Munich - 17th Annual Meeting German Society for Gene Therapy ...

In cooperation with 

17 th Annual Meeting of the 

German Society for Gene Therapy 

(DG-GT e.V.) 

The German Society of Virology, Study-Group 

«Viral Vectors and Gene Therapy» 

The DFG Research Priority Program 1230 

«Mechanisms of Gene Vector Entry and Persistence» 

The British Society for Gene Therapy 

Nanosystems Initiative Munich 

Helmholtz Zentrum München 

October 7 – 9, 2010 

LMU Campus Großhadern 

Munich

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Imprint 

Editors: 

Manfred Ogris 

Verena Brand 

Pirmin Burth 

Cover Layout: 

Pirmin Burth 

17 th Annual Meeting of the German Society for Gene Therapy (DG-GT e.V.), October 7-9, 2010, 


Table of Content 

Welcome 3 

General Information 4 

Organizing Committee 6 

Approach and Maps 6 

Meeting Program 9 

Sponsors 15 

Abstracts 16 


Munich 

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Welcome 

Dear members and friends of the German Society for Gene Therapy, 

on behalf of the German Society for Gene Therapy we cordially welcome you to our 17 th Annual 

Meeting held at the Chemistry and Pharmacy Campus of the University of Munich. The meeting is 

organized in conjunction with the British Society for Gene Therapy, the Study Group Viral Vectors of 

the German Society for Virology, the Research Priority Program SPP1230, the Nanosystems Initiative 

Munich (NIM) and the Munich Helmholtz Society. 

We have sought to put together an exciting and diversified program presenting state-of-the-art 

advances in the field of gene therapy, a field that has considerably evolved within recent years. The 

first day (Thursday, Oct., 7 th ) is dedicated to the Educational Sessions. Out of the portfolio of areas 

contributing to the progress of gene therapeutical approaches, we have chosen this year viral and 

non-viral vector design, tumor imaging and clinical aspects of gene therapy. The meeting is opened 

with an inaugural talk by our key note lecturer Mark Kay (Stanford University) on Thursday evening 

and is continued on Friday and Saturday with internationally highly recognized experts in areas like 

vector development, cancer gene therapy, treatment of metabolic diseases and others. In addition, 

21 oral presentations were selected out of >100 submitted abstracts. During coffee- and lunch 

breaks, >60 posters are presented. Also the socializing part will be addressed accordingly with a 

Bavarian evening on Thursday offering ‘a saubere Brotzeit’ with classics from the Bavarian cuisine 

and brewery and a social event on Friday evening themed ‘Sports Night’. We hope that you will all 

enjoy the meeting and take the chance to discuss science and to relish Munich. 

Thank you all for coming to Munich! 

Manfred Ogris Hildegard Büning Anja Ehrhardt 

Andrew Baker Wolfgang Hammerschmidt Ernst Wagner 



General Information 

Organization and contact 

PD Dr. Manfred Ogris 

Phone +49 (0)89 2180 77842 

Fax +49 (0)89 2180 77791 

Email: dg-gt2010@cup.uni-muenchen.de 

Butenandtstr. 5-13, 81377 München, Germany 

Venue 

Ludwig-Maximilians-Universität München 

Dekanat der Fakultät für Chemie und Pharmazie 

Butenandtstr. 5-13 

81377 München 

Registration 

The registration desk in the foyer of house F is open on: 

Thursday October 7 th from 12:00 pm to 06:30 pm 

Friday October 8 th from 08:00 am to 06:30 pm 

Saturday October 9 th from 08:00 am to 02:00 pm 

The registration fee includes: 

- access to scientific sessions 

- program and abstract book 

- coffee/tea during the morning and afternoon breaks 

- lunch during the poster sessions at noon 

- Post Oktoberfest event 

Oral Presentations 

All presentations will take place in the Buchner lecture hall (see map). The lecture hall is equipped 

with computers (Windows, NOT Mac) and a projector for PowerPoint presentations. For those 

people who use Mac please bring your own notebook. For those who use Windows please bring 

your presentation on a memory stick. 

There is a possibility to prepare the presentations in the Liebig lecture hall (next to Buchner lecture 

hall). It’s equipped with the same computers and projectors for PowerPoint presentations 

The Liebig lecture hall is open: 

Thursday October 7 th from 01:00 pm to 06:30 pm 

Friday October 8 th from 08:00 am to 10 am, from 1 pm to 06:30 pm 

Saturday October 9 th from 08:00 am to 02:00 pm 



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Posters 

The posters will be displayed in the foyer of house F (see map) from Friday 11:00 am to Saturday 

03:00 pm. 

Poster authors are present at their posters during poster sessions (see program). 

The posters can be handed over at the registration desk on Thursday or should be mounted on 

Friday until 10:30 am and can be removed on Saturday at 03:00 pm. 

Post Oktoberfest Event 

The Post Oktoberfest Event will take place in front of the Buchner lecture hall on Thursday, October 

7 th . 

Speakers Dinner 

The speakers dinner will take place at the Restaurant 181 on the top of the Olympiaturm. There will 

be a shuttle bus from the campus to the restaurant at 07:30 pm (return at 10:30 pm). 

Social Event “Sports Night” 

The social event is NOT included in the registration fee. Please wear your name badge! 

The social event will take place at Primafila in Laim (near Sportpark Nymphenburg) on Friday, 

October 8 th , 08:30 pm. 

Approach: 

There will be a shuttle bus from the campus to the restaurant at 08:00 pm. 

By public transport: 

From the campus take the subway U6 in the direction of Garching Forschungszentrum and get off at 

Holzapfelkreuth. Then take bus 51 in the direction of Olympia Einkaufszentrum and get off at Laim. 

Pass the underground passage and follow Wotanstraße, then turn left and walk along Margarethe- 

Danzi-Straße until you reach the Sportpark Nymphenburg and Primafila. 

You can also go there by S-Bahn and get off at Laim (see map). 



Organizing Committee 

PD Dr Manfred Ogris 

PD Dr Hildegard Büning 

PD Dr Anja Ehrhardt 

Prof Dr Andrew Baker 

Prof Dr Wolfgang Hammerschmidt 

Prof Dr Ernst Wagner 

Approach and Maps 

How to reach us by car 

• From the Nürnberg highway: get onto the Mittlerer Ring (direction Autobahn Lindau), then 

onto the Lindau highway to the exit Blumenau, keep going to Gräfelfing from where 

Würmtalstrasse will lead you to our campus. 

• From the Stuttgart highway: from the end of the highway in Obermenzing turn off to Pasing, 

from Pasing drive in the direction of Gräfelfing, then turn left to Großhadern. 

• From the Salzburg or Garmisch highways: drive onto the "Mittlerer Ring" in the direction of 

Großhadern and Stuttgart, then, in Großhadern turn to Gräfelfing and follow Würmtalstrasse 

which passes by our campus. 

Destination address for navigation system and parking 

Marchioninistraße, 81377 München 

Follow the parking signs at “Klinikum Großhadern” 

By train and public transport 

By IC, EC, ICE to Munich Central Station (München Hauptbahnhof). From there, take the subway U1 

or U2 to Sendlinger Tor. Change to U6 in the direction of Klinikum Großhadern and get off at 

Großhadern. Take the stairway to your left and keep left. You are now on Würmtalstraße. Now you 

can walk along Würmtalstraße until the campus comes into view (large modern buildings) on the left 

(about 10 to 15 minutes) or take the bus 266 or 268 in the direction of Planegg to Waldhüterstraße 



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By plane and public transport 

From the Munich airport take the S-Bahn S8 (or S1) to Marienplatz. Change to the subway U6 in the 

direction of Klinikum Großhadern and get off at Großhadern. Take the stairway to your left and keep 

left. You are now on Würmtalstraße. Now you can walk along Würmtalstraße until the campus 

comes into view (large modern buildings) on the left (about 10 to 15 minutes) or take the bus 266 or 

268 in the direction of Planegg to Waldhüterstraße. 

Ticketing for public transport 

You can use: 

- MVV-3-days ticket for the inner district (=white zone) from the moment of validation until 6 

am on the fourth day (3-Tages-Ticket Innenraum, 12,80€). 

- MVV-1-day ticket for the inner district (=white zone) from the moment of validation until 6 

am the following day (1-Tages-Ticket Innenraum, 5,20€). 

All tickets must be validated once in a blue ticket-cancelling machine before starting with your trip, 

except for tickets from the bus and tram ticket machines. These are already validated. 



Meeting Program 

Educational Session 

THURSDAY OCTOBER 7 

13:00 – 14:30 Educational Session Part 1 

Session Chair: Christian Kupatt, Klinikum Großhadern, LMU Munich 

• Inv 1 Christina Rauschhuber (Department of Virology, Max von Pettenkofer-Institute, 

Munich, Germany) 

Design of Recombinant Adenoviral Vectors for Gene Therapy: Improvements and 

Challenges 

• Inv 2 Martina Anton (Klinikum rechts der Isar, TUM, Munich, Germany) 

Lentiviral vectors in gene and cell therapy approaches 

• Inv 3 Christian Kupatt (Klinikum Großhadern, LMU, Munich, Germany) 

AAV based vectors for cardiovascular diseases 

14:30 – 15:00 Coffee break 

15:00 – 17:00 Educational Session Part 2 

Session Chair: Carsten Rudolph, Von Haunersches Kinderspital, LMU, Munich 

• Inv 4 Carsten Rudolph (Von Haunersches Kinderspital, LMU, Munich) 

Introduction into nonviral gene delivery – physical and chemical delivery methods 

• Inv 5 Christine Spitzweg (Klinikum Großhadern, LMU, Munich) 

Imaging in cancer gene therapy 

• Inv 6 Len Seymour (University of Oxford, UK) 

Gene therapy around the globe 

• Inv 7 Josef Rosenecker (Von Haunersches Kinderspital, LMU, Munich) 

Clinical experience with gene therapy in pediatric patients 

17:00 – 18:00 Coffee break 



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General Meeting 

18:00 – 18:15 Opening Remarks and Opening ceremony 

18:15 – 19:05 Keynote Lecture 

Session Chair: Hildegard Büning, University of Cologne 

• Inv 8 Mark Kay (School of Medicine, Stanford University, USA) 

Gene transfer approaches for gene addition, knockdown and cellular reprogramming 

in vivo 

19:05 – 20:30 Post Oktoberfest Event 

20:30 – 22:30 Speakers dinner 

FRIDAY OCTOBER 8 

9:00 – 11:00 Vector Development 

Session Chairs: Florian Kreppel, University of Ulm 

Ernst Wagner, Department of Pharmacy, LMU, Munich 

• Inv 9 Len Seymour (University of Oxford, UK) 

Delivery issues for oncolytic viruses 

• Inv 10 Andrew Baker (University of Glasgow, Scotland, UK) 

Modification of the adenovirus capsid: integrating virus biology and vector 

engineering 

• Selected Talk from abstracts: 

Or 1: Sigrid Espenlaub (Department of Gene Therapy, University of Ulm, Germany) 

Analysis of intracellular particle trafficking and bioresponsive bonds for Ad vector 

shielding by capsomer specific fluorescent labeling 

Or 2: Jessica Sallach (Clinic I of Internal Medicine and Center for Molecular Medicine 

Cologne, University of Cologne, Germany) 

Primary human keratinocyte-selective AAV2-based targeting vectors 

Or 3: Nadja Noske (Department of Virology, Max von Pettenkofer-Institute, LMU, Munich, 

Germany) 

Development of novel PhiC31 integrase fusion proteins for improving efficacy and 

safety of transgene insertion in therapeutic applications 

Or 4: Keiji Itaka (Division of Clinical Biotechnology, Graduate School of Medicine, The 

University of Tokyo, Japan) 

Biocompatible polyplex nanomicelle for safe and effective gene transfer 

11:00 – 11:15 Coffee Break and Poster Session 



11:15 – 12:00 Vector Development 

• Inv 11 Michael Gait (MRC Laboratory of Molecular Biology, Cambridge, UK) 

Peptide-enhanced delivery of oligonucleotide analogues targeting Duchenne 

muscular dystrophy 


Or 5: Frauke M. Koenig (Department of Chemistry, LMU, Munich, Germany) 

EGF receptor targeting of polyplexes with the short artificial peptide GE11 studied by 

live cell imaging 

12:00 – 13:30 On the Route to Clinical Application 

Session Chair: Christoph von Kalle, National Center for Tumor Diseases , Heidelberg 

• Inv 12 Marinee Chuah (Vesalius Research Center, Leuven, Belgium) 

Emerging transposon technology for gene transfer and IPS applications 

• Inv 13 Simon Waddington (Imperial College London, UK) 

Perinatal gene therapy for lethal genetic diseases 


Or 6: Stylianos Michalakis (Department of Pharmacy – Center for Drug Research, LMU, 

Munich, Germany) 

Restoration of cone vision in the CNGA3–/– mouse model of congenital complete lack 

of cone photoreceptor function 

Or 7: Anna Paruzynski (Department of Translational Oncology, National Center for Tumor 

Diseases (NCT) and German Cancer Research Center (DKFZ), Hannover, Germany) 

High throughput integration site analysis reveals a polyclonal lineage-specific 

integration site distribution in a successful WAS gene therapy trial 

13:30 – 14:30 Lunch Break and Poster Session 

14:30 – 16:00 Gene Therapy 

Session Chairs: Boris Fehse, University Medical Centre Hamburg-Eppendorf 

Manuel Grez, Georg-Speyer-Haus, Frankfurt 

• Inv 14 Robin Ali (Institute of Ophthalmology, London, UK) 

Gene therapy for inherited retinal dystrophies 

• Inv 15 Harald Petry (Amsterdam Molecular Therapeutics (AMT), Netherlands) 

Alipogene Tiparvovec: the first gene therapy for a general metabolic disorder 


Or 8: Teresa Trenkwalder (Klinikum Großhadern, LMU, Munich, Germany) 

Enhanced therapeutic neovascularization via AAV2.9/Thymosin β4: Evidence for a 

Myovascular Crosstalk 

Or 9: Abdullah Cim (King’s College London, School of Medicine, London, UK) 

Nonviral Delivery of the rat PDX1 gene to rat liver for the in vivo transdifferentiation 

of liver cells to pancreatic β-cells 



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16:30 – 17:15 Gene Therapy 

• Inv 16 Manuel Grez (Georg-Speyer-Haus, Frankfurt, Germany) 

Gene therapy of chronic granulomatous disease: the past and the future 


Or 10: Ute Modlich (Hannover Medical School, Hannover, Germany) 

Correction of Mpl deficiency by lentiviral vectors with lineage-specific expression 

17:15 – 18:45 Cancer Gene Therapy 

Session Chairs: Dorothee von Laer, Innsbruck Medical University 

Dirk Nettelbeck, DKFZ Heidelberg 

• Inv 17 Roberto Cattaneo (Mayo Clinic, Minnesota, USA) 

Viruses as cancer therapeutics: three points of attack 

• Inv 18 Caroline Breitbach (Jennerex, Inc., San Francisco, USA) 

Targeted and armed oncolytic poxviruses: a novel multi-mechanistic 

therapeutic class for cancer 


Or 11: Alexander Muik (Georg-Speyer-Haus, Frankfurt am Main, Germany) 

LCMV-Pseudotyped VSV-based systems for treatment of malignant glioma 

Or 12: Jennifer Altomonte (Klinikum rechts der Isar, TU München, Munich, Germany) 

18:45 – 19:00 Award Ceremony DG-GT Forscherpreis 

19:00 – 20:00 DGGT General Assembly 

20:30 – open end Social Event themed “Sports Night” 



SATURDAY OCTOBER 9 

9:00 – 9:45 Cancer Gene Therapy 

• Inv 19 John Bell (Ottawa Hospital Research Institute, Canada) 

Oncolytic vaccinia virus for the treatment of cancer 


Or 13: Geoffrey K. Grünwald (Klinikum Grosshadern, LMU, Munich, Germany) 

An α-fetoprotein promoter driven, conditionally replicating adenovirus that expresses 

the sodium iodide symporter (NIS) for radiovirotherapy of HCC 

9:45 – 10:45 Tumor Biology and (Cancer) Stem Cells 

Session Chair: Axel Schambach, Hannover Medical School 

• Inv 20 Norman Maitland (YCR Cancer Research Unit, University of York, UK) 

Prostate cancer stem cells: a new target for therapy 


Or 14: Kerstin Knoop (Klinikum Grosshadern, LMU, Munich, Germany) 

Tumor stroma-specific NIS gene delivery using mesenchymal stem cells 

Or 15: Axel Schambach (Department of Experimental Hematology, Hannover Medical 

School, Hannover, Germany) 

Monitoring and excising reprogramming factors: a novel lentiviral expression system 

for reprogramming strategies 


11:15 – 13:15 Cancer Immune Therapy and T-cell Therapy 

Session Chair: Wolfgang Uckert, Max Delbrück Center for Molecular Medicine, Berlin 

• Inv 21 Renata Stripecke (Hannover Medical School, Germany) 

Lentiviral vector-induced dendritic cells for melanoma immunotherapy 

• Inv 22 Hinrich Abken (Uniklinikum Köln, Germany) 

Arming immune cells to fight cancer 

• Inv 23 Farzin Farzaneh (Kings College, London, UK) 

Immune gene therapy for acute myeloid leukaemia 


Or 16: Matthias Leisegang (Max-Delbrück-Center for Molecular Medicine, Berlin, Germany) 

MHC-restricted fratricide of recipient lymphocytes expressing transgenic T-cell 

receptors specific for the apoptosis-inhibitor protein survivin 

Or 17: Eliana Ruggiero (German Cancer Research Center and National Center for Tumor 

Diseases, Heidelberg, Germany) 

Integration site analysis of reprogrammed T-cells in a mouse model of T-cell receptor 

gene therapy developing graft versus host disease 



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13:15 – 14:00 Lunch Break and Poster Session 

14:00 – 16:00 Pharmacology and Toxicology 

Session Chair: Hildegard Büning, University of Cologne 

• Inv 24 Anne Galy (Genethon, Evry, France) 

Preclinical safety and efficacy data leading to a clinical trial for the gene therapy of 

Wiskott Aldrich Syndrome 

• Inv 25 Klaus Cichutek (Paul-Ehrlich-Institut, Langen, Germany) 

Lentivector transduction of novel cell targets 


Or 18: Katarina Farkasova (Pharmaceutical Biotechnology, LMU, Munich, Germany) 

Luciferase-based dual bioluminescence imaging of tumor metastases after systemic 

transgene delivery with a synthetic gene carrier 

Or 19: Niels Heinz (Experimental Hematology, Hannover Medical School, Hannover, 

Germany) 

Retroviral and transposon-based Tet-regulated all-in-one vectors with reduced 

background expression and improved dynamic range 

Or 20: Simone J. Scholz (German Cancer Research Center and National Center for Tumor 

Diseases, Heidelberg, Germany) 

High-throughput integration site analysis for vector biosafety assessment in CGD 

gene therapy 

Or 21: Margaret R. Duffy (Glasgow Cardiovascular Research Centre, University of Glasgow, 

Glasgow, UK) 

Modification of the FX serine protease domain ablates HSPG engagement by Ad5-FX 

complexes 

16:00 – 16:15 Poster Award and Concluding Remarks 



Sponsors 

Gold Sponsors Silver Sponsors 

Thanks for the support to our sponsors! 



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Abstracts 

Invited presentations 16 - 27 

Oral presentations 27 - 40 

Poster presentations 41 - 78 

Abstract author index 79 - 82 

All abstract are published in the September 2010 issue of Human Gene Therapy and can be 

downloaded via this link: http://www.liebertonline.com/doi/abs/10.1089/hum.2010.804 



DG-GT 2010 Invited Presentations 

Inv 1 

Design of Recombinant Adenoviral Vectors 

for Gene Therapy: Improvements and 

Challenges 

Christina Rauschhuber 

Department of Virology, Max von Pettenkofer- 

Institute, Munich, Germany 

Over the past decade recombinant adenoviral 

vectors (rAdVs) became one of the most 

prominent gene therapeutic vector systems used 

in preclinical and clinical approaches. Adenoviral 

vectors exhibit several targets for modifications, 

thus they are useful for a variety of applications. 

Different generations of rAdV based on human 

adenovirus serotype 5 were generated starting 

with first and second generation adenoviruses, 

which lack one or two viral genes to more 

sophisticated technologies such as high capacity 

adenoviral vectors (HD-Ad) deleted for all viral 

coding sequences. Besides these types of 

vectors which will be discussed in detail, 

oncolytic adenoviruses exhibiting restricted 

replication in tumor tissues have to be mentioned 

but are not the major focus of this lecture. 

Common for all rAdV is their ability to efficiently 

transduce a broad range of dividing and nondividing 

cells and that they can be produced at 

high titers. However, in contrast to first and 

second generation adenoviruses, HD-AdVs have 

a significantly larger packaging capacity for large 

trangene expression cassettes and they display 

long-term transgene expression in preclinical 

studies. Nevertheless, the biggest challenge in 

adenoviral gene therapy is to circumvent the 

immune response against the vector. As HD-AdV 

lacks all viral coding sequences, there is no 

response to de novo synthesized viral proteins 

but immune response directed against the 

incoming viral capsid components may impair the 

outcome of a gene therapeutic approach. Thus, 

improvements with respect to the administration 

route and methods to modify the surface of the 

virion are the major focus of ongoing research 

and will be discussed within this lecture. 

Session: Educational Session 

Inv 2 

Lentiviral Vectors in Gene and Cell Therapy 

Approaches 

Martina Anton 



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Institute of Experimental Oncology and Therapy 

Research, Technische Universität München, 

Munich, Germany 

Retroviruses are evolutionally optimized for 

transfer of their genetic material into cells and 

integrating their genome into the host cell 

genome. They are thus well suited for long-term 

stable gene expression. Recombinant vectors 

based on gammaretroviruses (MLV) have been 

widely used in the past in preclinical research as 

well as clinical trials. However, during clinical 

trials of SCID, severe side effects occurred 

leading to induction of leukaemia in five cases. 

More recently researchers have tried to harness 

lentiviruses, another genus of the retroviridae and 

developed recombinant vectors based on Human 

Immunodeficiency Virus (HIV-1), Simian 

Immunodeficiency Virus (SIV), Feline (FIV) 

Immunodeficiency Virus, equine infectious 

anaemia virus (EIAV) and others. Whereas 

commonly used gammaretroviruses like MLV 

depend on cell division and breakdown of the 

nuclear membrane, lentiviruses and their vectors 

can also infect and transduce non-proliferating 

cells. The general concepts on how these 

pathogens can be converted into efficient and 

safe gene delivery tools for cell modification, the 

correction of inherited or acquired diseases will 

be introduced. Packaging concepts and targeting 

of lentiviral vectors (LV) will be discussed, as well 

as self inactivating (SIN) LV that are thought to 

be less likely to activate or disrupt neighbouring 

genes upon integration. Additionally important 

areas of research focus on how lentiviral vectors 

can be modified to avoid integration and thus 

reduce risk of insertional mutagenesis or to target 

integration to specific sites in the genome. An 

overview on construction and use of lentiviral 

vectors as tools in molecular biology, 

transcriptional targeting and de-targeting of LV, 

regulation of LV-mediated gene expression, preclinical 

as well as recent and future clinical 

applications will be presented. 

Session: Educational Session

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Inv 3 

AAV-Based Vectors for Cardiovascular 

Diseases: Therapeutic Potential in Chronic 

Ischemia 

C. Kupatt, R. Hinkel 

Klinikum Großhadern, Medizinische Klinik I und 

Poliklinik, Ludwig-Maximilians-University Munich, 


Therapeutic neovascularization of chronic 

ischemic muscle tissue is a treatment option for 

otherwise no-option patients, which are not 

suitable for interventional or surgical 

interventions. Protein application of proangiogenic 

factors, such as VEGF or bFGF 

provided vessel growth and perfusion gains in 

small and large animal studies. However, in 

patient studies no clearcut success was achieved 

by pro-angiogenic protein application. Similar 

discrepancies were idetified for plasmid therapy, 

e.g., by liposomal transfection: significant 

improvement in preclinical experiments was 

followed by mixed results in human. Even 

overexpression of pro-angiogenic genes via 

adenoviral vector, leading to a transgene 

overexpression for 5-7 days, failed to significantly 

improve perfusion of an ischemic myocardium 

(Yla-Herttuala, 2007, Lavu JMCC 2010) Since 

prolonged transgene expression currently 

appears as a critical variable for pro-angiogenic 

gene therapy, long-acting adeno associated viral 

vectors might no-option patients. Adenoassociated 

virus (AAV), a member of the 

parvovirus family, is a non-pathogenic DNA virus, 

which transduces dividing and non-dividing cells 

and leading to a long-term overexpression, 

displaying a favorable immunogenic profile 

compared to adenoviral vectors (Gruchala, 

2004). Recently, we identified the pseudotyped 

virus strains AAV2/6 (carrying a partial genome 

of AAV2 and the envelope of AAV6) and AAV2/9 

as highly efficient vectors in large animals (pigs, 

Raake et al., JACC 2008, Kupatt et al., JACC 

2010), the latter sufficing to resolve hibernating 

myocardium after chronic coronary artery 

occlusion. Moreover, the long-lasting transgene 

expression after AAV transduction may require 

inducible transgene activity. We established the 

combination of AAV vectors and a Tet-off 

transgene system, where the pro-angiogenic 

factor Thymosin β4 was used to provide 

neovascularization being active only 2d a week. 

In a model of chronic hindlimb ischemia, pulsed 

pro-angiogenic activity appeared non-inferior to 

constitutive transgene expression up to 4 weeks 

after vector application. Taken together, the 

combination of of AAV vector and an efficient 

pro-angiogenic transgene may offer therapeutic 

potential in patients with ischemic 

cardiomyopathy or peripheral artery disease and 

exhausted conventional options. 


Inv 4 

Introduction into Nonviral Gene Delivery – 

Overview of Physical and Chemical Delivery 

Methods 

Carsten Rudolph 

Ludwig Maximilians University, Munich, Germany 

Physicochemical methods for gene delivery have 

been intensively investigated during the last 

decades and first products have been approved 

for veterinary use. Although transfection rates 

achieved with nonviral delivery systems are 

frequently lower and gene expression is of only 

short duration when compared with viral vectors, 

they offer the advantage of being less 

immunogenic, less restricted to the size of the 

delivered transgene and being less expensive 

with respect to production. The currently used 

repertoire of physicochemical gene delivery 

methods have evolved from various basic 

concept using either naked DNA or complexed 

with gene transfer agents. Moreover, a variety of 

methods have been established which make use 

of different physical phenomena to introduce 

DNA into cells. In this lecture, basic concepts of 

various physicochemical gene delivery methods 

making use of either nanoparticle formation or 

physical forces for transfection will be discussed. 

In addition, advantages and restrictions of the 

physicochemical gene delivery methods will be 

discussed. 


Inv 5 

Imaging in Cancer Gene Therapy 

Christine Spitzweg 

Department of Internal Medicine II, Klinikum 

Großhadern, Ludwig Maximilians University, 


The field of gene therapy has made considerable 

strides in the last decade by the development of 

new vectors and an increasing repertoire of 



therapeutic genes. Non-invasive monitoring of 

the in vivo distribution of viral and non-viral 

vectors as well as biodistribution, level and 

duration of transgene expression have been 

recognized as critical elements in the design of 

clinical gene therapy trials. Besides 

bioluminescence imaging using luciferase, 

reporter genes that have been evaluated for 

possible human studies are the herpes simplex 

thymidine kinase and the dopamine D2 receptor. 

Cloning of the sodium iodide symporter (NIS), 

that mediates the active transport of iodide in the 

thyroid gland and represents the molecular basis 

for radioiodine scintigraphy, has provided us with 

one of the most promising reporter genes 

available today. NIS represents a nonimmunogenic 

protein with a well-defined body 

distribution that mediates the transport of readily 

available radionuclides such as 131 I, 123 I, 124 I, 

99m 188 211 

Tc, Re or At, which can be used for 

gamma camera scintigraphic imaging, SPECT 

and PET imaging. Several research groups 

including our own have studied the potential of 

NIS as reporter gene in various applications, 

demonstrating that in vivo imaging of radioiodine 

accumulation correlates well with the results of ex 

vivo gamma counter measurements as well as 

NIS mRNA and protein analysis. NIS was 

successfully used to monitor in vivo 

biodistribution of synthetic vectors and 

mesenchymal stem cells as gene delivery 

vehicles after systemic application as well as 

replication-competent viral vectors using 

123 99m 

conventional I- or Tc-gamma camera 

imaging or 99m Tc-SPECT/CT fusion imaging. In 

addition, PET imaging using 

124 I provides 

significant advantages for exact localization and 

quantitative analysis of NIS-mediated radioiodine 

accumulation due to enhanced resolution and 

sensitivity. Taken together, currently available 

data clearly demonstrate the enormous potential 

of NIS as a novel reporter gene, that in its dual 

function as reporter and therapy gene also allows 

therapeutic radionuclide application. 


Inv 6 

Gene Therapy Around the Globe 

Len Seymour 

Department of Clinical Pharmacology, University 

of Oxford, Oxford, UK 

Gene therapy is a whole new approach to 

medicine. Instead of designing drugs to treat the 



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symptoms of a disease, gene therapy seeks to 

identify the root genetic cause of a disorder and 

to treat it at that level. For diseases caused by 

mutations in ‘single genes’, this provides the 

possibility of treating the disease very effectively 

by providing healthy copies of the mutant gene, 

allowing production of normal proteins and 

restoring the healthy phenotype. Accordingly this 

approach has exciting potential for treatment of 

many gene-based diseases. Despite its promise, 

however, gene therapy is often limited by the 

difficulty of delivering therapeutic genes 

effectively into the diseased cells. Normally 

scientists try to do this using viruses as genevectors; 

however, even viruses find it challenging 

to access all diseased cells within a body. This is 

the central challenge of gene therapy. Where 

delivery has been successfully addressed, the 

results have been very encouraging. For 

example, children with bone marrow-based 

immune deficiencies can now be treated very 

effectively by introducing normal genes into their 

bone marrow, a procedure that is normally 

performed in the test tube before reintroducing 

the bone marrow to the patient. This approach 

has a success rate comparable to or higher than 

bone marrow transplantation, and can be applied 

to all patients (with no requirement to identify a 

matched bone marrow donor). Similarly a recent 

study has shown excellent treatment of 

adrenoleukodystrophy (the disease featured in 

the film ‘Lorenzo's Oil’) using this approach. 

