalkerKursa 2003 ; Ogris 2003; Boeckle 2005; Walker 20052005
Compacting Compacting PolycationPolycation
++ ++ ++ ++
Endosomal Endosomal releaserelease
** ** **
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Transport Transport DomainDomain
(melittin)(melittin)
++
Non-specific Non-specific interactions:interactions:Non-target cellsNon-target cellsBloodBlood
Shielding Shielding AgentAgentPEGPEG
++
Receptor Receptor mediated mediated uptake into uptake into target cellstarget cells
TargetingTargetingLigandLigand
(Tf, EGF)(Tf, EGF)
++
Bioresponsive Bioresponsive DisassemblyDisassembly
**
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****
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********
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******
** ******** ** **
**
Cellular Uptake of Synthetic VirusesCellular Uptake of Synthetic Viruses
NUCLEUSNUCLEUS
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Endosomal Release
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Endocytosis* *
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Endosome
NuclearTrafficking
DNAsiRNAsiRNA
HUH-7 cells / GFP-tagged tubulinGFP-tagged tubulin
Vesicle, virusVesicle, virus
Single Particle MicroscopySingle Particle MicroscopyDNA/PEI PolyplexesDNA/PEI Polyplexes
+
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***
** *
* *
**** *
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**
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*
*
****+
*
*+
+++
+
+ + ++ +
+++ +
In vivo Luciferase Imaging In vivo Luciferase Imaging
PEI /DNAPEI /DNA complexes (unshielded, positive charge)
gene expression in lungs and tail (application site)
I. Hildebrandt et al. Gene Ther. 2003
+
** ** **
***
** *
* *
**** *
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**
**
*
*
****+
*
*+
+++
+
+ + ++ +
+++ +
EGF Polyplexes for Systemic TargetingEGF Polyplexes for Systemic Targeting
Wolschek 2002; Prasmickaite, 2003
mouse 1 – pCMVLucmouse 1 – pCMVLucmouse 2 – controlmouse 2 – control
2 days
EGFEGFPEG
HUH-7 s.c. HUH-7 s.c. SCID miceSCID mice
0.8-1 µm<+4 mV
Cellular Uptake of Synthetic VirusesCellular Uptake of Synthetic Viruses
NUCLEUSNUCLEUS
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Endosomal Release
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DNA siRNAsiRNApICpIC
Endosomal Escape: Endosomal Escape: Membrane-active Polymers or/and PeptidesMembrane-active Polymers or/and Peptides
Endosomolytic polymers: e.g. polyethylenimine (PEI)Endosomolytic polymers: e.g. polyethylenimine (PEI) proton sponge effect,proton sponge effect, discovered by J.P. Behr (Boussif et al. 1995)discovered by J.P. Behr (Boussif et al. 1995)
DD-melittin, N-terminal -melittin, N-terminal
PEIPEI
Plank 1992 ; Ogris 2001; Boeckle 2005Plank 1992 ; Ogris 2001; Boeckle 2005
SourceSource peptidepeptide optimal pHoptimal pH
Influenza virusInfluenza virus HA-2HA-2 acidicacidic
Bee venomBee venom MelittinMelittin neutral
INF6: GLFG AIEG FIEN GWEG WEGn IDGW WYGG CG
Melittin-SH: CIGA VLKV LTTG LPAL ISWI KRKR QQ
Endosomolytic peptides:Endosomolytic peptides:(as alternative or in combination)(as alternative or in combination)
EGFR-targeted Poly IC:EGFR-targeted Poly IC: Intracellular DistributionIntracellular Distribution
A. Shir, M. Ogris, E. Wagner, A. Levitzki A. Shir, M. Ogris, E. Wagner, A. Levitzki PLOS MedicinePLOS Medicine 2006 2006
+Mel
-Mel
+Mel
-Mel
Fluorescein -labeled polyIC
pH-Specific Endosomal PEG DeshieldingpH-Specific Endosomal PEG Deshielding
Endosomalacidification
extra - intra- cellular
+
++
+
pH responsive linker- stable at pH 7, labile at pH 5.5
NH
NN C
H
PEG polyplex deshieldingpH 5: less than 1 hr / 37°CpH 7: no deshielding in 6 hrs /37°C
Bioreversible hydrazone linker
