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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Cellular Uptake of Synthetic VirusesCellular Uptake of Synthetic Viruses

NUCLEUSNUCLEUS

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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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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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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

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+8

+5

+6

+7

EGFREGFR-targeting-targeting

Renca-EGFRRenca-EGFR

Non Non shieldedshielded

StableStableshieldedshielded

Reversibly Reversibly shieldedshielded

log

RL

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+5

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+7

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pH-reversible PEG Shield: pH-reversible PEG Shield: in vivoin vivo

DNA

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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

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rgan

non shielded stable shielded reversibly shielded

G. Walker et al, Mol. Ther. 2005

Therapeutic Applications ....Therapeutic Applications ....

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

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0 5 10 15 20 25days after tumor setting

tum

orvo

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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