However, the choice of viral vectors is important, 

since treatment of these disorders requires lifelong 

expression of the therapeutic genes, and 

early generations of viruses used have been 

found to damage the DNA upon inserting into the 

genome, leading to leukaemia. Accordingly 

scientists are now seeking to identify safer 

viruses, to enable this approach to be used 

widely. Another approach to efficient delivery is in 

the field of retinal blindness, where viruses 

expressing therapeutic genes can be identified 

directly into the diseased area. Again, very 

encouraging results have been seen, with clear 

improvements in sight resulting. Gene therapy 

can also apply to the use of genetic vaccines, 

siRNA, and tumour-killing ‘oncolytic’ viruses, all 

areas where we are seeing rapid progress. As 

knowledge increases rapidly, so the day when 

genetic medicines are a routine part of life comes 

steadily closer. 

Session: Educational Session

19 | 

Inv 7 

Clinical Experience with Gene Therapy in 

Pediatric Patients 

Josef Rosenecker 

Department of Pediatrics, Ludwig Maximilians 

University, Munich, Germany 

The division of infectious diseases at the 

Department of Pediatrics, University of Munich, 

takes care for three patients who were treated 

with gene therapy protocols. This presentation 

will highlight their clinical follow up after gene 

therapy. The first patient was diagnosed with 

SCID-X1 in early infancy. No HLA-identical 

sibling was avialable and no matched bone 

marrow donor was available. Therefore, the 

patient was transferred to Hospital Necker- 

Enfants Malades, Dr. Fischer, where an ex vivo 

retroviral mediated gene therapy was performed. 

The lymphocyte development after gene therapy 

showed a successful reconstitution of the 

immune system. After gene therapy the patient 

developed a clonal T-cell proliferation, but is now 

in good clinical condition. The second patient was 

diagnosed with ADA-SCID and was transferred to 

Hospital San Raffaele, Dr. Aiuti, Milano, where an 

ex vivo gene therapy protocol was performed. 

After gene therapy the patient showed 

reconstitution of the immune system and is now 

also in good clinical condition. A third patient was 

diagnosed of chronic granulomatous disease and 

at the age of 5 years was having severe infection 

with Aspergillus nidulans of the lung and of the 

thoracic vertebral column. He showed 

progressive tetraparesis. Antimycotic therapy 

showed only limited success. No HLA-identical 

sibling was avialable. No matched unrelated 

donor found. In this situatuion the patient was 

transferred to Zurich, where an ex vivo gene 

therapy protocol has been performed. After gene 

therapy the patient showed 20% gene corrected 

cells in the PBL for about 4 weeks, then less than 

1%. He showed progressive recovery and plays 

no football and goes to school. 


Inv 8 

Gene Transfer Approaches for Gene Addition, 

Knockdown and Cellular Reprogramming In 

Vivo 

Mark A. Kay 

Departments of Pediatrics and Genetics, 

Stanford University, Stanford, CA, USA 

Vectors that allow for DNA directed-RNA 

transcription can be used to treat a broad number 

of diseases. Our laboratory has been developing 

both minicircle plasmid based vectors and 

recombinant adenoassociated viral (rAAV) 

vectors for the purpose of developing platforms 

for gene addition, knockdown, and cellular 

reprogramming in vivo. These platform 

technologies are being tested in mice, dogs, and 

humans with hemophilia B (Factor IX deficiency), 

mouse models of Hepatitis C Virus Infection, and 

mouse models of juvenile onset insulin 

dependent diabetes mellitus, as examples of 

gene addition, knockdown, and cellular 

reprogramming strategies, respectively. Minicircle 

DNA plasmid vectors are devoid of all bacterial 

plasmid backbone DNA providing 20 to 1000 

times more persistent transgene expression 

compared to routine plasmids when transfected 

into quiescent cells in vivo. The mechanistic 

differences compared to routine plasmid DNA 

vectors are beginning to be elucidated. From a 

practical standpoint, we have developed a 

simplified method for minicircle vector 

preparation that is nearly equivalent to a routine 

plasmid preparation making it feasible for these 

DNAs to replace routine plasmids for all 

mammalian expression studies. Novel 

recombinant AAV vectors with altered 

transduction properties and/or site-specific 

integration into the ribosomal DNA locus are 

being derived. These expanded vector properties 

increase their utility for treating serious diseases 

in people. We will present our current studies 

using these improved vectors for treating the 

three platform diseases stated above. 

Session: Keynote Lecture 

Inv 9 

Delivery Issues for Oncolytic Viruses 

Len Seymour 

Department of Clinical Pharmacology, University 

of Oxford, Oxford, UK 

While there are several examples where gene 

therapy approaches are looking promising, they 

all reflect situations where the gene vectors can 

be introduced efficiently into target cells – either 

by direct injection or by transduction ex vivo. 

Indeed, cancer gene therapy can be very 

effective if tumour-killing viruses are injected 



directly into tumours. Unfortunately three quarters 

of people who develop cancer in the West go on 

to die from metastatic disease. In this situation it 

is not possible to inject gene therapy vectors into 

all the tumour nodules, and intravenous 

‘systemic’ therapy is required. However, the 

human bloodstream represents a very aggressive 

environment for most gene therapy vectors. 

Therapeutic microbes delivered via the 

bloodstream encounter many host defences and 

anatomical barriers that must be surmounted to 

enable their access to disseminated cancers. 

This is particularly true for adenovirus type 5 in 

humans, where most recipients have powerful 

pre-existing adenovirus-neutralising activity. We 

have recently shown that human (but not murine) 

erythrocytes provide an additional barrier by 

sequestering adenovirus onto the Coxsackie and 

Adenovirus Receptor and (via antibodies) 

complement receptor 1. Coating adenovirus with 

a layer of hydrophilic polymer can prevent this 

interaction and allow virus to circulate free in the 

plasma, showing passive targeting to 

disseminated tumours and mediating good 

anticancer efficacy. Entry of polymer-coated virus 

particles into the tumour mass is a product of 

fluid transfer, and is directly proportional to the 

area under the plasma concentration-time curve. 

Increasing extravasation of fluid through tumourassociated 

endothelium using permeabilityenhancers 

such as Tumour Necrosis Factor 

alpha can improve virus particle entry into 

tumours over 100-fold, reaching as high as 10% 

injected dose (virus particles) per tumour. This 

provides the possibility for highly efficient 

targeting to tumours and good anticancer 

efficacy. An alternative approach is to target 

agents to infect tumour-associated vasculature. 

However while this provides a vulnerable target 

to traditional gene therapy approaches, 

endothelial cells do not normally support 

‘oncolytic’ viruses. One approach to overcoming 

this problem is to encode syncytium-forming 

proteins within the endothelial, to enable transcomplementation 

of virus replication by tumourassociated 

factors. 

Session: Vector Development 

Inv 10 

Adenovirus Vector Engineering for Gene 

Therapy 

Andrew H Baker 

University of Glasgow, Glasgow, UK 



| 20 

Adenoviral vectors are used frequently for gene 

therapy but much of their potential is limited by 

their tropism for liver and spleen, leading to 

effects on virion, sequestration, limited gene 

transfer to alternate tissue and toxicity. Recent 

studies have elucidated the mechanism 

underlying much of this tropism and the 

consequence of this infectivity profile. 1–4 We have 

shown the precise role of coagulation factor X 

(FX) in mediating liver uptake of adenovirus in 

rodent models in recent years. This occurs 

through a nM interaction between the FX Gla 

domain and the hypervariable regions of the Ad5 

hexon trimer. We modeled our cryoelectron 

microscopy data from the Ad5:FX interaction. We 

observed specific contact points within the hexon 

in hypervariable regions 5 and 7. Creation of 

novel vectors with deletions and amino acid 

mutations in these regions had dramatic effects 

on FX-mediated gene delivery in vitro and in vivo. 

We have also assessed the impact of HSPG 

structure on adenovirus uptake. These findings 

highlight the fundamental importance of the 

hexon:FX interaction dictating in vivo tropism as 

well as novel avenues for vector retargeting to 

alternate sites in vivo. 1 Parker AL et al., Blood 

(2006); 2 Waddington SN et al., Cell, (2008); 

3 4 

Kalyuzhniy, O et al., PNAS (2008); Di Paola, N 

et al., Immunity (2009). 


Inv 11 

Peptide-Enhanced Delivery of Oligonucleotide 

Analogues Targeting Duchenne Muscular 

Dystrophy 

Michael J Gait 1 , Amer F Saleh 1 , Andrey A 

Arzumanov 1 , Haifang Yin 2 , Matthew Wood 2 

1Laboratory of Molecular Biology, Medical 

Research Council, Cambridge, UK; 2 University of 

Oxford, Oxford, UK 

Duchenne muscular dystrophy (DMD) is an Xlinked 

genetic disease that affects 1 in 3500 male 

births. Mutations in the dystrophin gene result in 

aberrant splicing of the pre-mRNA and hence a 

truncated and inactive protein. Dystrophin 

connects actin to the dystrophin-associated 

complex at the sarcolemma membrane and the 

lack of dystrophin leads to progressive muscle 

degeneration and a significantly shorter life span. 

There is no effective treatment currently 

available. A number of potential gene therapy 

approaches are in development. Meanwhile a 

promising treatment has reached clinical trials,

21 | 

which involves use of antisense oligonucleotides 

(ON). ONs are targeted to bind to dystrophin premRNA 

and redirect splicing, resulting in “exon 

skipping” that restores the correct protein reading 

frame. The resultant expressed protein is shorter 

than natural dystrophin but can substitute very 

effectively. This phenotype is similar to Becker 

muscular dystrophy that shows generally only 

mild symptoms in patients. Two chemistry types 

for an exon 51 targeting ON are currently in 

clinical trials, ′ 2 -O-methyl phosphorothioates in 

Holland and Belgium and phosphoramidate 

morpholino oligonucleotides (PMO) in the UK. 

Early results in both cases have shown some 

dystrophin production in patients following 

systemic delivery. However, levels of dystrophin 

restored are variable and it is unclear yet whether 

a therapeutically useful effect will be reached in 

all muscle types at moderate dosage levels. To 

enhance activity, Arginine-rich peptides have 

been developed as conjugates of PMO targeting 

exon 23 that showed in an mdx mouse model of 

DMD substantially improved dystrophin 

production compared to naked PMO. We have 

been developing novel Arginine-rich Pip peptides 

that when attached to exon 23-targeted PMO 

have shown very high dystrophin generation in 

mdx mice, including in hard-to-reach heart 

muscle. These and other Arginine-rich peptides 

involving muscle-specific targeting domains are 

currently being evaluated as candidates for 

possible use as a peptide-PMO conjugate in a 

future clinical trial for DMD. 


Inv 12 

Hyperactive Transposons for Genetic 

Modification of Induced Pluripotent and Adult 

Stem Cells: A Novel Non-Viral Paradigm for 

Coaxed Differentiation 

Marinee K. L. Chuah 1 , Eyayu Belay 1 , Janka 

Mátrai 1 , Abel Acosta-Sanchez 1 , Ling Ma 1 , Mattia 

Quattrocelli 2 , Lajos Mátés 3 , Pau Sancho-Bru 2 , 

Martine Geraerts 2 , Bing Yan 1 , Joris Vermeesch 4 , 

Ermira Samara-Kuko 1 , Zoltán Ivics 3,5 , Catherine 

Verfaillie 2 , Maurillio Sampaolesi 2 , Zsuzsanna 

Izsvák 3,5 , Thierry VandenDriessche 1 

1 Vesalius Research Centrum KUL-VIB, Leuven, 

Belgium; 2 Stem Cell Institute, University of 

Leuven, Leuven, Belgium; 3 Max Delbrück Center 

for Molecular Medicine, Berlin, Germany; 4 Center 

for Human Genetics, University Hospital 

Gasthuisberg, Leuven, Belgium; 5 University of 

Debrecen, Debrecen, Hungary 

Adult stem cells and induced pluripotent stem 

cells (iPS) hold great promise for regenerative 

medicine. The development of robust non-viral 

approaches for stem cell gene transfer would 

facilitate functional studies and potential clinical 

applications. We therefore generated hyperactive 

transposases derived from Sleeping Beauty, 

using an in vitro molecular evolution and 

selection paradigm. These hyperactive 

transposases resulted in superior gene transfer 

efficiencies and expression in mesenchymal and 

muscle stem/progenitor cells, consistent with 

higher expression levels of therapeutically 

relevant proteins including coagulation factor IX. 

Their differentiation potential and karyotype was 

not affected. Most importantly, relatively efficient 

stable gene transfer could be obtained in bona 

fide hematopoietic stem cells that are capable of 

hematopoietic reconstitution and multi-lineage 

gene marking (Mates, Chuah et al., Nature 

Genetics, 41(6):753–761, 2009). Moreover, 

stable transposition could also be achieved in iPS 

which retained their ability to differentiate along 

neuronal, cardiac and hepatic lineages without 

causing cytogenetic abnormalities. Most 

importantly, transposon-mediated delivery of the 

myogenic PAX3 transcription factor into iPS 

coaxed their differentiation into MYOD + 

myogenic progenitors and multinucleated 

myofibers, suggesting that PAX3 may serve as a 

myogenic “molecular switch” in iPS. Hence, this 

hyperactive transposon system represents an 

attractive non-viral gene transfer platform with 

broad implications for regenerative medicine, cell 

and gene therapy. 

Session: On the Route to Clinical Application 

Inv 13 

Perinatal Gene Therapy for Lethal Genetic 

Diseases 

Simon N Waddington 1 , Ahad A. Rahim 2 , Citra 

Mattar 3 , Andrew M.S. Wong 4 , Klemens Hoeffer 4 , 

Suzanne M.K. Buckley 5 , Jonathan D. Cooper 4 , 

Jerry Chan 3 

1 University College London, London, UK; 

2 Institute for Women's Health, University College 

London, London, UK; 3 National University of 

Singapore, Singapore; 4 Paediatric Storage 

Disease Laboratory, Kings College London, 

London, UK; 5 Department of Haematology, 

University College London, London, UK; 

A number of inherited neurological diseases are 

characterised by neurodegenerative changes at 



or around birth. Prognosis remains dismal and for 

several of these diseases, including Acute 

Neuronopathic Gaucher Disease there is no 

treatment and palliative care remains the only 

option. Owing to the aggressive nature of this 

and related diseases fetal or neonatal gene 

therapy may be the only potential means of 

treatment. Recently, AAV9 has been shown to 

cross the blood brain barrier following 

intravenous injection into neonatal mice, cats and 

macaques. We have investigated this further by 

studying vector tropism after intravenous injection 

into fetal mice. In utero intravenous injection of 

single-stranded (ss) and self-complimentary (sc) 

AAV9 expressing green fluorescent protein 

(GFP) into mice (embryonic day 16) resulted in 

efficient global transduction of neurons in the 

CNS. Furthermore, there was a stark contrast in 

cell type transduction when compared to 

neonatal administration, confirmed by scanning 

confocal microscopy. The efficiency varied 

depending upon ss or sc configuration of the 

vector. Examination of injected mice by 

fluorescent microscopy, immunohistology, and 

GFP ELISA revealed extensive and efficient 

transduction in the visceral organs in addition to 

muscle, bone, eye, and skin. Gene expression 

was also seen in the peripheral nervous system. 

Similarly, extensive transduction was also 

observed following intravenous administration of 

AAV9 to the fetal macaque. The combination of 

CNS and visceral organ transduction is well 

suited to the study, and potential treatment for 

diseases such as acute neuronopathic Gaucher 

disease where both CNS and visceral pathology 

require targeting and a suitable mouse model is 

available. 


Inv 14 

Clinical Trial of Gene Therapy for Early Onset 

Severe Retinal Dystrophy Resulting from 

Defects in RPE65 

Robin R Ali 1 , JWB Bainbridge 1 , AJ Smith 1 , FW 

Fitzke 1 , GE Holder 1 , A Stockman 1 , SS 

Bhattacharya 1 , GS Rubin 1 , S Yzer 2 , I van den 

Born 2 , AT Moore 1 

1 Division of Molecular Therapy, UCL Institute of 

Ophthalmology and UCL/Moorfields Eye Hospital 

Biomedical Research Centre for Ophthalmology, 

London, UK; 2 Rotterdam Eye Hospital, 

Rotterdam, The Netherlands 



| 22 

Early-onset severe retinal dystrophy caused by 

defects in the gene encoding the retinal 

isomerase RPE65 is associated with poor vision 

at birth and complete loss of vision in early 

adulthood. In a phase I/II dose-escalation trial, 

we have delivered subretinally recombinant 

adeno-associated virus (rAAV) vector expressing 

RPE65 under the control of an RPE65 promoter 

in 9 human subjects with early onset severe 

retinal dystrophy associated with mutations in 

RPE65. We have examined systemic vector 

dissemination and immune responses following 

vector delivery, assessed visual function pre- and 

post-vector delivery using a range of 

psychophysical techniques, and performed 

detailed electrophysiology and retinal imaging 

studies. There have been no serious adverse 

effects of surgical delivery of vector in the 

subjects enrolled to date. We have detected no 

systemic dissemination of vector genome. 

Although we have detected an increase in 

systemic neutralising antibodies to AAV capsid in 

two subjects, there have been no evidence of 

immune responses to RPE65 protein. We have 

measured significant improvements in retinal 

sensitivity by microperimetry and dark-adapted 

perimetry, and improved performance in a test of 

visually-guided mobility. The outcomes in the first 

9 subjects to date suggest that subretinal delivery 

of rAAV vector can be safe in humans in the 

short term and can improve retinal sensitivity. 

These findings support further clinical studies in 

subjects with RPE65 deficiency and the 

development of gene therapy for other inherited 

retinal disorders. 

Session: Gene Therapy 

Inv 15 

Alipogene Tiparvovec: the First Gene Therapy 

for a General Metabolic Disorder 

Harald Petry 1 , D. Gaudet 2 , A. Carpentier 3 , E. 

Stroes 4 , J. Twisk 1 , S. Greentree 1 

1Amsterdam 

Molecular Therapeutics (AMT), 

Amsterdam, The Netherlands; 2 Department of 

medicine, Université de Montreal, Quebec, 

Canada; 3 Department of Endocrinology, 

University of Sherbrooke, Quebec, Canada; 

4 

Academisch Medical Center (AMC), Amsterdam, 

The Netherlands 

Gene therapy is coming of age. Correction or 

supplementation of genetic defects through gene 

therapy is amenable to both monogenic disorders 

as well as to diseases with a key protein playing

23 | 

a key role in the pathology. Alipogene tiparvovec 

(Glybera ® , AMT-011) is the first gene therapy 

product for a metabolic disease in late stage 

clinical development. The product contains a 

gain-of-function variant of the human lipoprotein 

lipase gene in an AAV1 based vector (AAV1- 

LPLS447X). Alipogene tiparvovec has been 

developed for the long term correction of 

lipoprotein lipase deficiency, to control or abolish 

symptoms and prevent complications in adult 

patients clinically diagnosed with lipoprotein 

lipase deficiency (LPLD). LPLD is a rare, 

seriously debilitating autosomal inherited 

monogenic disorder of lipid metabolism. 

Deficiency of LPL function results in 

chylomicronaemia. Recurrent pancreatitis is 

known as the most frequent, potentially lethal, 

complication; other severe sequelae include 

diabetes and increased tendency for 

atherosclerosis. During the presentation the 

various steps to develop a gene therapy 

medicinal product will be discussed. Alipogene 

tiparvovec in LPLD will be used to demonstrate 

that persistent gene transduction after one-time 

intramuscular administration is feasible, and 

results in long-term clinical improvements in the 

‘chylomicronaemia syndrome’ caused by LPLD. 


Inv 16 

Gene Therapy for Chronic Granulomatous 

Disease: the Past and the Future 

Manuel Grez 1 , CGD Consortium 2 

1Georg-Speyer-Haus, Frankfurt am Main, 

Germany; 2 Frankfurt, Heidelberg, Idar Oberstein, 

London, Zürich 

In our recent gene therapy trial for X-CGD we 

demonstrated reconstitution of superoxide activity 

in phagocytic cells and elimination of preexisting 

infections in two treated patients. However, an 

unexpected expansion of myeloid progenitors 

occurred five months after transplantation. Both 

patients developed a myelodysplastic syndrome 

(MDS) caused by insertional activation of 

MDS1/EVI1 followed by clonal progression and 

the gradual loss of chromosome 7. P1 died 27 

months after gene therapy of MDS in 

combination with severe septicemia, the latter 

resulting from loss of bacterial killing activity in 

transduced cells. P2 underwent allogeneic stem 

cell transplantation. Forced overexpression of 

MDS1/EVI1 or EVI1 in human cells disrupted 

normal centrosome duplication, linking 

MDS1/EVI1 activation to the development of 

genomic instability. Optimized SIN 

gammaretroviral vectors have been shown to 

have an enhanced safety profile. From several 

SIN-gammaretroviral vectors expressing 

gp91phox, we selected a construct containing the 

promoter of the human c-fes gene for detailed 

preclinical studies. We used X-CGD mice for 

monitoring safety and functional reconstitution of 

NADPH oxidase activity in primary and 

secondary animals. The SINfes vector was safe, 

was resistant to CpG methylation and 

reconstituted superoxide activity to clinical 

relevant levels. We plan to use this vector for the 

next Phase I gene therapy trial. 


Inv 17 

Reprogrammed Measles Viruses as Cancer 

Therapeutics Three Points of Attack 

Roberto Cattaneo 

Department of Molecular Medicine, Mayo Clinic, 

and Virology and Gene Therapy track, Mayo 

Graduate School, Rochester, USA 

Soon after viruses were recognized more than 

100 years ago, tumor regressions occasionally 

documented after accidental infections suggested 

the idea of using them to fight cancer. Early 

virotherapy clinical trials based on wild type 

viruses were unsuccessful, but recent ones 

based on genetically modified viruses are built on 

much stronger foundations. They foresee 

extensive monitoring of viral replication, gene 

expression, and host immunity. Therapeutic 

efficacy is being assessed by well-defined 

biological end points, and can be improved. For 

future clinical trials more specific and potent 

oncolytic viruses are developed based on three 

types of modification: targeting, arming, and 

shielding. Targeting introduces multiple layers of 

cancer specificity; arming amplifies locally the 

effects of approved cancer therapeutics; and 

shielding provides temporary relief from the 

immune response. We have shown that the 

envelope of measles (MV) and related 

paramyxoviruses can be targeted to many 

designated receptors. Retargeted MV envelopes 

have become preferred tools to pseudotype 

lentiviral and other gene transfer vectors. We 

have developed protease activation targeting, 

and innate immunity control targeting. We have 

armed MV with a prodrug convertase, and shown 

that this virus synergizes with the 



chemotherapeutic fludarabine to eliminate mantle 

cell lymphoma xenografts. MV-based 

therapeutics developed by the Russell and Peng 

groups at Mayo are being administered to 

patients who, having exhausted other therapeutic 

options, have enrolled in clinical trials of ovarian 

carcinoma, glioma, and myeloma. Survival and 

quality of life data are being collected and will be 

critical for the approval of new therapeutic 

products. Cattaneo, R. (2010) Paramyxovirus 

entry and targeted vectors for cancer therapy. 

PLoS Pathogens 6: e1000973. 

Session: Cancer Gene Therapy 

Inv 18 

Targeted and Armed Oncolytic Poxviruses: A 

Novel Multimechanistic Therapeutic Class for 

Cancer 

Caroline Breitbach 1 , John C Bell 1,2 , David H Kirn 1 

1 Jennerex Inc, San Francisco, USA; 2 Centre for 

Cancer Therapeutics, Ottawa Hospital Research 

Institute, Ottawa, Canada 

Engineered viruses have been developed for 

cancer therapy both as non-replicating gene 

therapy agents and as cancer vaccines. 

Oncolytic viruses, in contrast, were developed to 

replicate within and subsequently lyse, cancer 

cells. Clinical efficacy to date with each of these 

approaches has been limited by multiple factors 

including the inability to infect enough tumor cells 

in vivo locally within a tumor or systemically, and 

resistance of complex advanced tumors to a 

single mechanism-of-action (MOA). Over the last 

several years, however, a novel therapeutic class 

has emerged that combines the best features of 

all three approaches: targeted and armed 

oncolytic poxviruses. Recent preclinical and 

clinical results demonstrate convincingly that 

products from this therapeutic class can achieve 

highly selective and potent cancer destruction 

systemically through a multi-pronged MOA. 

Given recent clinical validation, we expect this 

therapeutic class to expand rapidly. 




| 24 

Inv 19 

Oncolytic Vaccinia Virus for the Treatment of 

Cancer 

John C. Bell 

Centre for Cancer Therapeutics, Ottawa Hospital 

Research Institute, Ottawa, Canada 

The creation of novel “cancer lysing” or 

“oncolytic” viruses over the last 10-15 years has 

led to the development of a number of innovative 

anti-cancer therapeutic products many of which 

have entered into clinical testing. We are 

examining a number of poxvirus backbones for 

their ability to selectively target and kill tumour 

cells. A derivative of the Wyeth Strain called JX- 

594 has engineered deletions, natural mutations 

and expresses a GM-CSF transgene, all of which 

contribute to its ability to infect and kill tumour 

tissues while leaving normal tissues unscathed. 

The results of our preclinical models and 

molecular studies have shown that JX-594 

attacks cancers using a multi-pronged approach 

that includes tumour vascular targeting, immune 

stimulation and direct cancer cell lysis. JX-594 

has now been tested in over 70 patients in a 

variety of different trial designs and consistently 

shows tumour targeting activity and a very strong 

safety profile. The results of our ongoing 

translational research efforts and clinical data 

from our phase I and II trials will be presented. 


Inv 20 

Prostate Cancer Stem Cells: A New Target for 


Norman J Maitland 

YCR Cancer Research Unit, Dept of Biology, 

University of York, Heslington, UK 

Human prostate cancer is characterised by 

cellular heterogeneity; perhaps one reason for 

the resistance of prostate cancer to many 

medical therapies. Once a prostate cancer has 

failed on hormone-based therapies, the 

prognosis for the patients is poor. Our approach 

to tissue heterogeneity in the prostate has been 

to identify and purify individual cell populations 

from human prostate cancers and matched 

normal tissues. We have fractionated cancer 

epithelium into a majority luminal/secretory 

population (which comprises more than 99% of 

the cancer mass) and a minor population

25 | 

consisting of committed basal cells, transamplifying 

cells and a stem cell population. The 

latter cells are responsible for tumour initiation 

and in vitro colony formation. Affymetrix 

microarray analysis of these populations confirms 

a distinct phenotype for each cell type and 

functional expression of fate influencing genes. 

Further analysis has revealed populations of 

genes whose expression is tightly linked to the 

differentiation process, while others are 

associated with the cancerous properties of the 

cells. The tumour origin of the identified cells has 

been confirmed by xenografting into a highly 

immuno-compromised mouse strain at orthotopic, 

kidney capsule and subcutaneous sites with 

demonstrable metastases. Genes associated 

with prostate cancer are expressed in the 

cultures and their functional significance 

assessed by SiRNA knock-down in stem cells 

and in their daughter cells. Epigenetic control, in 

contrast to permanent genetic alterations or 

gross alterations involving a reprogramming of 

cells to express specific transcription factor sets, 

seems to be a predominant mechanism for an 

adaptable stem cell in prostate. A better 

knowledge of the phenotype of the prostate 

CSC's should permit therapeutic targeting. 

However, whilst stem cell directed therapies (to 

treat less than 0.1% of the tumour mass) are 

unlikely to have an immediate effect on tumour 

volume, one consequence of targeting the wrong 

cell types within a heterogeneous tumour is to 

perturb the natural history of the tumour and 

results in an amplification of CSC's, in agreement 

with cancer relapse patterns in man. 

Session: Tumor Biology and (Cancer) Stem Cells 

Inv 21 

A Consortium for Clinical Development of 

Lentiviral Vector-Induced DCs for Melanoma 

Immunotherapy 

Renata Stripecke 1 , Sandra Kuhs 1 , Ralf Gutzmer 2 , 

Henk Garritsen 3 , Farzin Farzaneh 4 

1 Department of Hematology, Hemostasis, 

Oncology and Stem Cell Transplantation, 

Hannover Medical School, Hannover, Germany; 

2 Department of Dermatology, Hannover Medical 

School, Hannover, Germany; 3 Division of 

Transfusion Medicine, Braunschweig City 

Hospital, Braunschweig, Germany; 4 Department 

of Haematological Medicine, King's College 

London, Rayne Institute, London, UK 

Dendritic Cells (DCs) are key players in the 

development of adaptive immunity. Their use to 

boost anti-tumor responses have reached phase 

III clinical trials but ex vivo production is costly 

and difficult to standardize. A novel concept for 

DC production consists of overnight lentiviral 

vector (LV) transduction of growth factors and 

full-length antigens into monocytes that results 

into induction of “SMART-DCs” (Selfdifferentiated 

Myeloid-derived Antigenpresenting-cells 

Reactive against Tumors). This 

concept has been validated in the preclinical B16 

melanoma mouse model for potency and safety 

with MART-1 and TRP2 as melanoma-associated 

antigens, overexpressed in 90% of the metastatic 

melanoma (Koya et al., 2007; Pincha et al., 

submitted). Third generation self-inactivating 

tricistronic lentiviral vectors containing 

interspacing 2A elements co-expressing human 

GM-CSF, IL-4 and MART-1 or TRP-2 were 

constructed. Cytokine preconditioning and 16h 

transduction of CD14 + monocytes with high titer 

LVs resulted in persistent (3 weeks) autocrine 

production of GM-CSF (average 10 ng/ml), IL-4 

(average 12 ng/ml) and expression of MART-1 or 

TRP2 (detectable by intracellular staining). 

Transduced monocytes readily self-differentiated 

into SMART-DCs (CD209 + , MHCII + , CD80 + 

CD86+) and were stable in culture for 3 weeks. 

SMART-DCs/MART-1 were recognized by T cell 

clones specific for MART-1 in an HLA-restricted 

manner, assessed by IFN-γ-ELISPOT-Assay. 

PBMCs obtained from healthy donors that were 

primed/boosted in vitro with autologous SMART- 

DCs/MART-1 demonstrated the induction of 

MART-1-specific T cell responses assayed by 

IFN-γ-ELISPOT-Assay. SMART-DCs/MART-1 

and SMART-DCs/TRP2 are currently being 

tested for stimulation of T cell responses in 

melanoma patients. A consortium for clinical 

development of SMART-DCs has been formed: 

GMP-grade batch of the tricistronic vectors is 

planned for additional preclinical testing (optimal 

vector dose, number of integrated copies, 

toxicity), monocytes will be transduced with LVs 

in a closed GMP-grade bag system, development 

of Standard Operating Procedures and 

establishment of identity and potency markers 

are underway. 