++++
PC-PEG
++++
++++
DNA
G. Walker et al, Mol. Ther. 2005
DNA
Ligand plus pH-reversible PEG Shield Ligand plus pH-reversible PEG Shield
TfRTfR-targeting-targeting
K562K562
TfTf
G. Walker et al, Mol. Ther. 2005
or EGFor EGF
log
RL
U
+8
+5
+6
+7
EGFREGFR-targeting-targeting
Renca-EGFRRenca-EGFR
Non Non shieldedshielded
StableStableshieldedshielded
Reversibly Reversibly shieldedshielded
log
RL
U +4
+5
+6
+7
**
**
**
**
******
**
**
**
******
**
pH-reversible PEG Shield: pH-reversible PEG Shield: in vivoin vivo
DNA
** **
** **
** **
**
**
**** ** ******EGFEGF
EGFR-targeted reversibly shielded small polyplexes EGFR-targeted reversibly shielded small polyplexes mediate highest gene expression in sc HUH7 tumorsmediate highest gene expression in sc HUH7 tumors
1E+3
1E+4
1E+5
1E+6
1E+7
spleen kidney liver heart lung tumor
log
RL
U/ o
rgan
non shielded stable shielded reversibly shielded
G. Walker et al, Mol. Ther. 2005
Cancer gene therapy: GDEPTCancer gene therapy: GDEPT
CPA: Chemotherapy vs. GDEPTCPA: Chemotherapy vs. GDEPT
P450/CPA Gene TherapyP450/CPA Gene Therapy
• Human Hepatoma in Human Hepatoma in SCID miceSCID mice
0
200
400
600
800
1000
1200
1400
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0 5 10 15 20 25days after tumor setting
tum
orvo
lum
e
control
CPA
CYP/CPA
bGal/CPA
Synthetic RNA: a therapeutic approachSynthetic RNA: a therapeutic approach
EGF Receptor-Targeted Synthetic Double-
Stranded RNA Eliminates Glioblastoma, Breast
Cancer, and Adenocarcinoma Tumors in Mice
A. Shir, M. Ogris, E. Wagner, A. LevitzkiA. Shir, M. Ogris, E. Wagner, A. Levitzki
PLoS Medicine, 2006PLoS Medicine, 2006
dsRNA Complex FormationdsRNA Complex Formation
dsRNA
Targeting domain: EGF(select desired interaction)
Compacting domain: PEI
Shielding domain: PEG(block undesired interactions)
Endosomal domain: MelittinEndosomal domain: Melittin (intracellular release)(intracellular release)
++
++ ++dsRNA/ PEI
Targeted Poly (Targeted Poly (I•I• C) Eliminates C) EliminatesGlioblastoma Tumors From Mouse BrainsGlioblastoma Tumors From Mouse Brains
20 days tumors20 days tumors10 days tumors10 days tumors
No treatmentNo treatment No treatmentNo treatment conjugate controlconjugate control pIC/PEI-PEG-EGF+MelpIC/PEI-PEG-EGF+Mel
SurvivalSurvivalof mice (%)of mice (%)
0
100
0 20 40 60 80 100 120 140 160 180 200
Improve targeting Improve targeting - Overcome systemic barriersOvercome systemic barriers- Target tumor cells/tumor endotheliumTarget tumor cells/tumor endothelium
Enhance efficiencyEnhance efficiency- intracellular release by membrane-active peptidesintracellular release by membrane-active peptides- optimize into bioresponsiveoptimize into bioresponsive systems (e.g. PEG deshielding)systems (e.g. PEG deshielding)
Choice of therapeutic conceptChoice of therapeutic concept- Combined effects with chemotherapeuticsCombined effects with chemotherapeutics- Induction of immune responsesInduction of immune responses
Key Issues for Cancer Gene TherapyKey Issues for Cancer Gene Therapy
Ernst WagnerErnst Wagner
Sabine BoeckleSabine BoeckleJulia FahrmeirJulia FahrmeirCarolin FellaCarolin FellaKatharina v. Katharina v. GersdorffGersdorffJulia Klöckner Julia Klöckner Veronika KnorrVeronika KnorrNicole TietzeNicole Tietze
Collaborators:Collaborators:LMULMUChristoph Christoph BräuchleBräuchleKarla de Bruin Karla de Bruin Ralf BausingerRalf Bausinger
Jerusalem UJerusalem UAlexei ShirAlexei ShirAlexander LevitzkiAlexander Levitzki
EC FP6 ‘GIANT‘ DFG SFB486 ‘NanoMan‘ EC FP6 ‘GIANT‘ DFG SFB486 ‘NanoMan‘ Sanders Stiftung Dr. Mildred Scheel- Sanders Stiftung Dr. Mildred Scheel-
StiftungStiftung
Arkadi Zintchenko, postdoc Arkadi Zintchenko, postdoc Greg Walker, postdocGreg Walker, postdoc