Session: Cancer Immune Therapy and T-Cell 




Inv 22 

Arming Immune Cells to Fight Cancer 

Hinrich Abken 

Zentrum für Molekulare Medizin Köln und Klinik I 

für Innere Medizin, Uniklinik Köln, Cologne, 

Germany 

Cancer cells frequently escape T cell recognition 

by repression of the antigen processing 

machinery making them invisible for a cytolytic T 

cell attack. Strategies in adoptive immunotherapy 

during the last decade aimed to overcome the 

situation by engineering T cells with a 

recombinant chimeric antigen receptor (CAR) 

which uses a single chain fragment of variable 

region (scFv) antibody for target binding and the 

intracellular CD3zeta domain for T cell activation. 

To provide full T cell activation, the CD3zeta 

chain was fused with the CD28 costimulatory 

domain in one polypeptide chain; other 

costimulatory moieties are likewise used. We 

here discuss the impact of CD28 costimulation on 

the threshold in T cell activation and on the 

redirected anti-tumor response in the presence of 

TGF-beta and regulatory T cells. We moreover 

introduce a novel generation of CARs with 

combined CD28-OX40 costimulation in order to 

harness central memory T cells for a redirected 

anti-tumor attack. Finally, we demonstrate the 

power of a redirected T cell attack in eradicating 

established tumor lesions by eliminating minor 

tumor cell subsets. 



Inv 23 

Immune Gene Therapy for Acute Myeloid 

Leukaemia 

Farzin Farzaneh 1 , Lucas Chan 1 , Nicola 

Hardwick 1 , Wendy Ingram 1 , Ghulam Mufti 1 , Mark 

Aloysius 2 , Adrian Robins 3 , Nagy Habib 4 , Joti 

Bhalla 1 , Oleg Eremin 2 , Tye Gee Jun 1 , David 

Oppenheim 1 , James Wells 5 , Christopher 

Cowled 5 , Alistair Noble 5 

1 Department of Haematological Medicine, King's 

College London, Rayne Institute, London, UK; 

2 Section of Surgery, Biomedical Research Unit, 

Nottingham Digestive Diseases Centre, 

University of Nottingham, Nottingham, UK; 

3 Institute of Infection and Immunity, School of 

Molecular Medical Sciences, Nottingham 

University Hospitals, University of Nottingham, 



| 26 

Nottingham, UK; 4 Section of Surgery, 

Department of Surgical Oncology and 

Technology, Imperial College London, London, 

UK; 5 MRC and Asthma UK Centre in Allergic 

Mechanisms of Asthma, King's College London, 

Guy's Hospital, London, UK 

Immune mediated rejection of established 

tumours needs adequate stimulation of cell 

mediated immunity, as well as the suppression of 

inhibitory responses that are induced by chronic 

exposure to tumour associated antigens. 

Preclinical studies have demonstrated that 

tumour cells expressing immune costimulatory 

molecules and appropriate Th1 cytokines can 

induce immune mediated rejection of previously 

established tumours. This strategy is now being 

assessed in a Phase-I clinical trial for the 

vaccination of relapsed poor prognosis acute 

myeloid leukaemia (AML) with autologous AML 

cells that are genetically modified to express B7.1 

(CD80) and IL-2. We have also shown that 

vaccination of advanced solid tumour patients, 

with dendritic cells that are pulsed with 

telomerase (hTERT) peptides, can result in the 

expansion of antigen specific T cells with some 

evidence of a modest and short-lived therapeutic 

benefit. In a further recently started Phase-I study 

we are now evaluating the safety, and potential 

efficacy of, hTERT peptide loaded dendritic cell 

vaccination in combination with 

cyclophosphamide mediated inhibition of immune 

suppression by regulatory T cells (Treg). A much 

less demanding alternative to the use of dendritic 

or tumour cell vaccines is the direct vaccination 

with tumour associated antigens, provided that 

the vaccine could indeed induce cell mediated 

immunity. With this goal in mind we have recently 

developed a new vaccination strategy based on 

the combined adjuvants for synergistic activation 

of cellular immunity (CASAC). CASAC contains 

different combinations of defined molecules that 

act synergistically to induce dendritic cell 

activation. Subcutaneous vaccination with two 

doses of a single peptide (OVA or Trp2) plus 

CASAC, induces IL-12 secretion, stimulation of 

Th1-biased CD4 T-cells, and high levels 

(routinely 50% by tetramer staining) of antigen 

specific cytolytic CD8 T cells. The magnitude of 

CASAC mediated immune stimulation is 

substantially greater than can be achieved by 

other adjuvants (e.g., about 100-fold greater than 

complete Freund's adjuvant). The antigen 

specific CTL activity induced by CASAC 

mediated peptide vaccination allows the in vivo 

lysis of greater than 90% of antigen positive 

tumour cells in mouse tumour models, resulting 

in long-lasting immunity with a robust recall 

response. This strategy will shortly entre clinical

27 | 

studies for WT1 peptide vaccination of myeloid 

leukaemia patients. 



Inv 24 

Vector Safety Data Leading to a Clinical Trial 

for the Gene Therapy of Wiskott Aldrich 

Syndrome: A Perspective on Future 

Developments 

Anne Galy, Sabine Charrier, Otto-Wilhelm 

Merten, Didier Caizergues 

Genethon, Evry, France 

Hematopoietic gene therapy currently relies on 

the ex vivo gene correction of patient autologous 

hematopoietic stem cells. This approach has now 

been tested for over 10 years in pilot phase I/II 

trials in several inherited diseases. The domain is 

evolving and the use of lentiviral vectors derived 

from HIV-1 provide significant advantages in 

terms of genomic stability, biological properties 

but also in the area of pharmaceutical product 

characterization. The rare primary 

immunodeficiency Wiskott Aldrich syndrome 

(WAS) is a combined platelet and immune defect. 

In the perspective of developing a gene therapy 

for WAS, we have characterized an advanced 

generation rHIV lentiviral vector pseudotyped 

with VSVg and implemented a large-scale 

industrial process to produce and to control this 

vector under good manufacturing practices 

(GMP). Recently several European centers were 

approved to start gene therapy trials for WAS 

with this lentiviral vector. With a growing number 

of potential disease applications for lentiviral 

vector-mediated hematopoietic gene therapy, it 

seems that there is ground to integrate 

information on preclinical vector safety features 

particularly at the level of design, manufacture 

process or controls. We will discuss these 

various points through the different aspects 

leading to the development of WAS gene 

therapy. 

Session: Pharmacology and Toxicology 

Inv 25 

Lentivector Transduction of Novel Target 

Cells 

Klaus Cichutek 

Paul-Ehrlich-Institut, Langen, Germany 




DG-GT 2010 Oral Presentations 

Or 1 

Analysis of Intracellular Particle Trafficking 

and Bioresponsive Bonds for Ad Vector 

Shielding by Capsomer-Specific Fluorescent 

Labeling 

Sigrid Espenlaub 1 , Stephanie Corjon 1 , Tatjana 

Engler 1 , Carolin Fella 2 , Manfred Ogris 2 , Ernst 

Wagner 2 , Stefan Kochanek 1 , Florian Kreppel 1 

1Department of Gene Therapy, University of Ulm, 

Ulm, Germany; 2 Pharmaceutical Biotechnology, 

Department of Pharmacy, Ludwig-Maximilians- 


Adenovirus (Ad) vectors are widely used for gene 

therapy approaches. Due to the high abundance 

of the natural adenovirus receptors CAR/integrins 

on a wide variety of cells numerous methods 

have been developed to redirect the virions to 

specific receptors on target cell surfaces. An 

increasing number of recent publications 

evidenced that the success of targeting strategies 

does not only depend on receptor binding and 

cellular uptake, but also on intracellular trafficking 

processes. Therefore, improved knowledge on 

the intracellular fate of targeted Ad vector 

particles is mandatory for a rational design of 

targeted Ad vectors. However, while in principle 

potent tools are available for fluorescent labeling 

of Ad vectors in order to analyze post-receptor 

trafficking, these tools suffer from significant 

limitations: (i) capsids can so far only be labeled 

upon amino groups present in all proteins all over 

the particle surface and this imposes the risk of 

interference with particle infectivity, (ii) 

capsomere-specific labeling requires extensive 

genetic modifications and has only been 

demonstrated at protein IX, (iii) two-color labeling 

approaches are not available. Here we present a 

robust and straight forward labeling procedure 

based on a genetic-chemical approach that 

overcomes these limitations. It allows for specific 

labeling of the capsomeres fiber, protein IX, or 

hexon and permits two-color labeling. Using this 

technology we analyzed by means of live cell 

imaging two different bioresponsive bonds that 

potentially can be used for the reversible 

attachment of shielding/targeting moieties to the 

capsids: disulfide and hydrazone bonds. In our 

experiments we could not observe significant 



| 28 

reduction of disulfides within the observation time 

of 80 minutes post cell entry. Acid-responsive 

hydrazone bonds in contrast became quickly 

hydrolyzed after uptake of the virions (30 min) 

and may thus be favorable for the generation of 

bioresponsive vectors and potent tools to 

overcome the limitations in intracellular trafficking 

processes of targeted Ad vectors. 


Or 2 

Primary Human Keratinocyte-Selective AAV2- 

Based Targeting Vectors 

Jessica Sallach 1 , Fernando Laguzzi Larcher 2 , 

David Gomez Almarza 2 , Luca Perabo 1 , Michael 

Hallek 1 , Hildegard Büning 1 

1 Clinic I of Internal Medicine and Center for 

Molecular Medicine Cologne (CMMC), University 

Hospital Cologne, University of Cologne, 

Cologne, Germany; 2 Ciemat-Centro de 

Investigacion Energetica Medioambiental y 

Tecnologica, Moncloa-Aravaca, Madrid, Spain 

Vectors based on the adeno-associated virus 

(AAV), a non-enveloped DNA virus, have 

emerged as one of the leading gene transfer 

systems in gene therapy. However, transduction 

efficiency of clinical relevant cell types such as 

keratinocytes is insufficient and seems to depend 

on donor-specific factors. We therefore 

conducted AAV peptide display selection on 

human primary foreskin keratinocytes in order to 

identify AAV2-based targeting mutants with 

improved efficacy and specificity of gene transfer 

into keratinocytes. Aiming to force a ligandreceptor 

interaction that is independent of 

heparan sulphate proteoglycan (HSPG) binding, 

a cell surface molecule that is expressed on a 

wide range of cell types, we depleted our AAV 

peptide library of mutants capable of HSPG 

binding. Following five selection rounds on 

human primary keratinocytes obtained from 

different donors, viral genomes were sequenced 

to identify peptide sequence(s) that mediated 

viral infection. Interestingly, all selected mutants

29 | 

displayed an integrin binding motif as peptide 

insertion at amino acid position 587 of the AAV2 

capsid. Selected peptides were then introduced 

into the capsid of recombinant AAV vectors 

(rAAV) which were subsequently assayed for 

their transduction efficiency and cell type 

specificity. Compared to rAAV2 the targeting 

vectors showed an increased transduction 

efficiency of up to 15-fold in primary human 

keratinocytes and a dramatic decrease (up to 30fold) 

in off-target transduction. Inhibitor studies 

confirmed HSPG-independent, ligand-mediated 

cell entry. Further characterization of the entry 

mode and its consequences of host-cell 

interaction is ongoing and results will be 

discussed. 


Or 3 

Development of Novel PhiC31 Integrase 

Fusion Proteins for Improving Efficacy and 

Safety of Transgene Insertion in Therapeutic 

Applications 

Nadja Noske 1 , Nina Harms 1 , Toni Cathomen 2 , 

Anja Ehrhardt 1 

1 Department of Virology, Max von Pettenkofer- 

Institute, LMU, Munich, Germany; 2 Department of 

Experimental Hematology, Hannover Medical 

School, Hannover, Germany 

Integration systems are promising and attractive 

tools for the development of new gene 

therapeutic strategies. Based on site-directed 

recombination the bacteriophage derived PhiC31 

system provides outstanding opportunities for 

transgene integration into the host genome. 

However, due to unpredicted insertion events in 

the genome mediated by PhiC31, the 

optimization of activity and directing integration 

into specific target sites are important goals for 

the usage in therapeutic applications. Therefore, 

we constructed several fusion proteins of the 

integrase and different DNA binding domains 

(DBD) like the synthetic polydactyl zinc finger 

E2C and the AAV Rep protein. The motifs were 

either fused N- or C-terminal to the integrase 

separated by a short TS or a long GGS5 linker. 

Immunoblot and flow cytometry analysis revealed 

that the fusion proteins were expressed in 

comparable amounts after transfection into 293 

cells. Excision and integration activity retained up 

to 80% of the wildtype integrase (WT) when the 

binding motifs were attached to the C-terminus of 

the integrase separated by the 45-bp long GGS5 

interdomain. As a further step we generated 

fusion mutants lacking the native DBD of the 

integrase (ΔDBD). As expected, these were 

catalytically inactive when measuring 

recombination efficiencies between wild type 

PhiC31 integrase recognition sites. However, 

after performing initial colony forming assays to 

measure integration events mediated by the 

ΔDBD fusion constructs, we observed 31% 

activity of the WT. To further improve the PhiC31 

integrase system we plan to build up on our 

previous study in which we identified hyperactive 

variants of the integrase showing up to 5.5-fold 

increased integration efficiency (Liesner et al., 

2010). We also characterized critical amino acids 

involved either in attaching DNA or in the 

insertion process. Therefore, mutants which lost 

their native DNA binding properties and their 

restoration by fusing known binding motifs would 

be an interesting approach to develop novel tools 

for site-specific integration. In combination with 

hyperactive PhiC31 variants this approach could 

lead to a more specific and efficient PhiC31 

integrase system. 


Or 4 

Biocompatible Polyplex Nanomicelle for Safe 

and Effective Gene Transfer 

Keiji Itaka 1 , Kazunori Kataoka 2 

1 Division of Clinical Biotechnology, CDBIM, 

Graduate School of Medicine, The University of 

Tokyo, Tokyo, Japan; 2 Department of Materials 

Science and Engineering, Graduate School of 

Engineering, The University of Tokyo, Tokyo, 

Japan 

Gene delivery using cationic polymers has 

attracted much attention due to their potential 

advantages, such as large DNA loading capacity, 

ease of large-scale production, and reduced 

immunogenicity. We are developing a polyplex 

system forming micelle from polyethyleneglycol 

(PEG) - poly[N-[N-(2-aminoethyl)-2aminoethyl]aspartamide] 

(PEG-PAsp(DET)) and 

plasmid DNA. By highly-regulated structure of a 

diameter of ≈ 100 nm with a PEG palisade, the 

nanomicelle shows increased tolerance under 

physiologic conditions with the remarkably low 

cytotoxicity. A key feature for safe and effective 

gene transfer is the biodegradability of 

PAsp(DET). PAsp(DET) was revealed to undergo 

rapid degradation by Gel permeation 

chromatography (GPC) and electrospray 



ionization mass spectrometry (ESI-MS) 

measurements. In contrast, a derivative 

polycation, N-substituted polyglutamide 

(PGlu(DET)), showed no degradability, indicating 

that the degradation of PAsp(DET) was induced 

by a specific self-catalytic reaction between the 

PAsp backbone and the sidechain amide 

nitrogen. Degradation products of PAsp(DET) 

caused no cytotoxicity, even at high 

concentrations in the culture medium. Repeated 

transfection by administering the polyplexes for 

every 24 h showed that biodegradable 

PAsp(DET) provided a continuous increase in 

transgene expression, while non-degradable 

PGlu(DET) showed a decrease in transgene 

expression after 48 h, coupled with fluctuations in 

expression profiles of endogenous genes. The 

biocompatibility of nanomicelles plays a 

significantly role in in vivo applications. By 

obtaining sustained transgene expressions with 

minimized toxicity and inflammatory responses 

such as cytokine induction, we achieved 

successful therapeutic outcomes by gene 

introduction using nanomicelles, including in vivo 

bone regeneration by introducing osteogenic 

factors to bone defect area and intratracheal 

gene transfer of adrenomedullin to treat 

pulmonary hypertension. Hydrodynamic gene 

introduction to skeletal muscle was also well 

achieved compared with naked pDNA. We are 

now trying various applications for disease 

treatment and tissue regeneration using model 

animals. 


Or 5 

EGF Receptor Targeting of Polyplexes with 

the Short Artificial Peptide GE11 Studied by 

Live Cell Imaging 

Frauke M Koenig 1 , Nadia Ruthardt 1 , Ernst 

Wagner 2,3 , Manfred Ogris 2,3 , Christoph 

Braeuchle 1,3 

1Physical Chemistry, Department of Chemistry, 

Ludwig-Maximilians-University, Munich, 

Germany; 2 Pharmaceutical Biotechnology, 


University, Munich, Germany; 3 Center for 

Nanoscience (CeNS), Ludwig-Maximilians- 


For the selective gene therapy of cancer cells in 

the human body, a tumor specific targeting is 

required. The EGF receptor provides a promising 

target as it is overexpressed in many human 



| 30 

cancers. A number of anticancer therapeutics 

directed against the EGF receptor have been 

successfully developed so far. Polyplexes 

consisting of DNA complexed with a cationic 

polymer can be equipped with EGF as a ligand 

resulting in their fast, receptor-dependent uptake 

into cancer cells. However, full-length EGF has a 

high tendency to aggregate and is expensive in 

production. Furthermore, it activates the 

mitogenic signaling cascade of target cells, 

leading to undesirable cell proliferation. In this 

study, we therefore examined the alternative 

usage of GE11, a short artificial peptide with high 

affinity to the EGF receptor, for the specific 

targeting of polyplexes HUH7 cells. By live-cell 

imaging with highly sensitive fluorescence 

microscopy, we directly compared the 

internalization kinetics of GE11 polyplexes to the 

uptake of EGF coupled and untargeted 

polyplexes. To determine the extent of mitogenic 

signaling after polyplex binding to the receptor, 

phosphorylated signaling molecules were 

detected by western blotting. The correlation 

between receptor signaling and polyplex uptake 

was then examined in more detail by inhibitor 

studies with Erlotinib, a specific inhibitor of EGF 

receptor phosphorylation. Our results reveal that 

the uptake of GE11 polyplexes is slow compared 

to EGF polyplexes but much more efficient 

compared to untargeted polyplexes. After two 

hours of incubation, GE11- and EGF polyplexes 

reach similar internalization levels resulting in 

comparable transfection efficiencies on a long 

time scale. The activation of mitogenic signaling 

is significantly reduced for GE11- compared to 

EGF polyplexes. Further studies reveal that the 

different uptake kinetics of GE11 and EGF can 

be correlated to the activation of receptor 

signaling. Taken together, GE11 polyplexes are a 

promising candidate for clinical therapy as they 

show a high transfection efficiency, low 

aggregation properties and a reduced mitogenic 

activity. 

Session: Vector Development

31 | 

Or 6 

Restoration of Cone Vision in the CNGA3 −/− 

Mouse Model of Congenital Complete Lack of 

Cone Photoreceptor Function 

Stylianos Michalakis 1,2 , Regine Mühlfriedel 3 , 

Naoyuki Tanimoto 3 , Vidhyasankar 

Krishnamoorthy 4 , Susanne Koch 1,2 , M. Dominik 

Fischer 3 , Elvir Becirovic 1,2 , Lin Bai 1,2 , Gesine 

Huber 3 , Susanne C Beck 3 , Edda Fahl 3 , Hildegard 

Büning 4 , François Paquet-Durand 3 , Xiangang 

Zong 1,2 , Tim Gollisch 5 , Martin Biel 1,2 , Mathias W. 

Seeliger 3 

1 Department of Pharmacy – Center for Drug 

Research, Ludwig-Maximilians-Universität 

München, Munich, Germany; 2 Center for 

Integrated Protein Science Munich (CIPSM), 

Department of Pharmacy – Center for Drug 

Research, Ludwig-Maximilians-Universität 

München, Munich, Germany; 3 Division of Ocular 

Neurodegeneration and Division of Experimental 

Ophthalmology, Institute for Ophthalmic 

Research, Centre for Ophthalmology, Eberhard 

Karls-Universität, Tübingen, Germany; 4 Clinic I of 

Internal Medicine and Center for Molecular 

Medicine Cologne (CMMC), University Hospital 

Cologne, University of Cologne, Cologne, 

Germany; 5 Visual Coding Group, Max Planck 

Institute of Neurobiology, Martinsried, Germany 

Congenital absence of cone photoreceptor 

function is associated with strongly impaired 

daylight vision and loss of color discrimination in 

human achromatopsia. Here, we introduce rAAVmediated 

gene replacement therapy as a 

potential treatment for this disease in the 

CNGA3 −/− mouse model. We show that such 

therapy can restore cone-specific visual 

processing in the CNS even if cone 

photoreceptors had been nonfunctional from 

birth. The restoration of cone vision was 

assessed at different stages along the visual 

pathway. Treated CNGA3 −/− mice became able to 

generate cone photoreceptor responses and to 

transfer these signals to bipolar cells. In support, 

we found morphologically that treated cones 

expressed regular CNG channel complexes and 

opsins in outer segments, which previously they 

did not. Moreover, expression of CNGA3 

normalized cGMP levels in cones, reduced the 

inflammatory response of Müller glia cells that is 

typical of retinal degenerations and delayed cone 

cell loss. Furthermore, ganglion cells from 

treated, but not from untreated CNGA3 −/− mice 

displayed cone-driven light-evoked spiking 

activity, indicating that signals generated in the 

outer retina are transmitted to the brain. Finally, 

we demonstrate that this newly acquired sensory 

information was translated into cone-mediated 

vision-guided behavior. 


Or 7 

High Throughput Integration Site Analysis 

Reveals a Polyclonal Lineage-Specific 

Integration Site Distribution in a Successful 

WAS Gene Therapy Trial 

Anna Paruzynski 1 , Kaan Boztug 2 , Ali Nowrouzi 1 , 

Claudia R. Ball 1 , Anne Arens 1 , Christina Lulay 1 , 

Marie Böhm 2 , Sonja Naundorf 3 , Klaus Kühlcke 3 , 

Rainer Blasczyk 4 , Irina Kondratenko 5 , Laszló 

Maródi 6 , Hanno Glimm 1 , Christoph Klein 2 , 

Christof von Kalle 1 , Manfred Schmidt 1 

1Department of Translational Oncology, National 

Center for Tumor Diseases (NCT) and German 

Cancer Research Center (DKFZ), Hannover, 

Germany; 2 Department of Pediatric 

Hematology/Oncology, Hannover Medical 

School, Hannover, Germany; 3 EUFETS AG, Idar- 

Oberstein, Germany; 4 Institute for Transfusion 

Medicine, Hannover Medical School, Hannover, 

Germany; 5 Department of Clinical Immunology, 

Russian Clinical Children's Hospital, Moscow, 

Russia; 6 Department of Infectious and Pediatric 

Immunology, Medical and Health Science Center, 

University of Debrecen, Debrecen, Hungary 

The Wiskott Aldrich Syndrome (WAS) is a 

complex primary immunodeficiency disease 

affecting both the lymphoid and myeloid 

compartment and leading to the development of 

thrombocytopenia, eczema, autoimmunity and an 

increased risk of malignancies. In a German 

WAS clinical gene therapy trial, two patients were 

successfully treated by receiving autologous 

CD34 + cells transduced with a MLV-based vector 

encoding the WAS gene. By performing linear 

amplification-mediated PCR (LAM-PCR) 

combined with high-throughput sequencing, we 

were able to retrieve > 5700 unique integration 

sites (IS) for patient 1 (P1) and > 9500 unique IS 

for patient 2 (P2). In both patients, the genecorrected 

hematopoiesis showed a polyclonal IS 

pattern up to 3 years after gene therapy. The 

analysis of the clonal composition revealed a 

high clustering of IS in specific genomic regions 

with more than 70 common integration sites (CIS) 

of ≥ 10th order. Strikingly, this high clustering 

could be found amongst others within or close to 

the proto-oncogenes MDS1-EVI1 (P1: 81 IS; P2: 

94 IS), PRDM16 (P1: 10 IS; P2: 28 IS), LMO2 

(P1: 13 IS; P2: 29 IS) and CCND2 (P1: 11 IS; P2: 



18 IS). A quantitative analysis further revealed 

that the most active cell clones showed a vector 

integration upstream of CCND2 in P1 and within 

the MDS1 locus in P2 constantly contributing to 

the gene-corrected hematopoiesis. The analysis 

of sorted cell fractions showed a distinct IS 

distribution with the appearance of IS within or 

close to the MDS1-EVI1 and PRDM16 gene loci 

almost exclusively in the myeloid fraction, 

whereas IS within or close to the LMO2 and 

CCND2 gene loci were preferentially detected in 

the lymphoid compartment. Even though we 

detected several IS within or close to already 

known proto-oncogenes, our IS analysis shows a 

highly polyclonal reconstitution of the 

hematopoietic system until the latest time point 

analyzed (1108 and 1071 days after gene 

therapy, respectively) without any apparent signs 

of clonal outgrowth. The monitoring of the clonal 

inventory and a large scale tracking of individual 

clones is ongoing to further study the efficacy and 

safety of the vector used for this clinical WAS 

gene therapy trial. 


Or 8 

Enhanced Therapeutic Neovascularization via 

AAV2.9/Thymosin β4: Evidence for a 

Myovascular Crosstalk 

Teresa Trenkwalder 1 , Rabea Hinkel 1 , Shahana 

Sultana 1 , Georg Stachel 1 , Achim Pfosser 1 , 

Corinna Lebherz 1 , Ildiko Bock-Marquette 2 , 

Elisabeth Deindl 3 , Christian Kupatt 1 

1Internal Medicine I, Klinikum Großhadern, LMU 

München, Munich, Germany; 2 Southwestern 

Medical Center, Dallas, USA; 3 Walter-Brendel- 

Zentrum für Experimentelle Medizin, LMU 

München, Munich, Germany 

Thymosin β4 (Tβ4) is known to be involved in 

wound healing, coronary vessel formation and 

cardioprotection, alltogether implying an activated 

Protein Kinase B. We investigated the role of 

Thymosin β4 and AKT activation in angiogenesis 

and arteriogenesis using recombinant AAV2/9 

(rAAV) gene transfer in a model of chronic 

hindlimb ischemia. We hypothesise a 

myovascular crosstalk in which the Tβ4 induced 

stimulation of myocytes results in an enhanced 

neovascularisation. Methods: Mice (n = 8) were 

subjected to 3 × 10 12 r.AAV encoding for Tβ4, wt- 

AKT, AKT-DN, AKT-DN-Tet-off, GFP-Tet-off or 

Lac-Z 14 days before femoral artery ligation (d0). 

Recovery of blood flow was assessed by Laser 



| 32 

Doppler flowmetry on day 3, 7 and 14 after 

ligation. To further study the reversibility of the 

AAV system, we generated Tet-off vectors 

encoding for GFP and AKTDN. Doxycycline 

(Dox) was applied from day 3 to day 14 after 

femoral ligation and thereby disabeling gene 

transcription. Capillary density was assessed via 

immunostaining for PECAM-1 (c/mf). Results: 

rAAV-based overexpression of Tβ4 (0.80 ± 0.13) 

or wt-AKT (0.79 ± 0.02) showed an increase in 

perfusion in flow measurements on day 7 in 

comparison to the control group (0.59 ± 0.1). The 

application of AKT-DN (0.43 ± 0.12) resulted in 

distinctly lower perfusion. Furthermore, capillary 

density was significantly increased in mice 

treated with Tβ4 (1.71 ± 0.05 c/mf) or wt-AKT 

(1.67 ± 0.06 c/mf) in comparison to the AKT-DN 

group (1.14 ± 0.08 c/mf) which stayed on control 

level (1.1 ± 0.01 c/mf). Using the inducible Tet- 

System GFP-positive myocytes were only 

detectable in the hindlimb muscles in the 

absence of Dox. Injection of rAAV.Tet-off-AKT 

reduced perfusion in the ischemic limb (0.3 ± 

0.01) in the absence of Dox, whereas application 

of Doxycyline from day 3 on normalized recovery 

(0.73 ± 0.04). We conclude that rAAV-Tβ4 or 

rAAV.AKT-transduction of skeletal myoctes 

activates a myovascular crosstalk resulting in 

enhanced angiogenesis and arteriogenesis. The 

Tet-off system, switching off gene transcription in 

the presence of Doxycycline, elucidates the 

relevance and reversibility of early AKT-inhibition 

for functional neovascularization. 

Session: Gene Therapy

33 | 

Or 9 

Nonviral Delivery of the Rat PDX1 Gene to Rat 

Liver For the In Vivo Transdifferentiation of 

Liver Cells to Pancreatic Beta-Cells 

Abdullah Cim 1 , Greta Sawyer 1 , Xiahong Zhang 1 , 

Haibin Su 1 , Louise Collins 1 , Peter Jones 2 , 

Michael Antoniou 3 , Hans J. Lipps 4 , John Fabre 4 

1 Department of Hepatology and Transplantation, 

King's College London, School of Medicine, 

London, UK; 2 Beta Cell Development & Function 

Group, King's College London, School of 

Biomedical & Health Sciences, London, UK; 

3 Department of Medical and Molecular Genetics, 

King's College London, School of Medicine, 

London, UK; 4 Department of Cell Biology, 

University of Witten/Herdecke, Witten/Herdecke, 


Adenoviral vectors delivering single or multiple 

pancreatic transcription factors can induce liver 

cells or pancreatic exocrine cells to 

transdifferentiate towards pancreatic beta-cells. 

Nonviral approaches for gene delivery would be 

much more clinically applicable, but the level and 

longevity of gene expression necessary are 

unknown. We therefore evaluated five DNA 

expression constructs with different functional 

elements for the level and longevity of expression 

of the rat transcription factor pdx1 delivered 

hydrodynamically to rat liver. We also evaluated 

the degree of transdifferentiation towards a 

pancreas phenotype. Groups of three or four rats 

were harvested at days 1, 3, 7, 14 and 28. Pdx1 

from the plasmids and insulin 2 as a marker of 

transdifferentiation were assayed quantitatively 

using Taqman PCR. The pEPI plasmid, with a 

CMV promoter and a ≈2kb Scaffold/Matrix 

Attachment Region, gave strong pdx1 expression 

at day 1, but levels at day 3 were ≈150 fold lower. 

Substituting alpha1-antitrypsin promoters, with or 

without a ubiquitous chromatin opening element 

(UCOE), gave sustained pdx1 expression to day 

3, with a more gradual decline thereafter. 

However, actual levels of expression were 20 to 

300-fold lower than with the pEPI with CMV 

promoter. A CpG-depleted plasmid (InvivoGen) 

gave both high levels and sustained expression. 

Only the CpG-depleted plasmid induced a 

pancreatic phenotype in rat liver. Insulin 2 

expression 30 to 70-fold higher than day 1 liver 

was found in two of three rats at day 14. To 

conclude, nonviral delivery of pdx1 alone to liver 

in a plasmid giving sustained pdx1 expression 

gave weak insulin expression in some rats. 

Multiple transcription factors are currently under 

investigation. 


Or 10 

Correction of Mpl Deficiency by Lentiviral 

Vectors with Lineage-Specific Expression 

Ute Modlich 1 , Dirk Heckl 1 , Daniel Wicke 1 , 

Guntram Büsche 2 , Martijn Brugman 1 , Johann 

Meyer 1 , Axel Schambach 1 , Matthias Ballmaier 3 , 

Christopher Baum 1 

1Department of Experimental Hematology, 


2 Department of Pathology, Hannover Medical 

School, Hannover, Germany; 3 Department of 

Pediatric Hematology, Hannover Medical School, 

Hannover, Germany 

Congenital amegakaryocytic thrombocytopenia 

(CAMT) is a rare genetic disorder that is caused 

by inactivating mutations in the Thrombopoietin 

receptor MPL. Children with CAMT develop 

severe thrombocytopenia and aplastic anemia 

leading to early death. With the aim to develop a 

gene therapy for CAMT we retrovirally 

overexpressed Mpl. First experiments expressing 

Mpl C57Bl/6 wt mice caused severe adverse 

reactions (SAE), e.g., uncontrolled expansion of 

stem and progenitor cells (CMPD), stem cell 

alterations (MDS like phenotype) and increased 

frequency of insertional leukemias. To avoid 

those SAE, we developed self-inactivating 

lentiviral vectors that expressed Mpl by the 

lineage-specific human and murine Mpl or human 

GPIba promoter. For comparison we used the 

ubiquitously active PGK promoter. In vivo 

expression was restricted to megakaryocytes 

(CD41 + ) and hematopoietic stem cells (LSK 

CD34 − ). We established a murine model for 

CAMT gene therapy based on the transplantation 

of Mpl −/− hematopoietic cells into Mpl −/− mice. Mpl 

expressed by the PGK promoter caused death 6 

days post transplantation (6/7 mice) due to 

spleen rupture after erythroid hyperproliferation 

showing the extreme sensitivity to ectopic Mpl 

expression. Mice transplanted with bone marrow 

(BM) that express Mpl from the lineage specfic 

promoters survived long term while mice that 

were transplanted with eGFP transduced Mpl −/− 

BM cells showed increased lethality during 

establishment of long term hematopoiesis (>12 

weeks). Both early post-transplant complications 

were prevented when using the lineage-specific 

promoters. Furthermore platelet production was 

restored in ≈ 50% of transplanted mice (n = 23) 

and multinucleated megakaryocytes were found 

in the BM 6-7 months after transplantation. In 

addition, the number LSK cells was significantly 

increased in Mpl-corrected animals. The 



engraftment of secondary Mpl −/− recipients with 

Mpl corrected BM of primary mice gave further 

evidence for the correction of long term HSC. Our 

results show the potential of newly developed 

transcriptionally regulated lentiviral vectors to 

circumvent genotoxic and transgene related, 

potentially lethal adverse reactions. 


Or 11 

LCMV-Pseudotyped VSV-Based Systems for 

Treatment of Malignant Glioma 

Alexander Muik 1 , Inna Kneiske 1 , Tsanan 

Giroglou 1 , Gert Zimmer 2 , Stefan Momma 3 , 

Dorothee von Laer 4 

1Applied Virology and Gene Therapy Unit, Georg- 

Speyer-Haus, Frankfurt am Main, Germany; 

2 Institute of Virology and Immunoprophylaxis, 

Mittelhäusern, Switzerland; 3 Institute of 

Neurology, Johann Wolfgang Goethe University, 

Frankfurt am Main, Germany; 4 Section of 

Virology, Innsbruck Medical University, 

Innsbruck, Austria 

Malignant glioma is the most frequent primary 

brain tumor and still has a very poor prognosis 

despite advances in neurosurgical resection and 

adjuvant radio- and chemotherapy. A promising 

new approach is the use of vesicular stomatitis 

virus (VSV)-based systems for glioma-targeted 

oncolytic virotherapy and/or gene therapy. To 

improve glioma specificity and to abrogate the 

VSV G-mediated VSV-inherent neurotropism, we 

pseudotyped VSV with the LCMV glycoprotein 

(LCMV GP). Retargeting to glioma cells while 

sparing healthy neurons was successfully shown 

in vitro, using a panel of different brain tumor cell 

lines, human neural as well as brain tumor stem 

cells and human primary neurons. The reduction 

of neurotropism was confirmed in vivo in a rat 

model after intracranial injection of replicationdeficient 

VSV-WT and LCMV-GP pseudotype 

vectors, encoding the eGFP gene. Furthermore, 

we investigated different systems to address viral 

spread and microdistribution of LCMV GPpseudotyped 

VSV within the tumor. Besides the 

generation of a replication-competent LCMV GP 

pseudotype rVSV(GP) as a pure virotherapy 

approach, we were able to show that tumorinfiltrating 

adult stem cells can be used as a 

delivery system and packaging cell line for noncytopathic, 

replication-deficient VSV vectors 

encoding the HSV-TK as suicide gene. Efficacy 

of rVSVΔG–TK(GP) producing progenitor cells in 



| 34 

mediating tumor killing after ganciclovir treatment 

was shown in vitro in a G62 spheroid model. If 

the reduced neurotoxicity can be confirmed in our 

current studies in vivo, rVSV(GP) would be an 

extremely promising candidate for oncolytic 

virotherapy of brain cancer. 


Or 12 

TBA 

Jennifer Altomonte 1 , Enrico DeToni 2 , Irene 

Esposito 1 , Claus Hellerbrand 3 , Sabrina Marozin 1 , 

Oliver Ebert 1 

1II. Medizinische Klinik und Poliklinik, Klinikum 

rechts der Isar, TU München, Munich, Germany; 

2 Klinikum Großhadern, LMU; München, Munich, 

Germany; 3 Universita¨tsklinikum Regensburg, 



Or 13 

An α-Fetoprotein Promoter Driven, 

Conditionally Replicating Adenovirus that 

Expresses the Sodium Iodide Symporter (NIS) 

for Radiovirotherapy of HCC 

Geoffrey K Grünwald 1 , Kathrin Klutz 1 , Michael J 

Willhauck 1 , Nathalie Wunderlich 1 , Reingard 

Senekowitsch-Schmidtke 2 , Manfred Ogris 3 , Per S 

Holm 4 , Burkhard Göke 1 , Christine Spitzweg 1 

1 Department of Internal Medicine II, Klinikum 

Grosshadern, Ludwig-Maximilians-University 

Munich, Munich, Germany; 2 Department of 

Nuclear Medicine, Technical University Munich, 

Munich, Germany; 3 Pharmaceutical 

Biotechnology, Department of Pharmacy, 


Germany; 4 Institute of Experimental Oncology 

and Therapy Research, Klinikum rechts der Isar, 

TU Munich, Munich, Germany 

We recently reported iodide accumulation and 

therapeutic efficacy of 131 I in hepatocellular 

carcinoma (HCC) cells stably expressing the NIS 

gene under control of the tumor-specific αfetoprotein 

promoter (AFP) for transcriptional 

targeting. As a next crucial step towards clinical 

application of this promising targeting technology

35 | 

we investigated in the current study the in vitro 

and in vivo efficacy of NIS gene transfer using a 

replication-selective oncolytic adenovirus. For 

this purpose NIS gene transfer was performed in 

vitro and in vivo in human HCC cell (HepG2) 

xenografts, using a replication-selective oncolytic 

adenoviral vector carrying the NIS gene linked to 

a mouse AFP-promoter construct (Ad5-E3-AFP- 

NIS). In vitro experiments with HepG2 cells and 

control cell lines demonstrated high transduction 

efficiency and tumor selectivity of Ad5-E3-AFP- 

NIS. Functional NIS protein expression was 

confirmed by measurement of in vitro uptake of 

125 

I in HCC cells showing a 30-fold increase in 

perchlorate-sensitive iodide uptake activity as 

compared to control cell lines. A clonogenic 

assay with HCC cells infected with Ad5-E3-AFP- 

NIS (20 MOI) showed only 6.45 % survival after 

incubation with 0.8 mCi (29.6 MBq) 

131 I as 

compared to 100% survival of untreated HCC 

cells representing the high therapeutic potential 

of this gene therapy strategy. qPCR of NIS and 

fiber protein of the infected cells, a cytopathic 

effect assay and a spread assay showed 

corresponding results clearly demonstrating the 

replication selectivity due to the AFP promoter. In 

first in vivo experiments functional NIS protein 

expression after adenoviral NIS gene transfer 

was confirmed by 123 I gamma-camera imaging as 

well as qPCR. HepG2 cell xenografts in nude 

mice injected intratumorally with 5 × 10 8 PFU 

Ad5-E3-AFP-NIS accumulated approx. 11% ID/g 

(percentage injected dose per gram tumor tissue) 

48 h after virus injection with an average 

biological half-life of 5.5 h. These results clearly 

demonstrate the high potential of an α-fetoprotein 

promoter driven, conditionally replicating 

adenovirus for tumor-selective NIS gene delivery 

in liver cancer. 


Or 14 

Tumor Stroma-Specific NIS Gene Delivery 

Using Mesenchymal Stem Cells 

Kerstin Knoop 1 , Marie Kolokythas 1 , Kathrin 

Klutz 1 , Michael J Willhauck 1 , Nathalie 

Wunderlich 1 , Dan Draganovici 2 , Christian Zach 3 , 

Franz Josef Gildehaus 3 , Guido Böning 3 , Burkhard 

Göke 1 , Ernst Wagner 4 , Peter J Nelson 2 , Christine 

Spitzweg 1 

1Department of Internal Medicine II, Klinikum 

Grosshadern, Ludwig-Maximilians-University 

Munich, Munich, Germany; 2 Clinical Biochemistry 

Group, Medical Policlinic, Ludwig-Maximilians- 

University Munich, Munich, Germany; 

3 Department of Nuclear Medicine, Klinikum 

Großhadern, Ludwig-Maximilians-University 

Munich, Munich, Germany; 4 Pharmaceutical 


Ludwig-Maximilians-University, Munich, Germany 

The tumor-homing property of mesenchymal 

stem cells (MSC) has suggested their potential 

use as a delivery vehicle for cancer therapeutics. 

Tracking the delivery and engraftment of MSCs 

into human tumors is a crucial prerequisite for 

safety and effectiveness of MSCs as promising 

gene delivery vehicles. Due to its dual role as 

reporter and therapy gene, the sodium iodide 

symporter (NIS) allows non-invasive imaging of 

functional NIS expression by 123 I-scintigraphy and 

124 I-PET imaging, and thereby provides an 

excellent means to elucidate the capacity of 

MSCs for tumor targeting of therapeutic genes. In 

the current study we stably transfected human 

bone marrow derived CD34 − MSCs with a NISexpressing 

plasmid (CMV-NIS-pcDNA3) (NIS- 

MSC) followed by analysis of NIS expression and 

cytotoxicity of 131 I. In a human hepatocellular 

cancer (HCC) xenograft model (Huh7) we further 

investigated distribution and tumor recruitment of 

NIS-MSCs by I-123-scintigraphy and 124 I-PET 

imaging. NIS-MSCs revealed a 12-fold increase 

in perchlorate-sensitive iodide uptake activity as 

compared to wild-type MSCs. Further, Western 

Blot analysis confirmed NIS protein expression in 

NIS-MSCs. In mixed populations of NIS-MSC 

and HCC cells (ratio 1:1) a clonogenic assay 

showed a 50% reduction of HCC cell survival 

after 131 I application induced by its crossfire 

effect. After establishment of subcutaneous Huh7 

xenografts in nude mice, NIS-MSCs were 

injected via the tail vein. Gamma camera and 

PET imaging revealed active MSC recruitment 

into the tumor stroma as shown by tumorselective 

iodide accumulation (9.5% ID/g 123 I, 

biol. half-life 4h). Immunhistochemistry and ex 

vivo 123 I biodistribution analysis by gamma 

counter analysis confirmed active recruitment of 



NIS-MSCs into the tumor stroma while non-target 

organs showed no significant MSC recruitment. 

Taken together, our results convincingly show 

tumor-specific radioiodine accumulation after 

MSC-mediated NIS gene delivery demonstrating 

tumor stroma-selective MSC recruitment. These 

results open the exciting prospect of tumorspecific 

NIS-mediated radionuclide therapy of 

thyroidal or extrathyroidal tumors using MSCs as 

gene delivery vehicles. 


Or 15 

Monitoring and Excising Reprogramming 

Factors: A Novel Lentiviral Expression 

System for Reprogramming Strategies 

Axel Schambach 1 , Eva Warlich 1 , Johannes 

Kühle 1 , Tobias Cantz 2 , Martijn Brugman 1 , Tobias 

Mätzig 1 , Melanie Galla 1 , Adam Filipczyk 3 , 

Hannes Klump 4 , Hans Schöler 5 , Timm 

Schroeder 3 , Christopher Baum 1 

1 Department of Experimental Hematology, 


2 Junior Research Group Stem Cell Biology, 


3 Institute of Stem Cell Research, Helmholtz 

Zentrum München, Munich, Germany; 4 Institute 

for Transfusion Medicine, University Hospital 

Essen, Essen, Germany; 5 Department of Cell 

and Developmental Biology, MPI for Molecular 

Biomedicine, Münster, Münster, Germany 

Cellular identity is largely determined by 

transcription factor networks. The concerted 

action of 4 reprogramming factors (RFs: Oct4, 

Sox2, Klf4, c-Myc) leads to generation of induced 

pluripotent stem cells (iPSC), a promising 

resource for regenerative medicine and disease 

modelling. Here we constructed a modular 

versatile lentiviral vector system for mono- and 

polycistronic expression of these RFs (of either 

murine or human origin and optionally codonoptimized). 

To drive the reprogramming factors, 

we used the strong retroviral promoter SFFV, 

which enables high RF expression in most 

somatic cells and is frequently silenced in ES 

cells. To avoid the risks of residual permanent RF 

expression and insertional mutagenesis, we 

further improved our system by the inclusion of 

FRT, lox or rox sites to be able to remove the RF 

cassette through transient recombinase 

expression. Having the choice of 3 different 

recombinases offers the targeted shut off of RF 

at multiple desired time points. Using this system 



| 36 

and taking advantage of an Oct4-GFP reporter 

model, we were able to generate murine and 

human iPS cells with high efficiency. To learn 

more about the kinetics of iPSC reprogramming 

and to understand better the underlying 

mechanisms, we developed color-coded RF 

vectors to follow expression kinetics qualitatively 

and quantitatively in relation to Oct4-GFP. 

Thereby, we could document the dynamic 

conversion of fibroblasts to pluripotent cells by 

fluorescence microscopy combined with longterm 

single cell tracking and alternatively highdefinition 

structural analysis of single iPSC 

colonies. Filming the “birth” of iPSC generation, 

we obtained movies, which show the potential 

genetic mosaic of early iPSC colonies and also 

indicate the necessity of stochastic epigenetic 

changes during the process of iPSC 

development. With the growing insights in cellspecific 

transcription factor networks our versatile 

vector system will be a good candidate for direct 

reprogramming approaches into somatic cells 

(e.g., neurons). In summary, with the described 

vector system we have an efficient tool in hand 

for “excisable” and safe reprogramming 

strategies in regenerative medicine. 


Or 16 

MHC-Restricted Fratricide of Recipient 

Lymphocytes Expressing Transgenic T Cell 

Receptors Specific for the Apoptosis-Inhibitor 

Protein Survivin 

Matthias Leisegang 1 , Susanne Wilde 2 , Stefani 

Spranger 2 , Slavoljub Milosevic 2 , Bernhard 

Frankenberger 2 , Dolores J Schendel 2 , Wolfgang 

Uckert 1 

1 Max-Delbrück-Center for Molecular Medicine, 

Berlin, Germany; 2 Helmholtz Zentrum München, 


Expression of T cell receptors (TCR) as 

transgenic proteins in peripheral blood 

lymphocytes (PBL) enables T cells with defined 

tumor specificity to be generated in high numbers 

for patient-individualized therapy (TCR gene 

therapy). Hereby, the selection of tumorassociated 

antigens (TAA) that can be effectively 

targeted on tumor cells is of central importance. 

Survivin, a well-characterized inhibitor of 

apoptosis, has been suggested as a candidate 

TAA because of its potential role in oncogenicity 

and its broad expression in most tumors but 

absence from most normal adult tissues. To

37 | 

explore use of survivin as a target antigen for 

TCR gene therapy, we generated HLA-A2 allorestricted 

survivin-specific T cells with high 

functional avidity. We isolated several high 

affinity TCR recognizing peptides derived from 

survivin presented by HLA-A2 molecules. 

Following transfer of these TCR into HLA-A2- 

recipient lymphocytes, we found that the effector 

cells displayed excellent specific killing of HLA- 

A2 + survivin + tumor cells, indicating their 

therapeutic potential. Surprisingly, when these 

TCR were expressed as transgenic proteins in 

HLA-A2 + recipient lymphocytes, as they would 

be applied in the clinical setting, we discovered 

that the recipient lymphocytes underwent 

extensive apoptosis over time. This demise was 

caused by HLA-A2-restricted fratricide that 

occurred due to survivin expression in recipient 

lymphocytes, which created ligands for 

transgenic TCR recognition. Furthermore, we 

could demonstrate that cytotoxic T cell clones of 

various specificities derived from HLA-A2 + 

donors were directly killed by survivin-specific 

TCR-modified PBL. Therefore, survivin-specific 

TCR gene therapy would be limited to application 

in HLA-A2-mismatched stem cell transplantation. 

However, these results raise a general question 

regarding cancer vaccines targeting proteins that 

are also expressed in activated lymphocytes, 

since induction of high avidity T cells that expand 

in lymph nodes following vaccination or later 

accumulate at tumor sites might limit themselves 

by self-MHC-restricted fratricide and also 

eliminate neighboring T cells of other 

specificities. 



Or 17 

Integration Site Analysis of Reprogrammed T 

Cells in a Mouse Model of T Cell Receptor 

Gene Therapy Developing Graft Versus Host 

Disease 

Eliana Ruggiero 1 , Gavin M Bendle 2 , Ton N. 

Schumacher 2 , Hanno Glimm 1 , Christof von 

Kalle 1 , Manfred Schmidt 1 , Carsten Linnemann 1 , 

Laura Bies 1 

1German Cancer Research Center and National 

Center for Tumor Diseases, Heidelberg, 

Germany; 2 The Netherlands Cancer Institute, 

Amsterdam, The Netherlands, Amsterdam, The 

Netherlands 

The genetic modification of immune cells and the 

generation of recombinant vectors encoding 

cancer antigens have highlighted the great 

potential of immunotherapy in treating cancer. 

Since the ability of a T cell to specifically 

recognize an antigen relies on the T cell receptor 

(TCR), a tumor reactive T cell repertoire can be 

generated by the genetic introduction of 

transgenes encoding TCR directed towards 

specific tumor antigens. We applied our 

experience in the comprehensive genome wide 

retrieval of gammaretroviral insertions sites (IS) 

to analyze the integration site distribution and the 

potential influences on the clonal fate of 

transduced cells after TCR gene transfer in order 

to assess their safety for clinical trials. We 

performed IS analysis combined with highthroughput 

sequencing on mouse CD4 and CD8 

T cells transduced with a gamma retroviral vector 

encoding an ovalbumin-specific OT-I TCR. Mice 

receiving IL-2 administration after OT-I TCR 

transplantation developed Graft-versus-Host- 

Disease (GvHD). In total, we found 841 unique IS 

in OT-I transduced mice and 1063 unique IS in 

control GFP transduced mice with a higher 

clustering of IS in genes involved in the immune 

response (e.g., Cd28, Ctla4) in OT samples 

compared to the control samples. Results from 

the Ingenuity Pathways Analysis indicated the 

presence of specific gene classes, Hematological 

disease and Inflammatory response, 

preferentially targeted in mice transduced with 

the OT-I encoding vector compared to the mice 

transduced with the GFP vector (p = 7.84*10 −4 

and p = 2.13*10 −2 , respectively). In GvHD 

developing mice we observed a higher frequency 

of IS located in gene and in the surrounding 10 kb 

compared to the control counterpart (74% and 

65%, respectively; p = 3*10 −2 ) and an increased 

contribution of integration clones to genecorrected 

hematopoiesis, indicating the 

occurrence of a potential in vivo skewing. Further 

sequencing and statistical analyses are ongoing. 



Our results provide important insights in IS 

distribution, clonal diversity and identification of 

genes preferentially targeted by TCR specific 

gamma retroviral vectors. 



Or 18 

Luciferase-Based Dual Bioluminescence 

Imaging of Tumor Metastases After Systemic 

Transgene Delivery with a Synthetic Gene 

Carrier 

Katarina Farkasova 1 , Arzu Cengizeroglu 1 , Rudolf 

Haase 1 , Martina Anton 2 , Joachim Ellwart 3 , Ernst 

Wagner 1,4 , Manfred Ogris 1,4 

1 Pharmaceutical Biotechnology, Department of 

Pharmacy, Ludwig-Maximilians-University, 

Munich, Germany; 2 Institute of Experimental 

Oncology and Therapy Research, Klinikum 

rechts der Isar, TU Munich, Munich, Germany; 

3 Institute of Molecular Immunology, Helmholtz 

Center Munich, Munich, Germany; 4 Center for 



Noninvasive, biooptical in vivo imaging is a 

powerful method to study the biodistribution and 

pharmacodynamics of labeled drugs in the living 

organism. For studies on nucleic acid delivery, 

the use of luciferase gene allows the highly 

sensitive detection of transgene expression by 

bioluminescence imaging (BLI). Here we 

describe a method, which allows both monitoring 

the growth of tumor metastases and the 

visualization of transgene expression after 

intravenous delivery of nonviral gene vectors in a 

syngeneic mouse tumor model. Murine 

neuroblastoma cells (Neuro2A) were stably 

transduced with a firefly luciferase and enhanced 

green fluorescent protein (EGFP) encoding 

lentiviral vector. EGFP sorted cells were injected 

either intravenously or intrasplenic into A/J mice 

and the tumor growth was monitored by BLI after 

intraperitoneal injection of luciferin, the substrate 

for firefly luciferase. After development of well 

vascularized tumor metastases, polyplexes 

containing a novel plasmid encoding for 

membrane-bound gaussia luciferase (Santos et 

al., 2009, Nature Medicine 15, 338–344) and a 

polycationic, biodegradable gene carrier were 

applied via the tail vein. Gaussia transgene 

expression was monitored after intravenous 

injection of its substrate coelenterazine. Due to 

the different substrates applied for firefly and 



| 38 

gaussia luciferases, we could clearly distinguish 

between the firefly luciferase signal from the 

labeled tumor cells and the gaussia luciferase 

signal from cells expressing the gaussia 

transgene. After intravenous injection of 

polyplexes, gaussia transgene was only 

observed in areas were firefly luciferase labeled 

tumors were located, whereas in tumor-free 

animals no significant gaussia signal was 

observed. Due to a time lag between luciferase 

and coelenterazine injection, crosstalk between 

firefly and gaussia BLI signal was avoided. This 

study clearly shows, that polyplexes based on 

biodegradable polycations can selectively 

transfect well vascularized tumor metastases and 

dual BLI allows to distinguish between tumor 

localization and transgene expression. 


Or 19 

Retroviral and Transposon-Based Tet- 

Regulated All-In-One Vectors with Reduced 

Background Expression and Improved 

Dynamic Range 

Niels Heinz 1 , Rainer Loew 2 , Christopher Baum 1 , 

Bernhard Schiedlmeier 1 

1 Experimental Hematology, Hannover Medical 

School, Hannover, Germany; 2 EUFETS GmbH, 

Idar-Oberstein, Germany 

The constitutive expression of therapeutic genes 

might interfere with cell fate in vivo. Therefore, 

regulated transgene expression is of major 

interest for gene therapy. Such a system needs 

to provide tightly controlled inducible promoters, 

as shown for the tetracycline regulatory system 

(tet-system). However, its application requires the 

introduction of two components into the target 

cell genome: the tet-responsive transactivator 

and the regulated expression cassette. In order 

to render the tet-system valuable for approaches 

in gene therapy, preselection of transactivator 

expressing cells has to be avoided, thus both 

components for regulated transgene expression 

have to be provided by a single vector. Up to 

date, published All-In-One vectors led to 

regulatory windows of around 500-fold, even after 

selection for single cell clones, thereby displaying 

a relatively high signal-to-noise ratio. In this 

study, we develop optimized All-In-One vectors 

which display tight transgene regulation without 

the need for clonal selection. Improvements in 

the dynamic range could be achieved through 

modifications of the vector architecture combined

39 | 

with the introduction of newly developed tetresponsive 

promoters, Ptet. These optimizations 

increased the dynamic range to levels up to 

14,000-fold for retroviral and 25,000-fold for 

nonviral transposon vectors in non-clonal human 

cell lines, and up to 2,800-fold in murine 

hematopoietic cell lines. This improved regulation 

was based on a strong reduction of background 

expression in the off-state, even if cells were 

transduced at high multiplicity of infection, while 

induction remained at high levels. Our results 

indicate that an optimized signal-to-noise ratio of 

transgene expression in different target cells 

depends on vector architecture, the choice of the 

inducible promoter and the integration pattern. 


Or 20 

High-Throughput Integration Site Analysis for 

Vector Biosafety Assessment in CGD Gene 


Simone J. Scholz 1 , Margarita Diaz 2 , Stefan 

Stein 2 , Axel Schambach 3 , Hanno Glimm 1 , 

Christof von Kalle 1 , Manuel Grez 2 , Manfred 

Schmidt 1 

1German Cancer Research Center and National 

Center for Tumor Diseases, Heidelberg, 

Germany; 2 Institute for Biomedical Research, 

Georg-Speyer-Haus, Frankfurt am Main, 

Germany; 3 Experimental Hematology, Hannover 

Medical School, Hannover, Germany 

In several (pre)clinical gene therapy trials 

insertional mutagenesis by integrating 

gammaretroviral vectors was observed. In a 

clinical trial for the treatment of X-linked chronic 

granulomatous disease (X-CGD) insertional 

activation of MDS1/EVI1 has led to the clonal 

expansion of gene marked myeloid progenitor 

cells, triggering the development of monosomy 7 

and a myelodysplastic syndrome in both patients 

enrolled. Therefore, a new self-inactivating (SIN) 

vector containing an internal cellular promoter (cfes) 

and a codon-optimized transgene 

(SINfes.gp91s) was developed for the treatment 

of X-CGD improving the safety and efficacy of 

gene delivery (Manuel Grez, Georg-Speyer- 

Haus, Frankfurt). As part of the preclinical testing 

we performed high-throughput integration site 

(IS) analysis and deep sequencing (454/Roche) 

with associated bioinformatical tools on mice 

transplanted with gene modified lineage markernegative 

hematopoietic stem cells. As a control a 

full LTR SFFV-promoter driven vector 

(SF91eGFP) – as it was used in the previous trial 

– was analyzed in parallel. In total, we found 

1159 unique IS in SF91eGFP transduced mice (n 

= 4) and 2261 unique IS in SINfes.gp91s 

transduced mice (n = 5). While we did not 

observe any significant differences in the 

integration site distribution between the vectors, 

high-throughput sequencing revealed a less 

polyclonal IS pattern in SF91eGFP mice 

compared to the SINfes.gp91s transplanted mice 

(in average 281 vs. 442 unique IS/mouse). 

Furthermore, we found an increased frequency of 

(pre)dominant clones in mice transduced with the 

SF91eGFP vector than with SINfes.gp91s. 

Moreover, SF91eGFP integrants are more 

frequently found in/near EVI1/MDS1 than 

SINfes.gp91s integrants (9 of 1065 and 2 of 

2115, respectively; p = 6.7*10 −4 ). Finally, we could 

observe an increased in vivo skewing in 

secondary recipients of SF91eGFP transduced 

mice compared to SINfes.gp91s (p = 2.9*10 −6 ). 

Overall, our results showed no signs of obvious 

vector-induced side-effects (no loss of 

polyclonality, no obvious skewing) for the 

SINfes.gp91s vector. * contributed equally 


Or 21 

Modification of the Factor X Serine Protease 

Domain Ablates Heparan Sulfate 

Proteoglycan Engagement by Ad5-FX 

Complexes 

Margaret R. Duffy 1 , A.C. Bradshaw 1 , A.L. 

Parker 1 , S.A. Nicklin 1 , J.H. McVey 2 , A.H. Baker 1 

1Glasgow Cardiovascular Research Centre, 

University of Glasgow, Glasgow, UK; 

2 Thrombosis Research Institute, London, UK 

Adenoviruses are commonly used vectors in 

gene therapy clinical trials, the majority of which 

are based on serotype 5. Following intravascular 

administration in vivo Ad5 exhibits high 

sequestration to the liver and spleen. Recent 

studies show that hepatocyte transduction by 

Ad5 is mediated by a high affinity interaction 

between coagulation factor X (FX) and the 

adenovirus 5 hexon proteins bridging the 

adenovirus capsid to the cell hepatocyte surface 

putatively through interaction with heparan 

sulfate proteoglycan receptors (HSPGs). 

Pharmacological blockade of the heparin-binding 

exosite in the serine protease (SP) domain of FX 

prevents cell surface binding and gene transfer 

mediated through FX1. However the exact 



HSPG-interacting residues in the SP domain of 

FX require further elucidation. Previous studies 

have indicated that the basic residues Arg-316, 

Lys-319, Arg-349, Arg-390, Lys-394, Lys-463 and 

Arg-467 of FX constitute the exosite in the SP 

domain2. We therefore sought to generate a FX 

mutant and assess the importance of this exosite. 

A human FX plasmid construct with mutations at 

Arg-316, Lys-319, Arg-349, Arg-390, Lys-394, 

Lys-463 and Arg-467 was generated and 

confirmed by sequence analysis. Recombinant 

FX (mutant and wildtype) was produced by 

plasmid transfection into 293T cells in the 

presence of vitamin K with collection of the 

supernatant. Enzyme-linked immunosorbent 

assay was used to quantify the rFX. In vitro 

assays demonstrated that the exosite mutations 

blocked the ability of FX to enhance transduction 

of Ad5 thus confirming the importance of this 

exosite in HSPG engagement. [1] Waddington 

2008 Cell 132,397-409; [2] Rezaie 2000 JBC 

132,3320-3327. 




| 40

41 | 

DG-GT 2010 Poster Presentations 

P 1 

Development of a Stable Lyophilized 

Formulation for LPEI Polyplexes 

Julia C Kasper 1 , David Schaffert 2 , Manfred 

Ogris 2 , Ernst Wagner 2 , Wolfgang Frieß 1 

1 Department of Pharmacy, Pharmaceutical 

Technology and Biopharmaceutics, Ludwig- 

Maximilians-University Munich, Munich, 

Germany; 2 Department of Pharmacy, 

Pharmaceutical Biotechnology, Ludwig- 

Maximilians-University Munich, Munich, Germany 

Polyplexes based on linear polyethylenimine 

(LPEI) are efficient non-viral gene delivery 

systems but the requirement for freshly prepared 

formulations prior to administration due to the 

instability in aqueous suspension is a major 

drawback. Therefore, the aim was to establish an 

up-scaled and reproducible method for the 

preparation of standardized batches of welldefined 

LPEI polyplexes and to develop isotonic 

lyophilized polyplex formulations with long-term 

stability. Polyplexes consisting of pCMVLuc 

plasmid and 22 kDa LPEI were prepared by 

classical hand pipetting or using an up-scaled 

micro-mixer method at different mixing speeds. 

Freeze-thawing and lyophilization were 

performed on a laboratory scale freeze-drier. 

Several excipients at varying concentrations were 

tested for their cryo- and lyoprotectant potential. 

To evaluate long-term stability, lyophilized 

samples were stored at 2-8°C, 20°C and 40°C for 

6 weeks. The z-average diameter and 

polydispersity index (PdI) of the polyplexes was 

measured by dynamic light scattering. 

Transfection efficiency was evaluated in murine 

neuroblastoma (Neuro 2A) cells and metabolic 

activity as an indicator for cell toxicity was 

analyzed. By using the up-scaled micro-mixer 

method with varied mixing speeds, size (59–197 

nm) and PdI (0.05-0.19) of the polyplexes can be 

directly controlled. When formulated with 14% 

lactosucrose, 10% HP-beta-CD/6.5% sucrose or 

10% povidone/6.3% sucrose at isotonic 

concentrations, the increase in polyplex size was 

less than 50% after freeze-thawing, freeze-drying 

and during storage. Transfection efficiency and 

metabolic activity was high when polyplexes were 

formulated with lactosucrose or HP-beta- 

CD/sucrose but decreased when PVP/sucrose 

was used as stabilizer. The possibility to 

reproducibly manufacture large standardized 

batches of well-defined, transfection efficient 

polyplexes with long-term stability by using the 

established up-scaled preparation method 

followed by lyophilization is an important step 

closer from promising technology to clinical 

application. 


P 2 

Recombinant Influenza C Virus: A New 

Prototype of a Melanoma-Specific Oncolytic 

Virus 

Karin Pachler, Juliane Mayr, Reinhard Vlasak 

University of Salzburg, Department of Molecular 

Biology, Salzburg, Austria 

The ability to genetically engineer viruses 

contributes greatly to our understanding of their 

life cycle and allows creation of virus-derived 

vectors for vaccine development, gene therapy or 

oncolysis. In the past decade, several reversegenetics 

systems have been developed to 

manipulate the genomes of influenza A and B 

viruses. Here, we introduce a system for the 

generation of recombinant influenza C viruses 

from seven plasmids.The seven full-length viral 

cDNAs were cloned into an RNA pol I/pol IIbased 

bidirectional vector. Transfection of Vero 

cells and subsequent amplification on MDCK 

cells yielded viral HA titres of 128. With this 

efficient method, mutations or foreign genes may 

be easily incorporated into the influenza C virus 

genome. Next, the influenza C virus protein NS1 

was identified as a factor antagonising the 

cellular interferon (IFN) signalling, and the 

regions of NS1 necessary for IFN inhibition were 

mapped. Several melanoma cell lines have a 

defective IFN signalling and express the cellular 

receptor for influenza C virus, which is 9-O-acetyl 

sialic acid. Thus NS1-deficient recombinant 

influenza C viruses could be engineered to serve 



as gene delivery vectors and/or tumour-specific 

oncolytic viruses in the future. 


P 3 

Live-Cell Imaging and Single-Particle 

Tracking of Polyplex Internalization 

Nadia Ruthardt 1 , Karla de Bruin 1 , Ernst 

Wagner 2,3 , Christoph Bräuchle 1,3 

1Physical Chemistry, Department of Chemistry, 


Germany; 2 Pharmaceutical Biotechnology, 


University, Munich, Germany; 3 Center for 



We investigate the uptake of targeted and 

untargeted polymeric gene vectors (polyplexes) 

by highly sensitive fluorescence microscopic 

methods by live cell imaging on a single cell 

level. The epidermal growth factor receptor 

(EGFR) is overexpressed on a high percentage 

of human carcinomas and is therefore an 

attractive therapeutic target for tissue-specific 

targeting by non-viral vectors in cancer gene 

therapy. By comparing uptake kinetics and 

internalization dynamics, single particle tracking 

in combination with quenching experiments 

revealed typical three-phase dynamics of the 

uptake process independent of targeting. Phase I 

was characterized by slow, actin-cytoskeletonmediated 

movement of the particles with drift and 

included the internalization process. During 

phase II, particles displayed increased velocities 

with confined and anomalous diffusion in the 

cytoplasm. Phase III was characterized by fast 

active transport along microtubules. Targeting of 

polyplexes for receptor-mediated endocytosis by 

the EGF receptor resulted in shortening of phase 

I and strongly accelerated internalization. 

Targeted as well as untargeted particles were 

transported in early endosomes marked by Rab5- 

GFP and accumulated in late endosomes marked 

by Rab9-GFP. The endosomal release dynamics 

of polyplexes consisting of DNA condensed with 

the cationic polymers linear polyethyleneimine 

(LPEI), poly-(l)-lysine (PLL) or poly-(d)-lysine 

(PDL) were studied by photochemical release in 

living cells. Using double-labeled polyplexes, 

DNA and polymer were imaged simultaneously 

by dual-color fluorescence microscopy. Our 

results demonstrate that the characteristics of the 

cationic polymer significantly influence the 



| 42 

release behavior of the polyplexes. For 

LPEI/DNA particles, LPEI quickly spread 

throughout the cytosol, whereas DNA was 

released slowly and remained immobile 

thereafter. In case of PLL particles, both DNA 

and polymer showed quick release. PDL particles 

remained condensed upon photosensitizer 

activation. 


P 4 

Generation of the Novel, Synthetic Hybrid 

SCE-Promoter for Gene-Therapeutical 

Applications 

Rudolf Haase, Terese Magnusson, Ernst 

Wagner, Manfred Ogris 

Pharmaceutical Biotechnology, Department of 



A major factor for successful gene therapy is 

strong and enduring expression of the transgene. 

Commonly used promoters for expression of 

transgenes are the CMV-immediate early (CMV- 

IE), chicken β-actin, ubiquitin C or the EF1α 

promoter, each with their own advantages and 

disadvantages. Here we present the generation 

of novel synthetic hybrid promoter, based on the 

CMV-IE and the EF1α promoter. It was obtained 

by generating shuffled consensus sequences of 

both promoters, which were analyzed by 

promoter prediction software. One sequence, the 

shuffle CMV/EF1α promoter (SCE), was 

synthesized and tested in vitro and in vivo. The 

SCEP sequence features an identity of 83% with 

the CMV and 85% with the EF1α promoter. The 

sequence was CpG-free designed to circumvent 

inflammatory reactions towards CpG isles and 

promoter methylation leading to silencing. In 

several cell lines a 2-7 fold stronger expression of 

the SCE promoter compared to the EF1α was 

observed; when compared to CMV-IE, SCE 

promoter activity was almost similar. All 

promoters were within a CpG-free plasmid 

featuring a human CMV-IE enhancer and 

luciferase as transgene. In immunocompetent 

Balb/c mice we observed a ≈3 fold stronger 

transgene expression of SCEP compared to the 

EF1α promoter after hydrodynamic gene delivery 

to the liver for a time period of 30 days. Hence 

the SCE promoter is qualified for further 

applications in biotechnology or gene therapy, 

when strong and enduring transgene expression 

and evasion of promoter silencing is needed.

43 | 


P 5 

pH-Labile Degradable Pseudodendritic 

Polycations for In Vitro and In Vivo DNA 

Delivery into Cancer Cells 

Christian Dohmen, Thomas Fröhlich, Ruggero 

Foralosso, Ernst Wagner 




Polycationic dendritic structures show good 

properties for DNA delivery into cancer cells. For 

example branched polyethylenimines (bPEI) are 

known to have a good transfection ability but also 

high toxicity. Former work of our group (Russ et 

al., Gene Therapy 2008) indicated that 

pseudodendrimers containing degradable ester 

linkages have similar transfection efficiency but 

lower toxicity. Within this work we designed novel 

pseudodendrimers based on different polyamines 

such as polypropylenimine and oligoethylenimine 

as core structure. They were modified with the 

acid degradable bisacrylate acetal linker BAA 

and oligoethylenimine (OEI 800Da) as 

polycationic surface structure. We could show 

that these dendritic molecules have the ability to 

form stable polyplexes with DNA and are 

degradable under incubation at endosomal pH. 

Cell testing confirmed that this degradability 

leads to a lower intracellular toxicity, while 

keeping the transfection ability both in vitro as 

well as in vivo in a neuroblastoma mouse tumor 

model. 


P 6 

Biodegradable Pseudodendritic Oligoamines 

for siRNA Delivery 

Thomas Fröhlich, Alexander Philipp, Verena 

Ruß, Ernst Wagner 




Searching for efficient synthetic vectors for siRNA 

delivery, a series of pseudodendritic oligoamines 

with different hydrophilic/lipophilic properties was 

synthesized. The polymers consist of an 800 Da 

oligoethylenimine (OEI) core and various surface 

molecules (spermine, spermidine, OEI, etc.), 

attached to the core via ester bond containing 

linkers. Out of this series, the HD-S polymer most 

effectively facilitates siRNA delivery, silencing 

luciferase expression in stably transfected 

Neuro2A-luc cells. All presented polymers are 

biodegradable because of the incorporated ester 

bonds, which can be cleaved by esterases 

ubiquitously present in living organisms. 

Ineffective polymers like the HD-O can be 

transformed into successful delivery agents by 

crosslinking the surface amines of the polyplexes 

with DSP (Dithiobis-succinimidyl-propionate). 

This lateral stabilization of polyplexes boosts the 

delivery efficiency of HD-O, which is 

demonstrated on Neuro2A-luc cells and HUH-7luc 

hepatoma cells. Enhanced cellular uptake of 

the crosslinked particles is proven by FACS 

analysis and gene down regulation is analyzed 

on mRNA level by qPCR. The disulfide bond in 

DSP maintains the biodegradability of the 

polymers and ensures sufficient release of the 

siRNA out of the stabilized polyplexes due to 

cleavage of disulfide bonds in the reductive 

cytosolic environment. 


P 7 

Succinylated PEI as Carrier for siRNA 

Delivery in Tumor Xenograft Models: 

Evaluation by RT-qPCR 

Daniel Edinger, Alexander Philipp, Katarina 

Farkasova, Ernst Wagner 




Developing polymers for tumor targeted delivery 

of siRNA, succinylated 25 kDa PEI has emerged 

as highly effective carrier system. Mechanistic in 

vitro study demonstrated that lower toxicity of the 

polymer compared to standard 25 kDa PEI is 

mainly responsible for the enhanced siRNA 

delivery efficacy. Higher amounts of polymer can 

be applied, which result in improved endosomal 

escape characteristics. For in vivo knockdown 

studies in xenograft tumor models the 

establishment of accurate and reliable mRNA 

detection methods is important. For this purpose 

we optimized Reverse Transcription (RT) 

quantitative Polymerase Chain Reaction (qPCR). 

Variations in expression levels of reporter genes 



(luciferase), therapeutic genes (RAN, KSP) and 

housekeeping genes (ACTB, GAPDH, AHA) 

were evaluated in tumor samples in vitro and in 

vivo. Human and mouse specific primer probe 

sets were designed. Those species specific 

primer probe sets offer the possibility to 

accurately detect levels of the human tumor 

target RNA in xenograft tumor models in the 

presence of mouse tissue. In our studies ACTB 

has emerged to be a more reliable housekeeping 

gene than GAPDH due to less variation in mRNA 

levels. Knockdown of therapeutic genes (such as 

KSP), which are cell cycle dependant, showed 

higher variations in analysis of cell culture and in 

vivo tumor samples. In addition silencing target 

genes which affect tumor survival complicate the 

evaluation due to cell death of transfected cells. 

Thus, targeting of non-essential gene targets 

seems to be more feasible for measuring 

silencing efficiency. 


P 8 

Magnetic Microbubbles: Magnetically 

Targeted and Ultrasound-Triggered Vectors 

for Gene Delivery In Vitro 

Dialechti Vlaskou 1 , Olga Mykhaylyk 1 , Pallab 

Pradhan 1 , Christian Bergemann 2 , Alexander L. 

Klibanov 3 , Karin Hensel 4 , Georg Schmitz 4 , 

Christian Plank 1 

1 Klinikum rechts der Isar, TU München, Institute 

für Experimentelle Onkologie, Munich, Germany; 

2 Chemicell GmbH, Berlin, Berlin, Germany; 

3 Department of Biomedical Engineering, Division 

of Cardiovascular Medicine, University of 

Virginia, Charlottesville, USA; 4 Institute of 

Medical Engineering, Dept. for Electrical 

Engineering and Information Sciences, Ruhr- 

University Bochum, Bochum, Germany 

In recent years microbubbles technology has 

gained lot of interest in the field of gene and drug 

delivery. Based on the “Magnetofection” concept 

[1], besides the development of magnetic 

acoustically active lipospheres [2, 3], we have 

been able to prepare lipid monolayer shelled 

microbubbles loaded with highly positively 

charged naked magnetic nanoparticles 

(composed of iron oxide) through electrostatic 

and matrix affinity interactions. These magnetic 

microbubbles show strong ultrasound contrast. 

Treatment of cancer cells with these 

microbubbles using ultrasound exhibited strong 

dose-dependent cytotoxic effects, although 



| 44 

ultrasound alone, lipid microbubbles alone, 

magnetic nanoparticles or magnetic 

microbubbles alone at the corresponding 

concentrations did not affect the cell viability. On 

the other hand, when these magnetic 

microbubbles were mixed with plasmid DNA 

encoding a reporter gene, we achieved gene 

delivery to cultured adherent cells only when 

ultrasound was applied. Gene transfer efficiency 

was strongly dependent on the application of a 

gradient magnetic field to sediment the 

microbubbles on the target cell membranes. 

From the preliminary experiments we conclude 

that magnetic microbubbles could be used as 

magnetically targeted diagnostic agents for realtime 

ultrasound as well as magnetic resonance 

imaging. At the same time, such magnetic 

microbubbles may be useful for therapeutic 

purposes such as in cancer therapy, vascular 

thrombolysis and gene therapy. However, further 

improvements are required to control their 

cytotoxicity. References [1] C. Plank, U. 

Schillinger, F. Scherer, C. Bergemann, J-S. 

Remy, F. Krötz, M. Anton, J. Lausier, J. 

Rosenecker. The magnetofection method: using 

magnetic force to enhance gene delivery. Biol. 

Chem. 2003; 384 (5): 737–747. [2] Plank C, 

Vlaskou D, Schillinger U, Mykhaylyk O, Brill T, 

Rudolph C, Huth S, Krötz F, Hirschberger J, 

Bergemann C. Localized nucleic acid delivery 

using magnetic nanoparticles. European Cells 

and Materials. 2005; 10(Suppl. 5):8. [3] Vlaskou 

D, Mykhaylyk O, Giunta R, Neshkova I, Hellwig 

N, Krötz F, Bergemann C, Plank C. Magnetic 

Microbubbles: New Carriers for Localized Gene 

and Drug Delivery. Mol Ther. 2006; 13(Suppl. 

1):S290. 


P 9 

Sleeping Beauty-Mediated Transposition in 

Human Cells Is Enhanced in RNA Interference 

Knockdown Cells 

Christina Rauschhuber, Anja Ehrhardt 


Institute, LMU, Munich, Germany 

Integrating nonviral vectors based on 

transposable elements and bacteriophage 

derived integrases are widely used for genetically 

engineering mammalian cells, in functional 

genomics and therapeutic gene transfer. For the 

Sleeping Beauty (SB) transposase system it was 

demonstrated that convergent transcription

45 | 

driven by the SB transposase inverted repeats 

(IRs) in eukaryotic cells occurs. This may lead to 

formation of double-stranded RNAs potentially 

presenting targets for the RNA interference 

(RNAi) machinery and finally to silencing of the 

transgene. Therefore, in the present study we 

generated and characterized RNAi knockdown 

mammalian cell lines and analyzed transposition 

in these cells. RNAi knockdown HEK293 cell lines 

were generated by stably introducing the RNAi 

suppressor protein P19 from the tomato bushy 

stunt virus which binds and inhibits 21 nucleotide 

long double stranded siRNAs. In order to 

characterize our RNAi knockdown cell lines we 

used the marker gene HoxB8 which, in 

differentiated cells, is suppressed by the miRNA 

miR196a. Using a quantitative Real-Time PCR 

based approach we detected up to 10-fold higher 

HoxB8 mRNA levels in the RNAi knockdown cell 

lines compared to the parental cell line clearly 

demonstrating that P19 is a sufficient inhibitor of 

the RNAi pathway. After performing colony 

forming assays measuring SB transposition 

events we could show that transposition was 

increased up to 4.5-fold in our RNAi knockdown 

cell lines compared to parental HEK293 cells, 

providing clear evidence for the first time that SB 

transposition is regulated by the RNAi machinery 

in mammalian cells. In contrast another 

transposable element, the Frog Prince 

transposon that displays only modest 

transcriptional activity, a 1.2-fold increase in 

transposition events was observed. To 

investigate integration of non-viral vectors in a 

more global manner, we also quantified 

bacteriophage-derived integrase PhiC31 

mediated integration events, and found that 

integration was enhanced up to 3-fold. In 

conclusion, our results indicate that transposition 

in mammalian cells and potentially also the 

PhiC31 integrase system are regulated by the 

endogenous RNAi machinery. 


P 10 

Micro RNA Knockdown Significantly 

Enhances Adenovirus Replication and Vector 

Production 

Christina Rauschhuber, Martin Hausl, Anja 

Ehrhardt 



The use of microRNAs (miRNAs) fundamentally 

improved the generation of viral vectors for gene 

therapy. For instance the introduction of miRNA 

target sequences into lentiviral vectors excluded 

expression of the respective protein from an 

unwanted tissue (Brown et al., 2006). However, 

the effect of the RNA interference (RNAi) 

pathway itself on gene therapy vectors remains 

an obstacle. In this study we used our recently 

established RNAi knockdown cell line B6 

(Rauschhuber and Ehrhardt, submitted) to 

analyze the influence of the RNAi pathway on 

adenoviral vectors. We first investigated 

replication of wildtype adenovirus 5 (wtAd5) and 

first generation adenovirus (FgAd) on a genome 

level. An up to 10-fold increase in viral genome 

copy numbers 24 hours and 48 hours post 

infection could demonstrate that there is 

significantly enhanced viral DNA replication 

under RNAi knockdown conditions. To analyze 

whether we can also achieve higher viral titers in 

the B6 cell line, we infected with a commonly 

used FgAd expressing firefly luciferase 

(FgAdluc). After re-infection of HEK293 cells we 

measured up to 5-fold increased luciferase 

activity indicating a higher titer of FgAdluc 

derived from B6 cells. To reach even stronger 

effects on virus replication we hypothesized that 

the amount of the RNAi suppressor protein P19 

may be the limiting factor. Thus, we applied our 

novel adenovirus cloning technology based on 

bacterial artificial chromosomes and generated a 

recombinant adenovirus expressing p19 under 

the control of the fiber promoter for which the 

amount of P19 directly correlates with adenovirus 

genome replication. We detected up to 10-fold 

increased genome copy numbers compared to 

wtAd5 and the corresponding control virus. 

Following our new method of adenovirus type 

switch we also generated a helper-virus 

expressing p19 for helper-dependent adenovirus 

(HC-AdV) production. In contrast to a 

conventionally used helper-virus we detected up 

to 12-fold increased HC-AdV titers. In conclusion, 

we provide new insights into regulation of 

adenoviral vectors by RNAi and our RNAi 

knockdown cell line B6 could be used as a novel 

producer cell line for adenoviral vectors. 




P 11 

A Transient Cell Cycle Arrest Enhances AAV 

and ZFN-Mediated Gene Targeting 

Shamim H Rahman, Katharina Gellhaus, Kaweh 

Montazami-Astaneh, Regine Heilbronn, Toni 

Cathomen 

Department of Experimental Hematology, 

Hannover Medical School, Hannover, Germany 

Vectors based on adeno-associated virus (AAV) 

are efficient tools to modify complex genomes by 

gene targeting. Gene targeting is based on the 

homologous recombination (HR) pathway, which 

is mainly available during the G2 phase of the 

cell cycle. We have shown that insertion of a 

DNA double strand break (DSB) in the target 

locus stimulates gene targeting up to 1000-fold, 

most likely by activating the HR repair pathway. 

To assess the effect of the cell cycle on AAV and 

DSB-mediated gene targeting, we established 

three human cell lines (HeLa, HT-1080, U-2 OS) 

that carry a copy of a mutated eGFP gene 

flanked by recognition sites for the meganuclease 

I-SceI and zinc-finger nucleases (ZFNs). After 

treatment with different cytostatic drugs (including 

hydroxyurea, vinblastine, indirubin), cells were 

transduced with AAV vectors that encode the 

nuclease and an HR donor with the purpose of 

rescuing eGFP expression by gene targeting. 

The cell cycle profile, the extent of cytotoxicity 

and the frequency of gene targeting were 

assessed by flow cytometry. We show that a 

transient cell cycle arrest before the creation of a 

DSB in the target locus increased AAV-mediated 

gene targeting up to 6-fold. Although this effect 

could be partially attributed to an increase in AAV 

transduction, a significant increase in gene 

targeting was also observed by plasmid-based 

eGFP rescue, implying that a drug-induced 

transient cell cycle arrest is a general means to 

augment DSB-mediated gene targeting. 

Depending on the cell line and the AAV vector 

dose, we achieved targeted gene editing in up to 

12% of transduced cells. Interestingly, the ratio 

between AAV vectors used for gene targeting vs. 

illegitimate integration was strongly cell type 

dependent and ranged between 30:1 and 1:9. 

Also, drug-induced transient cell cycle arrest had 

only minor effects on this ratio. In conclusion, 

cytostatic drugs can be used to enhance DSBmediated 

gene targeting in different cell lines. 

The combined beneficial effects of site-specific 

DSBs and transient cell cycle arrest allowed us to 

lower the AAV vector dose – and hence 

illegitimate integration of the vectors – without 

compromising on the gene targeting efficacy. 


P 12 

Novel Heterospecific FRTs Enable Gene 

Targeting of Several Loci by Flp-Multiplex- 

RMCE 

Sören Turan, Juergen Bode, Johannes Kuehle, 

Axel Schambach 

Hannover Medical School (MHH), Experimental 

Hematology, Hannover, Germany 



| 46 

Since 48 bp Flp-recombinase sites (FRTs) 

represent unique targets in all eukaryotic 

genomes recombinase-mediated cassette 

exchange (Flp-RMCE) is not hampered by the 

occurence of genomic pseudosites. We explored 

the feasibility of exchanging two distinct 

genomically anchored cassettes, each flanked by 

an unique pair of heterospecific FRT sites. For 

successful RMCE novel constructed mutant 

FRTs had to meet two major prerequisites (a) 

given self-recognition potential comparable to a 

pair of FRT wildtype sites (b) negligible 

crossinteraction among heterospecific sites. We 

applied a two-step strategy to characterize 

various newly created FRT spacer mutants for 

these properties. As a result of our screening 

steps we identified combinations F3/F and 

F13/F14 of sites, which were successfully applied 

to simultaneous Flp-mediated genomic targeting 

(“multiplexing”) reactions. The emergence of 

novel heterospecific FRTs can play a key role for 

specified gene targeting approaches, for instance 

the sequential elaboration of larger gene domain 

subunits by RMCE. Reference: Turan S, Kuehle 

J, Schambach A, Baum, C and Bode, J (2010) 

RMCE-multiplexing: versatile extensions of the 

Flp-Recombinase-Mediated Cassette-Exchange 

technology. J. Mol. Biol. In press. 


47 | 

P 13 

Chemical Modification of the Adenovirus 

Capsid for Enhanced Viral Uptake into Target 

Cells 

Alexandra Vetter 1 , Kulpreet Singh Virdi 2 , Sigrid 

Espenlaub 3 , Wolfgang Rödl 1 , Ernst Wagner 1,4 , 

Per S. Holm 5 , Florian Kreppel 3 , Christina Scheu 2 , 

Christine Spitzweg 6 , Manfred Ogris 1,4 



Munich, Germany; 2 Physical Chemistry, 

Department of Chemistry, Ludwig-Maximilians- 

University, Munich, Germany; 3 Division of Gene 

Therapy, University of Ulm, Ulm, Germany; 

4 Center for Nanoscience (CeNS), Ludwig- 

Maximilians-University, Munich, Germany; 

5 Institute of Experimental Oncology and Therapy 


Munich, Germany; 6 Department of Internal 

Medicine II, Ludwig-Maximilians-University, 


Recombinant adenovirus type 5 (Ad5) is a highly 

efficient vector for cancer gene therapy 

approaches, but due to its natural tropism for the 

coxsackie- and adenovirus receptor (CAR) it 

lacks specificity for tumor cells and can induce 

severe inflammatory responses. Although 

present on a broad range of tissues, CAR is often 

down-regulated in several tumor types. 

Combining the high transfection efficiency of Ad 

with synthetic polymers can potentially allow detargeting 

from CAR positive tissues and retargeting 

to tumor cells. We have coated Ad5 

with cationic polymers like PAMAM dendrimer or 

linear polyethylenimine (LPEI) by electrostatic 

interaction with the negatively charged Ad capsid. 

Size and surface charge of polymer coated Ad5 

were measured by laser light scattering, particle 

shape by transmission electron microscopy 

(TEM). Uptake and intracellular transport was 

studied by fluorescence microscopy and flow 

cytometry using fluorescently labeled Ad 

(Alexa488) and polymer (Cy5). Transgene 

expression studies in vitro were carried out using 

either luciferase gene or the sodium iodide 

symporter gene (NIS), were the latter leads to 

cellular uptake of radioactive iodine. With TEM it 

was possible to show that the virus is 

encapsulated by the polymer forming a shell 

around the virus with a rough surface structure. 

The virus surface charge changed from a net 

negative value of naked virus −17mV) ( to + 4 mV 

when coated with PAMAM dendrimer, and the 

average particle size increased with increasing 

amounts of polycation added. Recharging the 

virus led to pronounced cellular binding and 

uptake of virus. On high CAR expressing cells 

transfection efficiency was reduced after polymer 

coating, whereas on cell lines with low CAR 

expression increased transgene expression was 

measured. In addition, we covalently attached a 

peptide ligand to the polymer binding to the 

epidermal growth factor receptor (EGFR). This 

further promoted uptake in EGFR positive tumor 

cells. The obtained data suggests that a 

combination of viral and nonviral gene vector 

systems could compensate the limitations each 

approach has on its own and lead to superior 

success in cancer gene therapy. 


P 14 

Generation of microRNA-Sensitive VSV 

Vectors for Safer Treatment of Hepatocellular 

Carcinoma and Hepatic Metastasis of 

Colorectal Cancer 

Sibylle Apfel 1 , Jennifer Altomonte 1 , Michael 

Stiess 2 , Frank Bradke 2 , Oliver Ebert 1 

1 II. Medizinische Klinik und Poliklinik, Klinikum 


2 Independent Junior Research Group Axonal 

Growth and Regeneration, Max Planck Institute 

of Neurobiology, Martinsried, Germany 

Vesicular stomatits virus (VSV) is an oncolytic 

virus, which shows promise as a therapeutic 

agent for cancer, however, toxic collateral effects 

on the brain and the liver occur when it is 

administered at high doses. We hypothesized 

that by employing a novel strategy, termed 

microRNA-engineering, we could eliminate offtarget 

replication and improve the therapeutic 

index. This approach involves the incorporation 

of target sequences of a specific miRNA (miRTs) 

into the viral genome, resulting in degradation of 

viral gene transcripts in cells in which this miRNA 

is highly expressed, without interfering with virus 

replication in cells not expressing the relevant 

miRNA. We screened a panel of candidate 

miRNAs to select for those with high expression 

levels in the brain and/or liver and low levels in 

primary human HCC and CRC and cell lines. We 

then confirmed the miRNA-mediated regulation of 

the candidates in a reporter gene assay. Four 

tandem repeats of perfect complementary miRTs 

were cloned into the ′ 3 -UTR of a luciferase 

reporter gene, and these plasmids were 

transfected into a panel of normal and tumor 

cells. We then selected only those miRTs which 

resulted in a reduction of luciferase expression in 

primary hepatocytes and neurons but not in 



tumor cells. Based on these preliminary 

experiments, we chose three miRTs which best 

fit our selection criteria, and created recombinant 

VSV vectors containing four tandem repeats of 

the miRTs in the 3′ -UTRs of crucial endogenous 

viral genes. After testing of these vectors in tumor 

and non-tumor cells in vitro, thorough toxicity and 

efficacy studies will be performed in mouse 

models of HCC and CRC. We ultimately aim to 

engineer next-generation viruses with one or 

more viral gene regulated by multiple miRTs. 

Because saturation of endogenous miRNAs in 

the target tissues could result in “leaky” virus 

replication, we hypothesize that the incorporation 

of multiple miRTs could overcome this limitation 

and result in an even safer vector. Thus, this 

strategy has the potential to provide a basis for 

clinical application of VSV vectors for the 

treatment of primary and metastatic liver disease, 

as well as other cancers, in the future. 


P 15 

Multifunctional Magnetic Liposomes for 

Cancer Therapy 

Pallab Pradhan 1 , Jyotsnendu Giri 2 , Andreas 

Steingoetter 3 , Christian Koch 1 , Olga Mykhaylyk 1 , 

Rinti Banerjee 4 , Dhirendra Bahadur 4 , Christian 

Plank 1 

1Institut für Experimentelle Onkologie und 

Therapieforschung, Klinikum rechts der Isar, 

TUM, Munich, Germany; 2 Dept. of Chemical 

Engineering, California Institute of Technology, 

Pasadena, USA; 3 Dept. of Nuclear Medicine, 

Klinikum rechts der Isar, Technical University of 

Munich, Munich, Germany; 4 Indian Institute of 

Technology, Bombay, Powai, Mumbai, India 

A multifunctional magnetic liposome formulation, 

which is designed to combine features of 

biological (folate receptor mediated) and physical 

(magnetic field assisted) drug targeting for use in 

magnetic hyperthermia triggered drug release for 

cancer therapy has been developed. The 

magnetic liposomes (MagFolDox) composed of 

DPPC/Cholesterol/DSPE-PEG2000/DSPE- 

PEG2000-Folate at 80:20:4.5:0.5 molar ratio 

released 52% doxorubicin at 43°C after 60 

minutes incubation in 50% fetal bovine serum. 

The MagFolDox, when physically targeted to 

tumor cells in culture by a permanent magnetic 

field yielded a several fold increase in cellular 

uptake of doxorubicin as compared to Caelyx ® (a 

stealth liposomal formulation of doxorubicin), 



| 48 

nonmagnetic folate-targeted liposomes and free 

doxorubicin in folate receptor expressing tumor 

cell lines. Consequently, the MagFolDox 

liposomes enabled improved tumor cell killing. 

Moreover, MagFolDox and magnetic 

hyperthermia (at 42.5-43.5°C) showed synergistic 

cytotoxic effect in KB cells. Also, the distribution 

of magnetic liposomes in vivo can be monitored 

by MRI due to their contrast enhancement 

properties. In vivo real time MRI monitoring in 

mice confirmed significantly higher blood 

circulation time for MagFolDox compared with 

free magnetic particles. This also confirmed that 

the biodistribution of the MagFolDox can be 

monitored in vivo by MRI. Thus, the 

multifunctionality of the magnetic liposomes has 

been successfully demonstrated. 


P 16 

Melanoma-Killing Adenovirus Derived by 

Direct Evolution 

Inés Fernández Ulibarri, Dominik Dorer, Dirk M 

Nettelbeck 

DKFZ, Heidelberg, Germany 

Viral oncolysis is a strategy for targeted treatment 

of cancer by tumor-restricted virus infection. 

Human Adenoviruses (Ad) are promising 

oncolytic agents, but showed insufficient 

therapeutic efficiency in clinical trials. Therefore, 

the key challenge for virotherapy research is to 

develop Ad with enhanced viral lysis. In this 

regard, I focus on developing effective oncolytic 

Ad for malignant melanoma by directed evolution 

in order to derive adenoviruses with increased 

lytic activity. First, we investigated the efficiency 

of a panel of Ad serotypes from species B, C, 

and D in melanoma cells in comparison to the 

most potent virus for this type of cancer, Ad5/3 

(Ad5 with the cell-binding domain of Ad3). Our 

results indicate that none of those serotypes are 

more potent than Ad5/3 in melanoma cells. 

However, the oncolysis of the melanoma cells is 

still strongly attenuated in comparison with 

bronchial epithelial cells, which are the natural 

host cells. Therefore, although Ad5/3 is the most 

potent Ad in melanoma an improvement is still 

needed. To enhance the lytic activity of Ad5/3, 

the viruses were treated with nitrous acid sodium 

(NaNO2) which induces random mutations in the 

virus genome. Subsequently, the originated 

mutations were fixed and the individual mutants 

were amplified in melanoma cells. Finally, with

49 | 

the objective of harvesting the most rapid spread 

viruses, the bioselection rounds of the 

mutagenized Ad5/3 were performed by 

passaging the supernatant (at early stage of 

infection) repeatedly on the melanoma cells. After 

the 20th round of bioselection, our results show 

that mutagenized Ad5/3 were 10-fold more potent 

in melanoma cells in comparison with the 

parenteral virus. This result suggests that the 

most potent virus generated after NaNO2 

treatment replicate, lyse the host and are 

released into the supernatant before the majority 

of virus population. Currently, I am characterizing 

the mutants to restrict their replication in 

melanoma cells by a tumor-specific promoter. In 

summary, our results indicated that directed 

evolution is a strategy which allows the 

enrichment of viruses' mutants with enhanced 

replication and spread for malignant melanoma 

cells. 


P 17 

Versatile and Efficient Genome Editing in 

Human Cells by Combining Zinc-Finger 

Nucleases with AAV Vectors 

Eva M Händel 1 , Katharina Gellhaus 2 , Kafaitullah 

Khan 1 , Christien Bednarski 1 , Tatjana I Cornu 2 , 

Felix Müller-Lerch 2 , Regine Heilbronn 2 , Toni 

Cathomen 1 

1 Department of Experimental Hematology, 


2 Institute of Virology (CBF), Charité Medical 

School, Berlin, Germany 

Zinc-finger nucleases (ZFNs) are an innovative 

tool for targeted genome engineering. They 

consist of a catalytic nuclease domain and a 

designed DNA binding domain and can be 

employed for either targeted deletion or gene 

disruption by harnessing the non-homologous 

end joining DNA repair pathway or, in the 

presence of a properly designed donor DNA, 

gene targeting by activating homology directed 

repair (HDR). Here, we demonstrate the 

versatility of ZFN-mediated genome editing when 

combined with vectors derived from adenoassociated 

virus type 2 (AAV2). For proof-ofconcept 

studies, we generated four different 

human reporter cell lines. For assessing gene 

disruption and targeted deletion of an integrated 

provirus, a lentiviral EGFP vector containing ZFN 

target sites in the LTRs was used to generate an 

EGFP-positive cell line. To evaluate therapeutic 

gene correction, cells harboring a mutated EGFP 

or Luciferase reporter were produced. The 

frequencies of targeted genome editing and the 

extent of cytotoxicity were determined by flow 

cytometry and/or luminometry at different time 

points after transduction. Disruption of the EGFP 

locus was achieved in 18% of cells infected with 

vectors encoding EGFP-specific ZFNs. Upon 

transduction with AAV vectors coding for LTRspecific 

ZFNs, the lentiviral provirus was excised 

in 10% of cells. Furthermore, after co-infection of 

cells with vectors that encode locus-specific 

ZFNs or serve as donors for gene targeting, 

AAV-mediated correction of the mutated marker 

genes restored reporter activity in up to 6% of 

cells. The frequency of genome editing was 

depending on the vector dose and the specificity 

of the ZFNs. Significant signs of cell death were 

not observed, suggesting minor toxicity 

associated with these approaches. In conclusion, 

the combined ZFN–AAV vector technology is a 

flexible tool to efficiently modify the human 

genome ad libitum. Because AAV vectors can 

transduce multiple human tissues, including 

induced pluripotent stem cells and mesenchymal 

stem cells, the ex vivo delivery of ZFNs and HDR 

donors via AAV vectors will be of great interest 

for the treatment of several disorders in 

regenerative medicine. 


P 18 

Progress in Minicircle Manufacturing and 

Performance Testing 

Marco Schmeer, Markus Blaesen, Ruth Baier, 

Martin Schleef 

PlasmidFactory GmbH & Co. KG, Bielefeld, 


Plasmid DNA is commonly used in vaccination, 

gene- or cell therapy but also as a basic 

substance in viral vector production. The 

dissemination of antibiotic resistance genes, as 

well as the uncontrolled expression of backbone 

sequences present in such plasmids may have 

profound detrimental effects. Additionally, 

unmethylated CpG motifs have been shown to 

contribute to silencing of episomal transgene 

expression. Therefore, an important goal in 

vector development is to produce supercoiled 

DNA lacking bacterial backbone sequences: 

Minicircle DNA. PlasmidFactory's minicircle 

production technology facilitates the large-scale 

production of highly pure minicircle DNA for 



applications in gene therapy and vaccination as 

well as, e.g., virus production. The production 

technology is based on two processes: (1) An 

inducible, sequence specific, and very efficient in 

vivo recombination process; (2) A 

chromatographic purification technology for the 

isolation of the minicircle DNA. The resulting 

minicircle DNA only consists of the gene of 

interest and a tiny residual sequence stretch 

including one of the two recombination sites. The 

chromatographic purification results in an 

exceptional purity, proven by various QC tests, 

which is extremely important since even small 

amounts of contaminants can produce dramatic 

experimental artefacts. For first efficacy studies, 

reporter genes for different types of analyses 

within various tissues, cells, animals and for 

testing the mode of administration (e.g., electro 

gene transfer, sonoporation, lipofection, 

magnetofection, etc.) have been used. Also 

biodistribution studies using these constructs 

have been performed. Additionally, minicircle 

constructs for vaccines, virus production, etc., are 

currently investigated. The results demonstrate a 

significant increase of gene expression of 

minicircle DNA in comparison to a standard 

plasmid. As a result of the work presented here, 

PlasmidFactory has launched several 

commercially available minicircle DNA products. 


P 19 

Minicircle – An Overview 

Martin Schleef, Marco Schmeer, Ruth Baier, 

Markus Blaesen 

PlasmidFactory GmbH & Co. KG, Bielefeld, 


For future gene therapy and even genetic 

vaccination approaches it is crucial to develop 

safe and highly efficient vector systems to be 

transferred into the target cells. Currently, viral 

and non-viral vectors are used, both having their 

advantages and limitations. The dissemination of 

antibiotic resistance genes, as well as the 

uncontrolled expression of backbone sequences 

present in plasmid DNA may have profound 

detrimental effects. Additionally, unmethylated 

CpG motifs have been shown to contribute to 

silencing of episomal transgene expression. 

Hence, it seems obvious that the removal of 

bacterial backbone DNA can greatly improve the 

safety and efficiency of plasmid DNA used in 

gene therapy and vaccination. Here, we give an 



| 50 

overview on earlier and recent approaches for 

the production, purification, and application of 

such minimal constructs, devoid of bacterial 

backbone sequences. Different approaches have 

been described so far, from plasmids where the 

antibiotics resistance gene has been replaced by 

another marker to minicircle DNA consisting only 

of the gene of interest and a tiny residual 

sequence stretch from the recombination. 

Minicircles can be produced by in vivo sitespecific 

recombination of a so-called parental 

plasmid resulting in a miniplasmid and the 

minicircle. This recombination can be achieved 

using different enzymes which need specific 

target sequences as recombination sites. The 

most important difference between these 

approaches is the efficiency of the recombination 

step as well as the purification procedure used in 

order to remove the miniplasmid (with the 

bacterial sequence motifs) and residual amounts 

of the parental plasmid if necessary. In addition 

to their improved safety profile, minicircles have 

been shown to greatly increase the efficiency of 

transgene expression in various in vitro and in 

vivo studies. Scale-up of such technology was 

recently possible in fermentation and results in an 

extremely pure DNA product. 


P 20 

Genetic Manipulation of High-Capacity 

Adenoviral Vectors Utilizing Bacterial 

Artificial Chromosomes and Rapid Small- 

Scale Vector Preparations 

Martin Hausl, Christina Rauschhuber, Richard 

Voigtlander, Zsolt Ruzsics, Anja Ehrhardt 



First-generation adenoviral vectors (FG-AdVs) 

are easy to produce and therefore representing 

the best option to analyse capsid-modifications or 

new expression systems. On the other hand 

high-capacity adenoviral vectors (HC-AdVs) 

lacking all viral coding sequences are the most 

attractive option for therapeutic approaches. 

Therefore, there is a great interest in developing 

a method for adoption of evaluated vectormodifications 

or expression systems in FG-AdVs 

towards HC-AdVs including fast generation of 

small scales of purified HC-AdV for initial 

experiments and subsequent large-scale 

amplification. Utilizing a novel platform based on 

homologous recombination of bacterial artificial

51 | 

chromosomes (BACs) we generated a BAC from 

a plasmid containing an HC-AdV genome by 

replacing the plasmid-backbone with the BACbackbone 

in a single cloning step. After this 

backbone-exchange we incorporated an eGFPexpression 

cassette, generated the respective 

HC-AdV and showed eGFP-expression. To adopt 

FG-AdVs to the BAC-platform we demonstrated 

that BACs with FG-AdV genomes can be 

constructed either by homologous recombination 

with adenoviral DNA isolated from purified virions 

or by backbone-exchange. Furthermore, we 

showed that our BAC technology can be used to 

switch from FG-AdVs to BACs with a helper virus 

(HV) genome for HC-AdV amplification, wild-type 

adenovirus or oncolytic adenovirus in a single 

cloning step. To highlight the impact of our BAC 

platform on adenovirus vectorology we 

exemplified adenovirus vector type switches by 

generating a HV with the established fiber mutant 

fib5/35 as well as a HV with an eGFP/Fluc 

expression cassette in the E3 region and a new 

hexon mutant with precisely exchanged 

hypervariable regions from human adenovirus 

serotype 12. All viruses were reconstituted and 

amplified. Notably, we observed that HC-AdVs 

can be first amplified in small amounts (4-6 × 20 

ml) and purified by column (Vivapure 

AdenoPACK 20, Sartorius). Yielded HC-AdV was 

used for initial proof-of-principle experiments and 

to optimize large-scale amplification making this 

technology amenable also for researchers 

without experiences in HC-AdV production. 


P 21 

A Novel Adenoviral Hybrid-Vector System 

Carrying a Plasmid Replicon for the Safe and 

Efficient Gene Therapy of Human Diseases 

Richard Voigtlander, Rudolf Haase, Martin Hausl, 

Armin Baiker, Anja Ehrhardt 



In dividing cells the two primary aims a gene 

therapy approach should accomplish are defined 

as the nuclear distribution and retention of 

therapeutic DNA. Because conventional 

monosystems fail to fulfil both tasks with equal 

efficiency, hybrid-vector systems are most 

promising in facing the challenges of modern 

molecular medicine. Our hybrid-vector system 

HDAdV-pEPito synergizes the helper-dependent 

adenoviral vector technology (HDAdV) with the 

plasmid pEPito containing the therapeutic gene 

and special DNA sequence 

SMAR(Scaffold/Matrix Attachment Region) for 

episomal retention and replication. Thus, our 

technique provides a powerful tool for stable 

maintenance of the transgene reducing the risk of 

insertional mutagenesis. Adenoviral hybridvectors 

HDAdV-pEPito and the respective SMAR 

deleted control (HDAdV-pEPito-ΔSMAR) were 

generated with a novel BAC-technology 

established in our laboratory (Hausl et al., in 

preparation). Both contain an eGFP-IRES-BSDcassette 

under control of promoter hCMV/EF1. In 

FACS analysis 83% of A549-cells infected with a 

MOI of 50 were positive for eGFP-expression. 

Since adenoviral vectors carry linear DNA, 

pEPito has to be released from their genomes 

with Flpe-recombinase to function as a plasmid. 

A specific PCR proved this recombination event 

for both constructs. The episomal status of 

recombined plasmids could be verified with 

bacterial rescue experiments at day 4 after 

coinfecting A549-cells with our constructs and 

Flpe-expressing virus HDAdV-mSB-Flpe. More 

bacterial colonies grew after transformation of 

genomic DNA containing HDAdV-pEPito with 

Flpe (n = 281) than of DNA containing HDAdVpEPito-ΔSMAR 

with Flpe (n = 25), indicating 

replication of excised plasmids. Ongoing 

experiments are the comparison of HDAdVpEPito 

and HDAdV-pEPito-ΔSMAR concerning 

their long-term persistence in mice and their in 

vitro establishment efficiency in a colony forming 

assay (CFA). Previous CFAs performed with the 

original plasmids inserted into our two viruses 

seemed to show a tendency towards SMARpositive 

pEPito (48 surviving colonies) compared 

to SMAR deleted pEPito-ΔSMAR (27 surviving 

colonies) after 4 weeks of blasticidin selection. 


P 22 

Transient Cold Shock Enhances Zinc-Finger 

Nuclease-Mediated Gene Disruption – Rule or 

Exception? 

Nadine Dannemann, Christien Bednarski, Anne- 

Kathrin Dreyer, Toni Cathomen 

Department of Experimental Hematology, 


Zinc-finger nucleases (ZFNs) are a promising tool 

for precise editing of the human genome and 

hence of great interest in the field of gene 

therapy. ZFNs are designer nucleases consisting 



of the catalytic domain of the FokI endonuclease 

and an engineered DNA binding domain. Three 

major platforms are available to generate zincfinger 

based DNA-binding domains: modular 

assembly, context-specific selection (OPEN), and 

the proprietary Sangamo platform. Recently, 

Doyon et al. (Nat Methods 7, 459-60; 2010) 

reported that transient hypothermia generally and 

robustly increased the frequency of ZFN-induced 

gene disruption. Here, we aimed to work out the 

mechanism behind enhanced ZFN activity at 

30°C and to elucidate whether enhanced ZFN 

activity upon cold shock is a generally applicable 

phenomenon. To this end, we employed six 

different ZFN pairs – Sangamo-based ZFNs 

directed against the human AAVS1, CCR5 and 

IL2RG loci and OPEN-based ZFN that recognize 

the CFTR, EGFP and HOXB13 genes – and 

compared their activities at 30°C and 37°C in 

vitro and in human cell lines. In vitro cleavage 

assays revealed no difference in ZFN activity 

when comparing ZFNs of the different platforms 

at 30°C vs. 37°C, implying that the temperature 

does neither affect DNA binding nor the catalytic 

activity. On the other hand, incubation of the cells 

at 30°C induced an M phase arrest and 

assessment of the ZFN levels by immunoblotting 

revealed increased protein levels, suggesting that 

the cold shock either enhances ZFN expression 

or stabilizes the enzyme. However, the higher 

ZFN levels in transfected cells did not correlate 

with a significant increase in gene disruption at 

30°C at the analyzed genomic loci, neither in 

HeLa nor in 293T cells. Moreover, analysis of 

ZFN-mediated EGFP disruption by flow 

cytometry in a U2OS.EGFP cell line did not 

reveal augmented knockout efficiency for cells 

that underwent transient cold shock treatment. In 

summary, our data suggest that although mild 

hypothermic conditions let to a robust increase in 

ZFN protein levels, enhanced gene disruption 

activity of ZFNs at 30°C may not be a general 

phenomenon but rather dependent on the cell 

type and the genomic target locus. 


P 23 

Development of Scaffold/Matrix Attachment 

Region (S/MAR) Minicircles for Enhanced and 

Persistent Transgene Expression in the 

Mouse Liver 

Orestis Argyros 1 , Suet-Ping Wong 1 , Constantinos 

Fedonidis 1 , Oleg Tolmachov 1 , Simon 

Waddington 2 , Steven Howe 3 , Charles Coutelle 1 , 

Richard Harbottle 1 



| 52 

1 Gene Therapy Research Group, Section of 

Molecular and Medicine, National Heart and Lung 

Institute, Imperial College London, London, UK; 

2 Department of Haematology, Haemophilia 

Centre and Haemostasis Unit, Royal Free and 

University College Medical School, London, UK; 

3 Molecular Immunology Unit, Institute of Child 

Health, University College London, London, UK 

We previously developed a Scaffold Matrix 

Attachment Region (S/MAR) plasmid system for 

in vivo application (1). We demonstrated about 

40% hepatocyte transfection and sustained in 

vivo luciferase transgene expression in the 

mouse liver for at least six months following a 

single hydrodynamic administration. 

Subsequently, we observed sustained transgene 

expression for the lifetime of the animal, but this 

dropped appreciably over time. It has been 

shown that bacterial sequences present in 

plasmid DNA are responsible for vector toxicity 

and silencing of expression in vivo. We therefore 

hypothesised that by eliminating these 

extraneous bacterial components from our 

vectors we might improve their performance in 

vivo. We describe here the development of a 

minimally sized S/MAR vector using Cre/Lox 

recombinase (2), to generate a minicircle devoid 

of bacterial sequences and comprising only an 

expression cassette and an S/MAR moiety. Upon 

administration to mice by hydrodynamic delivery 

we found it to be maintained episomally 

(Southern blot analysis) and to provide sustained 

levels of luciferase expression for up to three 

months (end of experiment), as measured by a 

bioluminescent imager (XENOGEN). In addition, 

luciferase expression did not decline over time, 

as seen with our original S/MAR vector (1) 

containing the bacterial backbone. At the final 

time point, luciferase expression from the 

minicircle vector was approximately two orders of 

magnitude higher than both the control minicircle 

vector (no S/MAR) and the original S/MAR vector 

containing the bacterial backbone. In a further 

experiment, we performed partial hepatectomy 

on S/MAR minicircle treated mice, and observed 

a rapid drop of expression, indicative of absence 

of plasmid replication. These promising results 

demonstrate the utility of minimally sized S/MAR 

vectors for persistent, atoxic gene expression in 

the liver. (1) Argyros et al., Gene Ther. (2008), 

15:1593-1605. (2) Bigger et al., J. Biol. Chem. 

(2001), Jun 22;276(25):23018-23027. 


53 | 

P 24 

Systemic Administration of Nonviral Vectors 

to Neonatal Mice 

Suet-Ping Wong 1 , Argyros Orestis 1 , Steven 

Howe 2 , Charles Coutelle 1 , Richard Harbottle 1 

1 Gene Therapy Research Group, Section of 

Molecular and Medicine, National Heart and Lung 

Institute, Imperial College London, London, UK; 

2 Molecular Immunology Unit, Institute of Child 

Health, University College London, London, UK 

Nonviral vectors have not been extensively 

investigated in neonatal mice due to the poor 

efficiency of the delivery methods available. 

Understanding the effects of nonviral vectors 

during early development is vital to develop safe 

gene therapy treatments where irreversible 

pathological processes may be avoided by early 

gene reconstitution. Here we describe a simple 

and effective method for the systemic 

administration of nonviral vectors via the superior 

temporal vein of mouse pups at 1.5 days of age. 

We show that injection of polyethylenimine (PEI) 

complexed plasmid DNA (pDNA) intravenously 

results in effective transfection in the liver, lung, 

heart and spleen. We also investigate the specific 

targeting of transgene expression to the 

proliferating neonate liver using a liver-specific 

S/MAR plasmid, which has previously been 

shown to confer long-term expression in adult 

mouse liver. Using bioluminescent imaging, a 

gradual increase in transgene expression was 

observed peaking at days 11–12, before the 

reduction of expression to background levels by 

day 25, suggestive of vector copy number loss. 

We conclude that nonviral vectors can 

successfully be used for systemic delivery to 

neonatal mice but that further optimization of the 

S/MAR nonviral systems is required for longerterm 

maintenance of expression during neonatal 

growth and development. 


P 25 

Therapeutic Angiogenesis – Nonviral Gene 

Therapy for Patients with Critical Limb 

Ischemia 

Alex Slobodianski 1 , Astrid Kathöfer 2 , Christoph 

Hartog 2 , Ziyang Zhang 1 , Jessica Frenz 2 , Peter 

Mailänder 2 , Hans-Günther Machens 1 

1 Klinikum rechts der Isar, TU München, Munich, 

Germany; 2 University Hospital of Schleswig- 

Holstein, Kiel, Germany 

Background: Therapeutic angiogenesis using 

angiogenic growth factors is expected to be a 

new treatment for patients with chronic limb 

ischemia. In our studies, we designed a GMPadapted 

process of therapeutic angiogenesis for 

medicinal purposes using fibroblasts to 

temporarily produce bFGF and VEGF165 local in 

ischemic tissue. Two different animal models of 

ischemia were used for evaluations. We also 

considered some important aspects for preclinical 

evaluation of gene transfer medical products. 

Methods: The eukaryotic expression vectors 

harboring VEGF and bFGF cDNAs were 

transfected into rat primary fibroblasts mediated 

by Nucleofection. The transfection efficiency as 

well as expression of genes in the transfected 

fibroblasts were detected by means of flow 

cytometry, immunohistochemistry, enzyme-linked 

immunosorbent assay (ELISA) and real-time 

PCR. Effects on clinical outcome after 

transfected cells administration were determined 

in an ischemic flap model and in a hindlimb 

ischemia model in rat. 

Results: Transfection efficiency of about 60% 

and viability nearly 80% were observed by 

transfection of rat primary fibroblasts. Specific 

assays and real-time PCR afford to determine 

any correlation between protein expression in 

vitro and local therapeutic effect of angiogenesis 

in target tissue. The established reproducible 

monitoring system allows to detect the 

distribution of gene therapeutics in the target 

tissue and to trace it in rats. A reduction in flap 

necrosis in flap model by nearly 40% was 

detected by planimetric measurements after two 

weeks if transfected cells were applied. 

Arteriogenesis and angiogenesis were improved 

in the rat hindlimb ischemia model two weeks 

after the transfected cells administration as it was 

confirmed by the computer analysis. 

Summary: Thus, in our studies we established a 

healing method Therapeutic Angiogenesis and 

demonstrated that transient expression of bFGF 

and VEGF165 induced therapeutic effects in 

iscemic tissue and improved tissue survival. Use 

of monitoring systems helps to recognize 

potential risks of gene therapeutics and to identify 

relations between the dosage and therapeutic 

effects in the target tissue for dose-finding 

studies. 




P 26 

Lentiviral Vector-Induced Dendritic Cells 

Coexpressing GM-CSF, IFN-α and NS3 for 

Immunotherapy of Hepatitis C 

Anusara Daenthanasanmak 1 , Rakesh Bakshi 1 , 

Sandra Kuhs 1 , Bala Sai Sundarasetty 1 , Gustavo 

Salguero 1 , Helmut Diepolder 2 , Michael Ott 3 , 

Heiner Wedemeyer 3 , Renata Stripecke 1 

1Department of Hematology, Hemostasis, 

Oncology and Stem Cell Transplantation, 


2 Klinikum der Universitaet Muenchen, Munich, 

Germany; 3 Department of Gastroenterology, 


Hepatitis C virus (HCV) is the main cause of 

chronic hepatitis and an effective therapeutic 

approach should effectively result in long-term 

immunologic eradication of the virus. 

Recombinant viruses and DNA vaccines are in 

the development for adjunct therapy along with 

pegylated interferon alpha (IFN-α) plus ribavirin. 

We propose to develop potent antigen-presenting 

cells (APCs), such as dendritic cells (DCs) 

capable to directly mimic the IFN-α therapy 

concurrently with optimized antigen presentation. 

We developed a novel technology for production 

of DC consisting of one overnight lentiviral vector 

(LV) transduction of GM-CSF and IL4 plus fulllength 

antigens into DC precursors that results 

into induction of “SMART-DCs” capable to selfdifferentiate 

directly into long-lived DCs in vivo, 

and thus bypass in vitro culturing. This concept 

has been tested and validated in preclinical 

human and mouse tumor models for potency 

(Koya et al., Molecular Therapy, 2007). Recently, 

we have demonstrated that monocyte-derived 

DCs transduced with lentiviral vectors expressing 

the HCV nonstructural cluster result into 

multiantigenic anti-HCV responses in vitro (Jirmo 

et al., Vaccine, 2010). Therefore, we are currently 

validating multicistronic LVs that coexpress GM- 

CSF, IFN-α and a conserved and potent HCV 

antigen (NS3) in inducing a novel modality of 

engineered APCs, namely self-differentiated 

myeloid derived and lentivirus-induced DCs 

(SMYLE-DCs). Cotransduction of GM-CSF and 

IFN-α genes into CD14 + monocytes with a 

bicistronic LV resulted high levels of GM-CSF (1- 

2 ng/ml) and IFN-α (4 ng/ml) production, which 

were persistently detectable for 21 days. The 

SMYLE-DCs showed similar morphological and 

immunophenotypic features of DCs (MHCII + , 

CD80 + CD86 + and CCR5+) and autonomous 

viability for 21 days of culture. The SMYLE-DCs 

coexpressing NS3 will be subsequently tested in 

in vitro assays and in humanized mouse models 

for the stimulation of CD4 + T cell responses 



| 54 

against NS3 class II epitopes, which have been 

correlated with virus clearance. Ultimately, we 

propose clinical development of programmed DC 

vaccines for HCV immunotherapy with high 

capabilities of standardization. 


P 27 

Resolution of Chronic Ischemia by Regional 

Application of Embryonic eEPCs or Thymosin 

β4 in a Preclinical Pig Model 

Rabea Hinkel 1 , Alexander Wuchrer 1 , Jan C 

Horstkotte 1 , Corinna Lebherz 1 , Antonis K 

Hatzopoulos 1 , Ildiko Bock-Marquette 2 , Michael 

DiMaio 2 , Peter Boekstegers 1 , Christian Kupatt 1 

1Internal medicine I, Klinikum Großhadern, LMU 

München, Munich, Germany; 2 Southwestern 

Medical Center, Dallas, USA 

In patients suffering from ischemic 

cardiomyopathy, lacking interventional or surgical 

treatment options, induction of neovascularisation 

via vascular growth factor application and cell 

therapy is a novel therapeutic approach. In a 

preclinical pig model model of chronic ischemia, 

we investigated the efficacy of regional 

application of murine eEPCs or an AAV-mediated 

longterm Thymosin β4 (Tβ4) overexpression. 

Methods: In vitro, human microvascular 

endothelial cells (HMECs) were embedded in 

matrigel and tube formation was obtained. Cells 

were stimulated with conditioned media (CM) 

from eEPCs ± Tβ4shRNA. Tubes like structures 

per low power field were evaluated. In pigs (n = 6) 

a reduction stent graft was implanted into the 

circumflex artery, leading to complete occlusion 

at day 28. Retroinfusion of saline, 5 × 10 6 murine 

eEPCs or recombinant AAV2/9 rAAV.Tβ4 (5 × 

10 12 viral particles) was performed at d28. At 

days 28 and 56, global myocardial function 

(LVEDP) was assessed. Regional myocardial 

function (SES) and post mortem angiography 

were obtained at day 56. Tissue samples from 

the ischemic and non-ischemic tissue were 

harvested for capillary staining. Results: In vitro, 

HMECs incubated with conditioned media of the 

eEPCs revealed an increase of tube formation 

which was comparable to the stimulation with 

VEGF (16 ± 1 control vs. 34 ± 2 VEGF stimulation 

and 31 ± 2 with eEPC CM tube-like structures/low 

power field). The reduction of Tβ4 expression in 

the eEPCs via shRNA abolished this effect (11 ± 

1). In the pig model, retroinfusion of eEPCs 

induced an increase of capillaries (200 ± 12 vs.

55 | 

112 ± 6 C/F in controls) and collaterals (4.4 ± 0.4 

vs. 1.2 ± 0.2 in controls), followed by improvement 

of the global and regional myocardial function 

(LVEDP: 14 ± 1 vs. 19 ± 1.2 mmHg in control 

group; SES 44 ± 4 vs. 10 ± 6% in control group). 

rAAV.Tβ4 displayed a similar gain of 

neovascularization and myocardial function. (223 

± 14 C/F; 6.4 ± 0.5 collaterals; 14 ± 0.5mmHg 

LVEDP; 56 ± 7% SES). Murine embryonic EPCs 

are capable of inducing angiogenesis and 

arteriogenesis in a Tβ4 dependent manner. The 

combination of AAV vector and Tβ4 may offer 

therapeutic potential in patients with ischemic 

cardiomyopathy. 


P 28 

AAV-Mediated VEGF-A and PDGF-B 

Overexpression Resolves Mal-perfusion in 

Chronic Ischemia: Role of Vessel Maturation 

Rabea Hinkel 1 , Achim Pfosser 1 , Michael 

Thormann 1 , Corinna Lebherz 1 , Alexander 

Wuchrer 1 , Franziska Globisch 1 , Chiraz El-Aouni 1 , 

Andrea Banfi 2 , Peter Boekstegers 1 , Christian 

Kupatt 1 

1 Internal medicine I, Klinikum Groβhadern, LMU 

München, Munich, Germany; 2 Cell and Gene 

Therapy, Institute for surgical Research and 

Hospital Management, Basel, Switzerland 

In patients suffering from ischemic 

cardiomyopathy or peripheral artery disease, 

lacking interventional or surgical treatment 

options, induction of neovascularization via 

vascular growth factor application is a novel 

therapeutic concept. Here we tested an approach 

by combining hVEGF-A with hPDGF-B and 

prolonging the expression period by using a AAV 

as viral vector. 

Methods: In rabbits (n = 5/group), 0.5 × 10 11 

rAAV.VEGF-A ± 1 × 10 12 rAAV.hPDGF-B were 

retroinfused at d7 of femoral artery excision. 

Angiography of both hindlimbs was performed for 

collateral score and frame count score (% d7). 

Capillary density was determined at d35. In pigs, 

a reduction stent graft was implanted into the 

circumflex artery, leading to complete occlusion 

at day 28, when retroinfusion of rAAV.VEGF- 

A/PDGF-B (2 × 10 12 particles each), rAAV.VEGF- 

A (1 × 10 13 particles) or rAAV.LacZ was 

performed. Global and regional myocardial 

function and perfusion were assessed at d28 and 

d56. 

Results: In rabbits, rAAV.hVEGF-A strongly 

induced angiogenesis (C/MF 1.32 ± 0.07 vs. 0.96 

± 0.08 in controls), but neither collateral growth 

(125 ± 7% d7 vs. 95 ± 6% d7) nor perfusion (136 ± 

12% vs. 107 ± 9%). hVEGF-A/hPDGF-B 

cotransfection, enhanced collateral growth (146 ± 

9%) and perfusion (163 ± 8%) at a similar 

capillary density (1.44 ± 0.10 CM/F). NG2 staining 

revealed a significant increase of pericytes after 

VEGFA/PDGF-B overexpression, in contrast to 

the VEGF-A group. In the pig model, retroinfusion 

of rAAV-VEGF-A/PDGF-B increased myocardial 

blood flow (1.5 ± 0.1ml/g vs. 1.0 ± 0,1ml/g in 

controls), based on collateral growth (9.5 ± 0.3 vs. 

5.5 ± 0.5 in controls) and capillary density (257 ± 

23 vs. 135 ± 6/field in controls). VEGF-A alone 

failed to enhance myocardial perfusion by the 

same capillary density (274 ± 12/field). 

rAAV.VEGF-A/PDGF-B improved global 

myocardial function (LVEDP: 14.7 ± 0.9 vs. 19.6 ± 

0.8mmHg in controls) and regional myocardial 

function in the RCx area (11 ± 3% SES vs. 2 ± 

6%, controls), an effect lacking in the 

rAAV.VEGF-A group (EF 37 ± 2%, SES 6 ± 3%). 

Long-term overexpression of hVEGF and PDGF 

substantially enhanced angio- and arteriogenesis 

in the ischemic region followed by an improved 

function, which is superior to hVEGF alone. 


P 29 

Inhibition of G-Protein Coupled Receptor 

Kinase 2 Prevents Myocardial Hypertrophy in 

a Mouse Model for Dystrophin-Deficient 

Cardiomyopathy 

Ralf Bauer, Stefanie Schinkel, Christian Volz, 

Hugo A Katus, Oliver J Müller 

Medizinische Klinik Heidelberg, Abteilung 

Kardiologie, Angiologie und Pneumologie, 

Heidelberg, Germany 

Severe cardiomyopathy is a common feature in 

Duchenne muscular dystrophy (DMD) and has 

critical impact on mortality of these patients. 

Although, to date efficient treatment strategies to 

prevent cardiac complications in DMD remain 

elusive, cardiac-specific adeno-associated virus 

(AAV)-based gene therapy strategies represent a 

promising tool. In DMD, absence of dystrophin 

causes instability of the dystrophin-glycoproteincomplex 

and increased vulnerability of 

cardiomyocytes towards contraction-induced 

damage. It is unclear to what extend impairment 

of the cardiac beta-adrenergic receptor (BAR) 



system contributes to progressive cardiac 

damage in DMD. It has been shown in transgenic 

mice that the inhibition of G-protein coupled 

receptor kinase 2 (GRK2) through the carboxylterminus 

of GRK2 (βARKct) has beneficial effects 

with an attenuation of heart failure symptoms. 

Therefore, we have investigated the efficiency of 

AAV9- mediated cardiac overexpression of 

βARKct to prevent the development of 

cardiomyopathy in dystrophin-deficient (mdx) 

mice. AAV-9 vectors containing βARKct cDNA 

under transcriptional control of the CMV-MLC 

promoter (AAV9/βARKct) were created. 10 12 

AAV9/βARKct vector particles were intravenously 

injected into the tail vein of 8 week-old mdx mice 

before the onset of any signs of cardiomyopathy. 

In some mice AAV9 with enhanced green 

fluorescent protein (AAV9/EGFP) was injected as 

control vector. At the age of 12 months 

echocardiography to assess contractility 

(fractional shortening (FS)) and diastolic left 

ventricular posterior wall thickness (PWTd) was 

performed. Untreated and AAV9/EGFP-treated 

mdx mice showed extensive myocardial 

hypertrophy and reduced contractility (PWTd 

16.5 + 0.04 and 16.3 + 0.14 mm; FS 45 + 9% and 

45 + 8%, respectively (values + standard error)). 

Treatment with AAV9/βARKct attenuated 

myocardial hypertrophy and improved 

contractility (PWTd 12.6 + 0.08 mm and FS 71 + 

3%) in mdx mice. We suggest that cardiac 

inhibition of GRK2 through AAV-mediated 

overexpression of βARKct is a valuable strategy 

limiting cardiac damage in muscular dystrophyassociated 

cardiomyopathy. 


P 30 

Non-Integrating Lentiviral Gene Transfer of 

E2F2 to Induce Corneal Endothelial Cell 

Replication 

Daniel Kampik, Alexander Smith, Ulrich F.O. 

Luhmann, Prateek Buch, Daniel F.P. Larkin, 

Robin R. Ali 

UCL Institute of Ophthalmology, London, UK 

The osmotic activity of corneal endothelial cells 

(CEC) maintains the clarity of the eye's cornea. 

CECs being non-replicative in humans, a 

decrease of CEC density due to aging, injury or 

inherited disease causes corneal swelling and 

loss of transparency. E2F2 is a transcription 

factor regulating progression from G1 to S phase 

of the cell cycle. We investigate whether gene 



| 56 

transfer of E2F2 is able to induce cell proliferation 

in CEC. We constructed a non-integrating HIVbased 

lentiviral vector delivering E2F2 under a 

CMV promoter (LNT-E2F2). A lentiviral vector 

delivering GFP (LNT-GFP) served as control. In 

293T cells, exposure to LNT-E2F2 resulted in a > 

200-fold increase of E2F2 mRNA compared to 

uninfected or LNT-GFP infected controls, as 

detected by quantitative PCR. E2F2 

overexpression was detected by Western blot 

and immunohistochemistry. Samples of human 

corneal tissue were exposed to either LNT-E2F2 

or LNT-GFP or left uninfected. Exogenous E2F2 

protein was detectable only in LNT-E2F2 infected 

samples by Western blot and 

immunohistochemistry. 5-bromodeoxyuridine 

(BrdU) incorporation and Ki-67 immunostaining 

was used to detect G1 to S phase progression or 

cells in any active cell cycle phase, respectively. 

CECs infected with LNT-E2F2 showed both BrdU 

and Ki67 nuclear staining. Lentivirus mediated 

E2F2 overexpression could be used to increase 

CEC density in donor corneas ex vivo before 

transplantation. Safety measures are now being 

investigated to enable clinical use. 


P 31 

Membrane-Anchored and Secreted C 

Peptides as HIV Entry Inhibitors 

Lisa Egerer 1 , Janine Kimpel 1 , Andreas Volk 2 , 

Joerg Kahle 3 , Marianne Hartmann 2 , Felix G. 

Hermann 3 , Sebastian Newrzela 2 , Dorothee von 

Laer 1 

1 Dept. of Hygiene, Microbiology and Social 

Medicine, Section Virology, Medical University 

Innsbruck, Innsbruck, Austria; 2 Applied Virology 

and Gene Therapy, Institute for Biomedical 

Research, Georg-Speyer-Haus, Frankfurt am 

Main, Germany; 3 Vision 7 GmbH, Frankfurt am 

Main, Germany 

C peptides derived from the HIV envelope 

glycoprotein gp41 (e.g., T-20, C46) are highly 

efficient inhibitors of virus entry. Here, we 

analysed a membrane-anchored (maC46) as well 

as a secreted (sC46) variant of the peptide C46 

for gene therapy of HIV infection. Membraneanchored 

or secreted C46 peptides were 

expressed from gammaretroviral or lentiviral 

vectors in B and T cell lines as well as in primary 

human T cells. Cultures were analysed for 

expression of the peptides and antiviral effect. 

Furthermore, a humanized mouse model was

57 | 

estabished to test these antiviral peptides in vivo. 

The sC46 peptides were expressed and secreted 

in different lymphoid cells and exerted high 

antiviral activity against a variety of HIV envelope 

glycoproteins. In mixed cell cultures peptides 

secreted from transduced cells produced a 

bystander effect and prevented infection of nonmodified 

cells. In the mouse model, we observed 

a substantial increase of maC46 + CD4 + T cells 

in blood as well as in spleen, apparently due to 

the selective pressure of ongoing HIV infection. 

This increase of CD4 + T cells was neither seen 

in uninfected control mice nor with a control 

vector. The clear accumulation of maC46 + cells 

in HIV-infected humanized mice indicates that 

these cells are protected from HIV infection in 

vivo. Further studies in this model with maC46 

and sC46 will allow us to analyze the conditions 

that determine efficacy of T cell based 

immuno/gene therapy for HIV-infection. 


P 32 

Self-Complementary rAAV8-iSAVE Vectors for 

Liver-Targeted Entry-Inhibitory HIV-1 Gene 


Andreas Volk 1 , Janine Kimpel 1 , Lisa M Egerer 1 , 

Hanna Janicki 2 , Hildegard Büning 2 , Dorothee von 

Laer 1 

1 Department of Virology, Medical University 

Innsbruck, Innsbruck, Austria; 2 Department I of 

Internal Medicine and Center for Molecular 

Medicine Cologne, Cologne, Germany 

Antiviral entry-inhibitory C-peptides, a novel class 

of antivirals in HIV standard therapy (HAART), 

are derived from the C-heptad motif in the 

extracellular domain of the gp41 transmembrane 

protein. C-peptides (such as T-20 or C46) bind to 

a fusion intermediate state of gp41 and thereby 

block viral entry with an IC50 in the low nM range. 

T-20 is the only marketed C-peptide and is 

administered twice daily via subcutaneous 

injection. Major drawbacks of this drug are (i) the 

inconvenient route of administration and (ii) rapid 

viral resistance emergence. To circumvent these 

drawbacks, our group focuses on the 

development of an entry-inhibitory gene therapy 

based on an elongated variant of T-20, the ‘in 

vivo secreted antiviral entry inhibitory C-peptide’ 

(iSAVE). In comparison to T-20, iSAVE also 

targets a very conserved membrane proximal 

region, which cannot be mutated without massive 

viral replication deficiencies. Additionally, a gene 

therapy ideally requires only one administration 

of antiviral and thus reduces the need for 

constant patient adherence. In our gene 

therapeutical model, iSAVE expression can be 

targeted to several sites of production. Besides 

the lymphatic tissue as sites of viral replication, 

the liver is a very promising organ for iSAVE 

expression, since (i) hepatocytes are prone to 

secrete high amounts of protein and (ii) the liver 

is known to be able to induce a tolerogenic 

response towards transgenes. Here, we present 

first results of iSAVE delivered by a selfcomplementary 

rAAV8 vector to the liver. iSAVE 

is expressed to moderate nM plasma 

concentrations, while its IC50 is in the low nM 

range promising a sustained antiviral effect upon 

in vivo challenge. Furthermore, no humoral 

response against the iSAVE peptide could be 

detected in immunocompetent mice, pointing to 

an potential tolerogenic transgene response. 

Currently, we are investigating the antiviral effect 

in a humanized mouse model for HIV-infection. In 

conclusion, scAAV8-iSAVE is a promising 

candidate for HIV gene therapy and will be 

further analyzed for its antiviral activity and 

transgene-induced immunologic responses. 


P 33 

Gene Vector Loaded Biphasic Calcium 

Phosphate Ceramic Induces Ectopic Bone 

Formation in Rats 

Christian Koch 1 , Andreas Kolk 2 , Balasz Vigh 1 , 

Bernd Gänsbacher 1 , Christian Plank 1 

1Institute of Experimental Oncology and Therapy 


Munich, Germany; 2 Department of Oral and 

Maxillofacial Surgery, Technische Universität 


TricOs is a resorbable bone substitute which is 

already used in clinical applications. This material 

is a biphasic calcium phosphate ceramic 

composed of a mixture of hydroxyapatite and βtricalcium 

phosphate which combines structural 

stability with osteogenic capacity. For faster and 

better healing of large bone defects the 

optimization of the biological properties of this 

substitute would be an advantage. This could be 

achieved by localized gene delivery using vectors 

coding, for e.g., a growth factor such as BMP-2. 

Based on our knowledge of localized gene 

delivery systems using copolymer protected gene 

vectors (COPROGs) and organic matrices such 



as collagen sponges or fibrin glue, we examined 

whether a combination of COPROGs and the 

inorganic calcium ceramic for matrix mediated 

gene delivery is possible. For the in vitro studies 

we used a novel reporter gene, Metridia 

luciferase, a protein which is secreted in the 

tissue culture supernatant. We could 

demonstrate that our gene vector formulation 

immobilized on TricOs revealed Metridia 

luciferase expression over a period of 48 days 

with a maximum at day 5. Detection of GFP 

expression exhibited a bright fluorescene of cells 

which colonized the TricOs granules. For in vivo 

studies we implanted TricOs granules loaded 

with COPROGs coding either for BMP-2 or firefly 

luciferase as control in the musculus latisimus 

dorsi in rats. At different time points the bone 

substitutes were explanted and the formation of 

newly formed bone was examined by micro-CT, 

conventional CT and PETscan. In contrast to the 

luciferase implants BMP-2 gene vector loaded 

granules exhibited a better turnover of the bone 

substitute into newly formed bone and a higher 

bone density. The finding that ectopic bone 

formation in the muscle of rats suggests that 

matrix mediated gene delivery is useful in bone 

regeneration applications. 


P 34 

Bioactivation of Collagen Scaffolds with 

Nonviral Gene Vectors to Enhance the 

Release of Proangiogenic Molecules in Tissue 

Regeneration 

Ann K Reckhenrich 1 , Christian Koch 1 , Ursula 

Hopfner 2 , Hans G Machens 2 , Christian Plank 1 , 

Tomas J Egana 2 

1Institute für Experimentelle Onkologie, Klinikum 


2 . Klinik für Plastische Chirurgie und 

Handchirurgie, Klinikum rechts der Isar, TU 


Background: Biodegradable scaffolds serve as 

structural matrix for cell guiding, promoting cell 

growth and proliferation. Moreover, scaffolds can 

also be used as carriers for bioactive molecules. 

The combination of nanobiotechnology and 

tissue engineering offers the opportunity to 

temporarily modify cells by nucleic acid 

transporting nanoparticles. Under such 

conditions, infiltrating cells will be temporarily 

modified to express genes of interest. In this 

study we developed a new approach to activate 



| 58 

biomaterials to release proangiogenic molecules 

by combining their use with a new class of nonviral 

gene vectors so called Copolymer-protected 

gene vectors (COPROGs). 

Methods: COPROGs were incorporated into 

commercially available collagen scaffolds, and 

the optimal concentration of DNA was 

determined in vitro by following the release of 

reporter proteins. After cell seeding into the 

scaffolds, distribution and attachment of the cells 

were evaluated by fluorescence microscopy and 

cell viability and proliferation by metabolic MTT 

assays. Finally, scaffolds were activated with 

vectors encoding for relevant growth factors (e.g., 

VEGF165) and their release was quantified by 

ELISA. 

Results: We have determined that the optimal 

amount of COPROGs per scaffold was 0.4 μg 

(relative to DNA) per milligram of scaffold. After 

seeding, results showed that cells were 

homogeneously distributed forming focal 

adhesions with the scaffold. Moreover, the 

presence of COPROGs did not affect cell viability 

and proliferation of seeded cells. The release of a 

reporter protein was detected for at least 2 weeks 

in vitro, with a peak of expression between days 

3 to 5. Similar results were obtained by the use of 

DNA encoding for VEGF, where a transient 

release of the protein was observed for at least 2 

weeks with a peak of expression at day 5. 

Conclusions: Combine use of collagen scaffolds 

and COPROGs, allows inducing local release of 

therapeutic proteins without cell toxicity in vitro. 

Such technologies may have tremendous impact 

in the field of tissue engineering and regenerative 

medicine. Preclinical studies are under current 

evaluation. 

Session: Gene Therapy

59 | 

P 35 

Evaluation of a Novel AAV/Transposase 

Hybrid-Vector System for Somatic Integration 

In Vitro and In Vivo 

Nadine Müther 1 , Lajos Mates 2 , Zsuzsanna 

Izsvak 2 , Zoltan Ivics 2 , Wenli Zhang 1 , Martin 

Hausl 1 , Nina Harms 1 , Nicola M. Wolf 1 , Anja 

Ehrhardt 1 

1Max von Pettenkofer-Institute, Department of 

Virology, Ludwig-Maximilians-University of 

Munich, Munich, Germany; 2 Max Delbrück 

Center for Molecular Medicine, Berlin, Germany 

Recombinant adeno-associated viral (rAAV) 

vectors predominantly persist as 

extrachromosomal genomes. However, in 

dividing cells, rAAV vector genome copy 

numbers and transgene expression levels decline 

rapidly. Herein, we inserted the hyperactive 

Sleeping Beauty (SB) transposase variants HSB5 

and the novel mutant SB100 into our AAV-based 

two-viral-vector system displaying 10- and 100fold 

increased integration efficiencies compared 

to wild-type SB, respectively. Our previous work 

showed that transposition only works sufficiently 

from circular substrates. However, once inside 

the cell rAAV genomes form various episomal 

DNA forms including circular and linear DNA 

molecules. Thus, in initial experiments we 

addressed whether Flp recombinase-mediated 

circularization of the transposon from rAAV 

genome is a precondition of our system. Our data 

suggested that Flp-mediated circularization may 

not be required for AAV-delivered transposase 

activity significantly reducing the complexity of 

our system. In order to avoid co-transduction of 

two AAV-vectors into the same cell we generated 

a stably SB100 expressing cell line. To analyze 

integration efficiencies we performed colony 

forming assays and found that after infection with 

an rAAV with a neomycin encoding transposon 

(MOI 10.000), the integration efficiencies in 

SB100 cells was 5-fold increased compared to 

the control cell line with inactive SB (mSB). This 

demonstrated that inserting the SB transposase 

system into rAAV increases integration efficiency 

significantly. We are in the process to determine 

sites of insertion after SB100-mediated 

integration from the rAAV vector into the host 

genome by using a plasmid rescue strategy. To 

address the question whether the new version of 

the rAAV hybrid-vector system also results into 

stable transgene expression levels in vivo, we 

coinjected C57Bl/6 mice with the rAAV 

transposon donor vector expressing the 

coagulation factor IX and HSB5 delivered by an 

adenoviral vector. After induction of rapid cell 

cycling in mouse liver, transgene expression 

levels were more stable in mice which received 

HSB5 compared to the control mSB group 

indicating that transposition works from rAAV. 


P 36 

AAV2.9 Thymosin β4 Regional Application 

Enhances Therapeutic Neovascularization 

and Requires AKT Activation and Capillary 

Sprouting in the Calf Muscle 

Rabea Hinkel 1 , Teresa Trenkwalder 1 , Elena 

Gottlieb 1 , Achim Pfosser 1 , Shahana Sultana 1 , 

Franziska Globisch 1 , Georg Stachel 1 , Corinna 

Lebherz 1 , Ildiko Bock-Marquette 2 , Christian 

Kupatt 1 

1 

Internal medicine I, Klinikum Großhadern, LMU 

2 

München, Munich, Germany; Southwestern 

Medical Center, Dallas, Texas, USA 

Thymosin β4, an endogenously occurring peptide 

of 5 kDa, is cardioprotective after ischemia and 

reperfusion in a Protein Kinase B (AKT) 

dependent manner. Furthermore it is essential for 

coronary vessel development and provides 

angiogenesis during wound healing of the adult 

organism. Here we tested if long-term 

overexpression of Thymosin β4 with an adenoassociated 

virus is beneficial for therapeutic 

neovascularisation in a chronic in vivo model of 

hindlimb ischemia. 

Methods: In rabbits (n = 5/group), femoral artery 

excision was performed at d0 and 5 × 10 12 

AAV2/9 expressing Tβ4 or Lac-Z were applied 

through intramuscular injection. Tβ4 was either 

injected over the whole limb or only into the calf 

muscles. An additional group received Tβ4 whole 

limb and 1 × 10 13 AAV 2/9 AKT-DN into the lower 

limb only (i.m.). At day 7 and day 35 angiography 

was performed for collateral quantification (% of 

day 7) and frame count score (% of day 7) and 

fluorescent microspheres were applied to 

determine the regional blood flow. At day 35 

tissue was harvested for determination of 

capillary density via immunostaining for PECAM- 

1 (c/mf). 

Results: rAAV-Tβ4 significantly increasd 

capillary density in the ischemic limb (1.6 ± 0.2 

c/mf) compared to the control group (0.96 ± 

0.1c/mf). Quantification of collateral growth (175 ± 

15% vs. 95 ± 6% in controls) and microspheres 

(157 ± 10 vs. 110 ± 6 in controls) yielded similar 

results after Tβ4 transfection. However 



cotransfection of AKT-DN blunted the effect (c/mf 

1.3 ± 0.2, collaterals 126 ± 11%; microspheres 

107 ± 3%). Moreover, calf muscle transduction of 

Tβ4 sufficed to induce collateral growth (176.84 ± 

4.1%) and gain of perfusion (160 ± 12.1%) to the 

same extent as whole limb Tβ4 transduction. 

Conclusion: Thymosin β4 long-term 

overexpression exerts therapeutic 

neovascularisation in chronic ischemia. The 

down-regulation of activated AKT in the lower 

limb only suppresses capillary formation and 

subsequent collateral growth. In contrast, 

stimulation of microcirculatory growth using Tβ4 

in calf muscles only sufficed to promote 

arteriogenesis in the upper leg. These results 

suggest a backward signalling from 

microcirculation to conductance vessel formation. 


P 37 

Extensive Methylation of SFFV Promoter 

Sequences Does Not Allow Lentiviral 

Transgene Expression In Vivo 

Friederike Herbst 1 , Claudia R Ball 1 , Francesca 

Tuorto 2 , Wei Wang 2 , Ulrich Kloz 2 , Franciscus van 

der Hoeven 2 , Frank Lyko 2 , Manfred Schmidt 1 , 

Christof von Kalle 1 , Hanno Glimm 1 

1 National Center for Tumour Diseases (NCT) & 

German Cancer Research Center (DKFZ), 

Heidelberg, Germany; 2 German Cancer 

Research Center (DKFZ), Heidelberg, Germany 

Lentiviral vectors (LV) possess a high stability of 

expression in vivo and are often used to 

investigate genes and their functions. Lentiviral 

gene transfer is considered to be a promising 

less time consuming technology to facilitate the 

generation of transgenic mice with a higher yield 

of transgenic offspring as compared to the 

commonly used DNA microinjection. We applied 

LV to generate a mouse model transgenic for 

SETBP1 and eGFP. Lentiviral particles were 

injected into the perivitelline space of early stage 

embryos. LV integration was detected in newborn 

animals (F0) using PCRs specific for either the 

SETBP1 transgene or for the WPRE element of 

the construct. Lentiviral integration sites were 

detected in 65% of 31 analyzed F0 mice by 

highly sensitive LAM-PCR. 5 out of 9 F0 mice 

showed germline transmission, revealing a total 

of 33% vector positive offspring. However, no 

ectopic transcription and overexpression of 

neither SETBP1 nor eGFP could be detected in 



| 60 

transgenic mice. We therefore analyzed the 

methylation status of the internal SFFV promoter 

(SFFVp) by bisulfite sequencing. 18 of 18 

analyzed CpGs within the promoter region were 

extensively methylated in F0 animals and in all 

progeny determined (n = 12). To exclude 

transgene effects on epigenetic silencing of 

SFFVp sequences in self-inactivating LVs, we 

transduced mES cells with 

LV.SFFV.Setbp1.IRES.eGFP or the 

corresponding eGFP-expressing control vector. 

eGFP expression decreased 1.8 fold and 3.5 fold 

after differentiation of ES cells infected with the 

transgene vector and SFFV driven control vector, 

respectively. Further, we compared the 

methylation status of SFFVp sequences of 

gammaretroviral (RV) and LV in peripheral blood 

of bone marrow transplanted mice (n = 7) 3 

months after transplantation. Strikingly, LV 

transplanted mice showed a higher degree of 

methylation. Here, we demonstrate that the 

commonly used SFFV promoter is highly 

methylated with remarkable strength and 

frequency during development in vivo and 

differentiation in vitro. We conclude that LV using 

an internal SFFV promoter are not suitable for 

the generation of transgenic mice or constitutive 

expression studies in hematopoietic cells. 


P 38 

Effect of Nogo-B Overexpression in Vascular 

Smooth Muscle Cell Migration and 

Proliferation Mediated by Integration-Deficient 

Lentiviral Vectors 

Helen Chick 1 , RA McDonald 1 , AB Kritz 1 , NM 

Kane 1 , R Alba 1 , WC Sessa 2 , AJ Thrasher 3 , AH 

Baker 1 

1BHF Glasgow Cardiovascular Research Centre, 

Division of Cardiovascular and Medical Sciences, 

University of Glasgow, Glasgow, UK; 

2 Department of Pharmacology and Program in 

Vascular Signaling and Therapeutics, Boyer 

Centre for Molecular Medicine, Yale University 

School of Medicine, New Haven, Connecticut, 

USA; 3 Molecular Immunology Unit, UCL, Institute 

of Child Health, London, UK 

Proliferation and migration of vascular smooth 

muscle cells (VSMCs) is a hallmark of neointima 

formation (NI) associated with acute vascular 

injury. Nogo-B is a member of the reticulon 4 

family of proteins. Nogo-B is highly expressed in 

vascular endothelial cells and VSMCs and

61 | 

regulates vascular remodeling. Previous studies 

have demonstrated that adenoviral-mediated 

overexpression of Nogo-B reduced NI after acute 

vascular injury in animal models. This effect was 

mediated through inhibition of VSMC migration 

and proliferation. Lentiviral vectors (LVs) are 

efficient at targeting VSMCs and the recent 

development of integration-deficient lentiviral 

vectors (IDLVs) offers additional potential for LVs 

in vascular gene delivery. Here, we have 

assessed Nogo-B overexpression in VSMCs 

mediated by IDLVs in vitro, as a potential 

therapeutic strategy for the prevention of NI in 

vivo. Immunofluorescence and western blot 

analysis indicated that IDLVs expressing Nogo-B 

efficiently increased Nogo-B expression in 

VSMCs, compared to IDLVs expressing GFP and 

no virus controls. Assessment of proliferation 

demonstrated that IDLVs overexpressing Nogo- 

B, led to a significant decrease in VSMC 

proliferation compared to IDLVs expressing GFP 

(multiplicity of infection (MOI) 25: 0.14 ± 0.0084 

absorbance (abs) at 570 nm vs. 0.28 ± 0.01 abs, p 

< 0.05). A wound-mediated cell migration assay 

demonstrated that IDLV delivery of Nogo-B 

significantly reduced the migration of VSMCs, 

compared to the IDLVs expressing GFP (MOI 25: 

55 ± 4.9 μm vs. 113 ± 7.6 μm, p < 0.05). Taken 

together, our study demonstrates that IDLVs are 

efficient in mediating overexpression of Nogo-B 

in VSMCs, leading to phenotypic effects on 

migration and proliferation. This has important 

implications for the use of IDLVs as a potential 

therapeutic vector for the prevention of NI during 

acute vascular injury. 


P 39 

Combining Oncolytic Adenoviruses with 

Expression of Therapeutic Antibodies 

Dominik E Dorer 1 , Roland Kontermann 2 , Dirk M 

Nettelbeck 1 

1 Helmholtz University Group Oncolytic 

Adenoviruses, DKFZ and Department of 

Dermatology, Heidelberg University, Heidelberg, 

Germany; 2 Institute for Cell Biology and 

Immunology, University of Stuttgart, Stuttgart, 


Oncolytic viruses which selectively kill tumor cells 

are emerging tools for cancer treatment. We seek 

to combine adenoviral oncolysis and therapeutic 

antibody expression in order to further increase 

anti-cancer efficacy through antibody-mediated 

toxicity. Therefore, we created a model system 

by arming our adenoviruses with a recombinant 

single-chain antibody directed against the well 

established carcinoembryonic cancer antigen 

(CEA) fused to the constant domain of IgG2a 

(scFvCEA-Fc). To ensure cancer cell specific 

replication, replication-competent viruses were 

engineered to bear a 24 bp deletion in the E1A 

region and a chimeric 5/3 fiber to further increase 

infectivity for various cancer entities. 

Furthermore, we used different genomic positions 

and genetic tools for our scFvCEA-Fc transgene 

insertion. Among these we investigated splice 

acceptor sites derived from the beta-actin gene, 

the adenoviral pIII gene, and the Ad40 long fiber 

gene and compared resulting transgene levels to 

expression via established tools such as an 

internal ribosomal entry site or a nonreplicating 

adenovirus containing a cytomegalovirus early 

promoter. Infection of various CEA + and CEA − 

cell lines with any of our recombinant 

adenoviruses led to detectable amounts of 

scFvCEA-Fc in cell culture supernatants and cell 

pellets. However, transgene levels were strongly 

varying depending on the genomic loci and 

genetic tool used, giving the highest amount with 

the Ad40 splice acceptor. The expressed 

scFvCEA-Fc was then further characterized in 

flow cytometry, SDS-page, ELISA, and 

cytotoxicity assays. Our results show that 

scFvCEA-Fc binds only to cells expressing CEA 

or to purified antigen in a dose-dependent way 

and that transgene expression/antibody mediated 

cytotoxicity does not interfere with adenoviral 

replication and hence oncolysis. Currently, we 

are investigating anti-tumoral efficacy of immune 

cells in presence of scFvCEA-Fc in vitro and 

eventually in vivo. In the end, we want to create a 

highly efficient oncolytic adenovirus platform that 

combines virus-induced tumor cell killing and 

expression of therapeutic antibodies with 

systemic activity directed against various cancer 

markers. 




P 40 

TNF Alpha Gene Therapy Synergizes with 

Liposomal Doxorubicin in the Treatment of 

Experimental Neuroblastoma and Hepatoma 

in Mice 

Baowei Su 1 , Ernst Wagner 1,2 , Manfred Ogris 1,2 



Munich, Germany; 2 Center for Nanoscience 

(CeNS), Ludwig-Maximilians-University, Munich, 


Tumor necrosis factor alpha (TNF) is a highly 

potent cytokine involved in endothelial cell 

activation, inflammation and tumor cell killing. As 

systemic application of TNF protein can be highly 

toxic, tumor localized expression of the cytokine 

after gene delivery is a potentially suited 

approach. We could recently show that systemic 

TNF gene delivery promoted tumor accumulation 

of liposomally encapsulated doxorubicine 

(Caelyx). Here we present the therapeutic effect 

of this treatment on subcutaneous Neuro2A 

murine neuroblastoma and enhanced Caelyx 

accumulation in HUH7 human hepatocellular 

carcinoma. A/J mice bearing subcutaneous, 

syngeneic Neuro2A tumors were treated with 

polyplexes based on a biodegradable polymer 

(G3-HD-OEI), HUH7 hepatoma xenografts in 

SCID mice received TNF polyplexes based on 

linear polyethylenimine carrying a peptide (GE11) 

selectively binding to the epidermal growth factor 

receptor, which is highly overexpressed in HUH7. 

When pretreating mice with TNF polyplexes, 

improved tumor accumulation of Caelyx was 

observed in both tumor models as compared to 

control polyplexes plus Caelyx. With the help of 

live bioluminescense imaging, enhanced tumor 

accumulation of Caelyx fluorescently labeled with 

the lipid DiR emitting in the near infrared was 

observed in living animals. This suggests a TNF 

mediated increase of tumor blood vessel 

permeability augmenting the accumulation of 

liposomal chemotherapeutics. In the Neuro2A 

model, three rounds of systemic TNF polyplex 

application and subsequent Caelyx treatment led 

to tumor growth inhibition lasting until eight days 

after the last treatment. Regrowth of treated 

tumors could be inhibited by a second treatment 

cycle indicating that no resistance towards 

combined TNF/Caelyx treatment occurred. 

Histological staining of cryosection showed 

significant decrease of CD31 expression in the 

re-treated group, which indicates a decreased 

vessel density caused by TNF/Caelyx treatment. 

In conclusion, combining liposomal 

chemotherapeutic with TNF gene therapy leads 

to a synergistic effect on tumor reduction in vivo. 




| 62 

P 41 

Harnessing Molecular Targets in Human 

Colon Cancer Cells for Tumor Specific RNAi 

and pDNA-Based Therapies 

Arzu Cengizeroglu 1 , Katarina Farkasova 1 , 

Daniela Deutsch 1 , Rudolf Haase 1 , Martina 

Anton 2 , Ernst Wagner 1,3 , Manfred Ogris 1,3 

1Pharmaceutical Biotechnology, Department of 


Munich, Germany; 2 Institute of Experimental 

Oncology and Therapy Research, Technische 

Universität München, Munich, Germany; 3 Center 

for Nanoscience (CeNS), Ludwig-Maximilians- 


A major challenge in cancer therapy is to achieve 

high levels of specificity and efficacy preventing 

cancer cells to escape apoptosis, which 

otherwise leads to uncontrolled proliferation. For 

targeted cancer gene therapy a tumor cell 

specific promoter would be essential, which is 

inactive in nontransformed tissue and at the 

same allows for high levels of transgene 

expression in tumor cells. Moreover, the tumorspecific 

activity of such a promoter can be utilized 

for tumor detection as well as measuring antitumoral 

effects. In gastrointestinal tumors, the 

constitutive activation of the wnt-pathway leads to 

malignancy due to high levels of the transcription 

factor beta-catenin in the nucleus, which in turn 

constitutively activates target genes causing 

enhanced proliferation, invasion and metastasis. 

Here we describe the use of the synthetic betacatenin 

dependent promoter CTP4 for specific 

expression of toxic transgenes, like the VP3 

subunit of the chicken anemia virus (Apoptin). In 

addition, CTP4 driven luciferase expression 

allowed us to evaluate the effect of RNAi 

mediated knockdown of wnt components on 

beta-catenin mediated transcription. Early 

passages of human colorectal cancer cell lines 

and control cell lines (HeLa and U87MG 

glioblastoma) were either transiently transfected 

with CTP4-luciferase plasmid or stably 

transduced with a lentiviral vector encoding for 

CTP4 driven luciferase. Only cell lines with a 

deregulated wnt/beta-catenin signaling showed 

transgene expression, whereas in control cell 

lines transgene expression was below the level of 

the control vector without promoter. Knockdown 

of beta-catenin with siRNA clearly reduced CTP4 

driven luciferase expression, and in a similar way 

other factors were targeted by siRNA

63 | 

transfection. For plasmid based therapies we 

were able to show that in wnt/beta-catenin 

deregulated colon carcinoma cells CTP4 driven 

apoptin expression led to more prominent cell 

killing when compared to control cells. Taken 

together, these results provide rationales for 

further preclinical in vivo testing of 

transcriptionally targeted plasmid vectors and 

siRNA based therapies for colorectal cancer. 


P 42 

Liver Cancer Protease Activity Supports MMP 

Activatable Oncolytic Measles Virus 

Michael D. Muehlebach 1 , Thomas Schaser 1 , 

Martina Zimmermann 2 , Sorin Armeanu 2 , Kay- 

Martin O. Hanschmann 3 , Michael Bitzer 2 , Ullrich 

Lauer 2 , Roberto Cattaneo 4 , Klaus Cichutek 1 , 

Christian J. Buchholz 1 

1 Division of Medical Biotechnology, Paul-Ehrlich- 

Insitut, Langen, Germany; 2 Innere Medizin, 

Medizinische Universitätsklinik, Tübingen, 

Germany; 3 Biostatistics Section, Paul-Ehrlich- 

Institut, Langen, Germany; 4 Molecular Medicine, 

Mayo Clinic, Rochester (MN), USA 

Primary and secondary cancers of the liver are a 

significant health problem with limited treatment 

options. We sought to develop an oncolytic 

measles virus (MV) preferentially activated in 

liver tumor tissue thus reducing infection and 

destruction of healthy tissue. Making cell entry of 

measles virus (MV) dependent on tumourassociated 

matrix metalloproteases (MMPs) 

restricts virus propagation to tumour cells and 

improves the virus safety in xenograft tumour 

models. We documented with specimen of 44 

patients that in primary tumor tissue urokinase 

type plasminogen activator (uPA) and especially 

matrix metallproteinase-2 (MMP-2) are 

significantly more active than in adjacent nontumorous 

tissue. When allocated to specific 

tumor entities, the overall tendency observed in 

the whole collective was confirmed. We then 

generated variants of the MV fusion protein by 

inserting different MMP-substrate motifs at the 

protease cleavage site, and identified the motif 

PQGLYA as the most efficient cleavage site as 

determined by syncytia formation on protease 

positive tumor cells. The corresponding MMPactivatable 

oncolytic MV-MMPA1 virus was 

rescued and shown to be strongly restricted on 

primary human hepatocytes and healthy human 

liver tissue, while remaining as effective as the 

parental MV in tumor tissue sections on tissue 

slices of 10 patients. Thereby, a strong trend (p = 

0.09) for improved tumor targeting of MV-MMPA1 

was revealed, which correlated to the upregulation 

of MMP-2 activity in tumorous tissue (r 

= 0.7388). These data underline the clinical 

potency of the MMP-activation concept to 

generate safer oncolytic viruses for the treatment 

of primary and secondary cancers of the liver. 


P 43 

Specific LNA-ASOs Against Oncogenic p73 

Isoforms Permit Therapeutic Cancer 

Targeting In Vivo 

Stephan Emmrich 1 , Weiwei Wang 2 , Katja John 1 , 

Wenzhong Li 2 , Brigitte M Pützer 1 

1 Department of Vectorology and Experimental 

Gene Therapy, Biomedical Research Center, 

University of Rostock, Rostock, Germany; 

2 Department of Cardiac Surgery, University of 

Rostock, Rostock, Germany 

Differential mRNA splicing and alternative 

promoter usage of the TP73 tumor suppressor 

gene results in the expression of multiple NH2truncated 

isoforms that act as oncogenes. 

Abundant levels of these p73 variants in a variety 

of human cancers correlate with adverse clinical 

prognosis and response failure to conventional 

therapies, underscoring their relevance as 

marker for disease severity and target for cancer 

intervention. With respect to an equally important 

role for amino-truncated p73 splice forms 

(DeltaTAp73) and DeltaNp73 (summarized as 

DNp73) in the tumorigenic process, we designed 

locked nucleic acid (LNA) antisense 

oligonucleotide (ASO) gapmers against individual 

species that were complementary to DeltaEx2 

and DeltaEx2/3 splice junctions and a region in 

exon 3B unique for ′ DeltaN and DeltaN. 

Treatment of cancer cells with these ASOs 

resulted in a strong and specific reduction of 

tumorigenic p73 transcripts and proteins, 

importantly, without abolishing the wild-type p73 

tumor suppressor form as observed with p73shRNA. 

The specific antisense oligonucleotides 

rescued cells from apoptosis inhibition due to 

overexpression of their corresponding aminotruncated 

p73 isoform and decreased tumor cell 

proliferation. Furthermore, ASO-116 against 

DeltaEx2/3 coupled to magnetic nanobead 

polyethyleneimine (MNB/PEI) carriers 

significantly inhibited malignant melanoma 



growth, which correlated with a shift in the 

balance between endogenous TAp73 and 

DeltaEx2/3 towards apoptotic full-length p73. Our 

study demonstrates the successful development 

of LNA-ASOs that selectively differentiate 

between the closely related p73 oncoproteins, 

and provide new tools to further delineate their 

biological properties in different human 

malignancies and for therapeutic cancer 

targeting. 


P 44 

Oncolytic Enhancement of VSV Through 

Inhibition of Host Proinflammatory Innate 

Immune Responses by Vector-Mediated 

Expression of NFκB Super-Repressor 

Jennifer Altomonte, Sabrina Marozin, Oliver 

Ebert 

II. Medizinische Klinik und Poliklinik, Klinikum 

rechts der Isar, TU München, Munich, Germany 

Vesicular stomatitis virus (VSV) vectors harboring 

the MD51 mutation in the endogenous matrix (M) 

protein are potent inducers of interferon (IFN) in 

non-neoplastic cells, making them safer oncolytic 

agents than the wild-type virus. However, we 

have observed substantial attenuation of 

intratumoral replication of these vectors in our 

immune-competent, orthotopic rat model of HCC, 

significantly limiting the therapeutic efficacy. 

Following a peak in rVSV(MD51) titers in infected 

tumors after only 24 hours, a rapid and 

logarithmic reduction occurs, which is 

coincidental with the massive infiltration of 

inflammatory cells to the infected tumor sites. We 

hypothesized that intratumoral virus replication, 

and hence the oncolytic potency of rVSV(MD51) 

could be significantly enhanced by blocking antiviral 

inflammatory responses triggered by 

induction of the NF-κB pathway following viral 

infection without substantially interfering with the 

vector's intrinsic interferon (IFN) induction 

capacity to protect non-tumor cells. To test this 

hypothesis, we incorporated srIkBalpha, the socalled 

NF-κB super-repressor into the full-length 

VSV(MD51) vector. In vitro characterization 

revealed that expression of srIκBalpha as an 

additional transcription unit in the rVSV(MD51) 

backbone successfully blocked virus-mediated 

activation of NF-κB in primary human 

hepatocytes. Although reporter assays 

demonstrated that srIκBalpha expression partially 

interferes with the robust IFN induction and 



| 64 

response to the rVSV(MD51) vector, the 

rVSV(MD51)-srIkB vector is nevertheless able to 

induce IFN significantly better than wild-type 

VSV. The advantage of such an intermediate 

vector is that it would maintain an improved 

safety profile over wild-type, while maintaining 

high levels of replication within the tumor. We 

predict that this strategy will prolong the kinetics 

of rVSV(M51R) replication, resulting in an 

improved oncolytic agent. We further hypothesize 

that a general reduction of inflammation could 

potentially allow us to safely administer higher 

doses of virus. These questions will be answered 

through comprehensive toxicity and efficacy 

studies, which are underway in HCC tumorbearing 

immune-competent rats. 


P 45 

Noninvasive In Vivo Monitoring of Tumor 

Responses to Oncolytic Viral Therapy of 

Hepatocellular Carcinoma in Rats by [ 18 F]- 

FDG-PET and MRI 

Jennifer Altomonte 1 , Andreas K Buck 2 , Rickmer 

Braren 3 , Oliver Ebert 1 

1 II. Medizinische Klinik und Poliklinik, Klinikum 


2 Nuklearmedizinische Klinik und Poliklinik, 

Klinikum rechts der Isar, TU München, Munich, 

Germany; 3 Institut für Röntgendiagnostik, 

Klinikum rechts der Isar, TU München, Munich, 


Oncolytic viruses are currently under intense 

development as alternative cancer therapies. 

Preclinical and clinical studies will benefit 

significantly from the development of noninvasive 

imaging modalities to repetitively monitor key 

parameters of tumor response. As oncolytic 

viruses can kill tumor cells by distinct 

mechanisms from conventional therapies, they 

require new approaches to accurately evaluate 

responses. We hypothesized that the 

combination of positron emission tomography 

(PET) and magnetic resonance (MR) imaging 

could informatively monitor the effects of 

vesicular stomatitis virus (VSV) treatment of HCC 

tumors. To this end, VSV or control buffer were 

infused through the hepatic arteries of multifocal 

HCC-bearing rats, and the animals were imaged 

by PET and MRI on days − 1, 1, and 3 posttreatment. 

In addition, a subset of animals was 

euthanized at each time-point in order to validate 

the imaging data with histology. For PET

65 | 

imaging, we utilized the 18 F-fluorodeoxyglucose 

([ 18 F]-FDG) tracer, which is an analog of glucose 

that is taken up by metabolically active cells, 

such as tumor cells. Analysis of the isocontour 50 

(50% of maximum) of tumor intensities revealed 

a significant decrease in [ 18 F]-FDG uptake 

following VSV treatment, indicating a reduction of 

metabolic activity, as compared to buffer controls. 

To determine whether this reduction correlated 

with increased tumor necrosis, histological 

sections of corresponding tumors were subjected 

to morphometric analysis of necrotic areas. 

Interestingly, a strong correlation of the two 

parameters existed only for the buffer control 

group. Further investigation by analysis of MRI 

and histological data revealed complex 

processes underway at early time-points postviral 

therapy, such as necrosis, inflammation, and 

repair of necrotic tissue, all of which result in 

changes in [ 18 F]-FDG uptake. Together, these 

experiments indicate that [ 18 F]-FDG-PET data 

should be carefully considered at early timepoints 

after VSV therapy, as changes in tracer 

uptake can be easily misinterpreted. This data 

strongly supports the rationale for multimodal 

imaging to assess tumor responses to oncolytic 

viral therapy. 


P 46 

Functional Analysis of Armed Oncolytic 

Adenoviruses for Nuclear Reporter Gene 

Imaging 

Youlia Kostova 1 , Klaus Mantwill 1 , Katja Dumler 1 , 

Anja Wolf 1 , Hans-Jürgen Wester 2 , Bernd 

Gansbacher 1 , Per S Holm 1 , Martina Anton 1 

1 Klinikum rechts der Isar der TUM, Institut für 

Experimentelle Onkologie und 

Therapieforschung, Munich, Germany; 2 Klinikum 

rechts der Isar der TUM, Nuklearmedizinische 

Klinik und Poliklinik, Munich, Germany 

Oncolytic adenoviruses (AdV) are recently 

gaining interest in gene therapy, as they have the 

ability to lyse tumour cells. Hence, we 

investigated whether a YB-1-dependent oncolytic 

AdV armed with the reporter and suicide gene 

herpes simplex virus thymidine kinase (HSV1sr39tk) 

displays an enhanced tumour cell killing 

through the combined action of oncolysis and 

HSV1-sr39tk-Ganciclovir (GCV) mediated 

cytotoxicity in human U87MG glioma cells, and if 

this AdV is suitable for in vivo imaging studies. As 

a nuclear reporter gene HSV1-sr39tk is favoured 

over the wild-type form because of its improved 

in vivo imaging properties. YB-1-dependent 

oncolytic AdV have so far not been combined 

with suicide genes in attempt to boost their 

therapeutic effect. U87MG cells were infected 

with armed oncolytic AdV expressing HSV1sr39tk 

in the E3 region and control AdV under 

varying conditions. DNA, RNA and protein levels 

of HSV1-sr39tk were determined by Southern 

blot, qRT-PCR and Western blot analyses, 

respectively. The oncolytic and cytotoxic effects 

of the virus were analysed by Sulforhodamine B 

staining or XTT assay and compared to the 

activity of other oncolytic and replication-deficient 

AdV. The enzymatic function of HSV1-sr39tk was 

measured by uptake of the [18F]-FHBG, a 

radiolabelled prodrug-analogue, 2 days after 

infection. Increased AdV DNA and HSV1-sr39TK 

DNA and protein levels over time were observed. 

The virus displayed an enhanced overlapping 

oncolytic and HSV1-sr39tk-GCV-mediated 

cytotoxic effect (>95%) in glioma cells, even at a 

low viral dosage and at GCV concentrations < 0.1 

μg/ml. The strongest cell killing was achieved if 

GCV was applied 2–3 days after infection. We 

also observed an increased bystander effect 

when infected cells were mixed with uninfected 

cells. Furthermore, HSV1-sr39TK displayed 

enhanced accumulation of radioactive tracer as 

compared to control AdV expressing HSV1sr39tk 

in the E3 region. The results of this study 

indicate that the HSV1-sr39TK expressing 

oncolytic adenovirus effectively induces tumour 

cell killing and radiotracer accumulation ([ 18 F]- 

FHBG) and might allow for in vivo PET imaging 

within a limited time frame. 




P 47 

Radioiodine Therapy of HCC Following 

Systemic Sodium Iodide Symporter (NIS) 

Gene Transfer Using EGF-Receptor Targeted 

Nonviral Gene Delivery Vectors 

Kathrin Klutz 1 , David Schaffert 2 , Geoffrey k 

Grünwald 1 , Michael J Willhauck 1 , Wolfgang 

Rödl 2 , Nathalie Wunderlich 1 , Christian Zach 3 , 

Franz Josef Gildehaus 3 , Reingard Senekowitsch- 

Schmidtke 4 , Ernst Wagner 2 , Burkhard Göke 1 , 

Manfred Ogris 2 , Christine Spitzweg 1 

1Department of Internal Medicine II, Klinikum 

Großhadern, Ludwig-Maximilians-University 

Munich, Munich, Germany; 2 Pharmaceutical 



Germany; 3 Department of Nuclear Medicine, 

Klinikum Groβhadern, Ludwig-Maximilians- 

University Munich, Munich, Germany; 

4 Department of Nuclear Medicine, Technical 

University, Munich, Munich, Germany 

We have recently demonstrated induction of 

significant tumor-selective uptake and therapeutic 

efficacy of radioiodine in neuroblastoma tumors 

after systemic nonviral polyplex-mediated NIS 

gene delivery. The aim of the current study was 

to evaluate the efficacy of novel nanoparticle 

vectors based on linear polyethylenimine (LPEI), 

shielded by polyethylene glycol (PEG), and 

coupled with the synthetic peptide GE11 as an 

epidermal growth factor (EGF) receptor-specific 

ligand for targeting the NIS gene to EGFRexpressing 

human HCC (Huh7) cells. We used 

LPEI-PEG-GE11 to form targeted polyplexes with 

a NIS-expressing plasmid (pCpG-EF1-NIS) to 

transfect Huh7 cells followed by analysis of 

functional NIS expression in vitro and in vivo. In 

vitro incubation of Huh7 cells with NIS-conjugated 

LPEI-PEG-GE11 resulted in a 22-fold increase in 

iodide uptake activity. After establishment of 

subcutaneous Huh7 tumors in nude mice, NISconjugated 

nanoparticle vectors or control 

vectors were injected via the tail vein followed by 

analysis of radioiodine biodistribution after i.p. 

injection of 123 I using γ-camera imaging and ex 

vivo γ-counting. While injection of control vectors 

did not result in tumoral iodide accumulation, 

Huh7 tumors showed a perchlorate-sensitive 

iodide uptake of 6–9 % ID/g 123 I with an eff. halflife 

of approx. 6 h. In contrast, non-target organs 

like liver, lungs and kidneys showed no 

significant iodide uptake activity. After application 

of the EGFR-specific antibody cetuximab 24 h 

prior to administration of NIS-conjugated LPEI- 

PEG-GE11 tumoral iodide uptake activity and 

NIS mRNA expression were markedly reduced 

confirming the specificity of EGFR-targeted 



| 66 

nanoparticle vectors. After 4 cycles of polymer 

application followed by therapeutic application of 

131 I, tumor growth was significantly reduced as 

compared to control groups. In conclusion, these 

results clearly demonstrate that systemic in vivo 

NIS gene transfer using novel synthetic 

nanoparticle vectors coupled with an EGFRtargeting 

ligand is capable of inducing tumorspecific 

iodide uptake, which represents a 

promising innovative strategy for systemic NIS 

gene therapy in metastatic cancers. 


P 48 

MSC-Mediated sTRAIL and CXCR4 Delivery in 

Combination with XIAP Knockdown in 

Pancreatic Cancer Cells Leads to Inhibition of 

Metastatic Growth of Pancreatic Carcinoma 

Sina Rupprecht 1 , Andrea Mohr 1 , Stella 

Albarenque 1 , Rui Yu 1 , Laura Deedigan 1 , Mairead 

Reidy 1 , Simone Fulda 2 , Ralf Zwacka 2 

1National Centre for Biomedical Engineering 

Science, Molecular Therapeutics Group, National 

University of Ireland, Galway, Ireland; 2 Children's 

Hospital Ulm, Ulm University, Ulm, Germany 

Disseminating tumour cells appear to be one of 

the biggest problems in oncological medicine. 

Here, we combined the tumour-specific 

apoptosis-inducing activity of TRAIL with the 

ability of mesenchymal stem cells (MSCs) to 

infiltrate both tumour and lymphatic tissues. 

Using this approach it was our aim to target 

primary tumours as well as disseminated cancer 

cells in a human pancreatic cancer mouse model. 

Targeting XIAP by applying RNAi inside the 

cancer cells further optimized this approach, 

because it is known that knocking down XIAP 

has an apoptosis sensitizing and anti-metastatic 

effect on targeted cancer cells. Furthermore, it 

has recently been published that the chemokine 

receptor CXCR4 and its ligand, the stromal-cellderived 

factor (SDF-1), enhanced the migratory 

abilities of MSCs, following the tumour-derived 

SDF-1 chemotactic axis. We generated MSCs 

expressing a trimeric soluble wild-type TRAIL 

(MSC.sTRAIL). MSC.sTRAIL triggered limited 

apoptosis in pancreatic cancer cells that were 

resistant to soluble recombinant TRAIL, which is 

most likely due to the enhanced effect of the 

direct, cell-mediated delivery of trimeric TRAIL. 

MSC.sTRAIL-mediated cell death was markedly 

increased by concomitant knockdown of XIAP by 

RNAi in the cancer cells. These findings were

67 | 

confirmed in xenograft models. Using 

MSC.sTRAIL on XIAP silenced xenografts it was 

possible to force the primary tumour to go into 

remission and to block the growth of lung 

metastasis in MSC.sTRAIL treated animals. 

Furthermore, to enhance the directed migration 

of sTRAIL-loaded MSCs towards tumour cells, 

these cells were adenovirally transducted to 

overexpress the chemokine receptor CXCR4. 

The natural secretion of SDF-1 by tumour cells 

builds up a chemotactic axis and it could be 

demonstrated that CXCR4 overexpressing 

MSC.sTRAIL utilize this gradient for enhanced 

migration towards the tumour cells. In summary, 

this is the first demonstration that a combined 

approach using systemic MSC-mediated delivery 

of sTRAIL and overexpression of CXCR4 

together with XIAP inhibition suppresses 

metastatic growth of pancreatic carcinoma. 


P 49 

YB-1 Dependent Oncolytic Adenovirus as a 

Strategy for the Treatment of Glioma Cancer 

Stem Cells 

Ulrike Naumann 1 , Per Sonne Holm 2 , Klaus 

Mantwill 2 , Janina Seznec 1 

1Hertie Institute for Clinical Brain Research and 

Center Neurology, Laboratory for Molecular 

Neuro-Oncology, Tübingen, Germany; 2 Institute 

of Experimental Oncology and Therapy 

Research, Klinikum rechts der Isar, TU Munich, 


Recent studies have demonstrated the existence 

of a small fraction of glioma cells endowed with 

features of primitive progenitor cells and tumorinitiating 

function. Such cells have been defined 

as glioma-like cancer stem cells (GCSC). GCCS 

contribute to glioma progression due o their 

properties to overcome cell death induced by 

chemo- and radiation-therapy, to self-renew and 

to promote angiogenesis. For this reason, GCSC 

might never be completely eradicated using 

standard therapy and will be responsible for the 

recurrence of glioma. It has been shown that the 

Y-box protein YB-1 is an oncogenic 

transcription/translation factor associated with the 

development of cancer. By upregulation of the 

detoxification membrane channel protein MRP 

and MDR, YB-1 induces drug resistance. Again, 

YB-1 itself is regulated via the stem cell-specific 

transcription factors SLUG, SNAIL and TWIST. 

Third, YB-1 is overexpressed in GCSC, but is 

barely detectable in normal human stem cells. 

Fourth, YB-1 plays an important role in the 

adenovirus half-life by targeting the adenoviral 

E2-late promoter. This knowledge promoted us to 

develop an YB-1 based glioma virotherapy. We 

investigated the therapeutic potential of the YB-1 

dependent oncolytic adenovirus Ad-Delo3-RGD 

in GCSC. We demonstrated that in vitro Ad- 

Delo3-RGD replicates in GCSC, kills highly 

temozolomide (TMZ)-resistant GCSC and 

moderately rendered these cells susceptible 

towards TMZ-treatment. In vivo, using an 

orthotopic mouse xenograft glioma model, 

infection of GCSC-derived tumors with Ad-Delo3- 

RGD significantly prolonged survival. Taken 

together all data, an YB-1 based adenovirotherapy 

might be a promising treatment 

strategy against glioma. 


P 50 

Tet-Regulated, Lentivirally Mediated BMP-2 

Expression in Primary Cells 

Daniela Wübbenhorst, Katja Dumler, Gabriele 

Wexel, Andreas Imhoff, Bernd Gansbacher, 

Stephan Vogt, Martina Anton 

Institute of Experimental Oncology and Therapy 



Therapy of cartilage defects is challenging due to 

poor self-healing capacity. A combined gene and 

cell therapeutic approach using transduced 

primary chondrocytes and pluripotent 

mesenchymal stem cells (MSC) was chosen. 

MSC are able to differentiate into a variety of cell 

types among them osteocytes and chondrocytes 

and are therefore considered for the treatment of 

osteochondral defects. Regulated gene 

expression of growth factor bone morphogenetic 

protein 2 (BMP-2) was achieved using the Tet-on 

System, delivered by VSV-G pseudotyped 

lentiviral SIN vectors (LV). Primary rabbit 

chondrocytes were infected with 1-vector Tet-on 

constructs, expressing the reverse Transactivator 

(rtTA) and eGFP or BMP 2 under control of the 

Tet-responsive element (TRE). Transgene 

expression was induced by doxycycline (dox). 

The efficacy of the Tet-on induction system was 

optimized using eGFP. Using BMP-2-expressing 

vectors resulted in secretion of 15–16 ng/ml 

BMP-2 into the medium. After withdrawal of dox 

BMP-2 expression decreased to background 

levels and was re-inducible for various cycles 



without loss of efficacy. Additionally, prolonged 

tet-regulated BMP-2 expression was feasible. 

Produced BMP-2 was functional as evidenced by 

proteoglycan synthesis. Transduction efficiency 

of rabbit bone marrow derived MSC by eGFP 

expressing LV was 65%. After lentiviral infection 

cells retained full differentiation potential. 

Transduction of MSC with the BMP-2 Tet-on 

vectors resulted in inducible BMP-2 expression 

with a 36-fold increase of BMP-2 expression on 

RNA level as well as a 113-fold increase on 

protein level compared to uninduced cells. The 

lentivirally delivered Tet-on System allows for 

regulated expression of BMP-2 in primary rabbit 

chondrocytes as well as MSCs. The amount of 

BMP-2 produced by chondrocytes after induction 

in vitro is sufficient for proteoglycan synthesis, a 

marker for chondrogenesis and levels are 

comparable to results achieved using a 

constitutively expressing retroviral vector. 


P 51 

Neutrality and Non-Neutrality in Clonal 

Development 

Lars Thielecke, Ingmar Glauche, Sebastian 

Gerdes, Ingo Roeder 

Institut für Medizinische Informatik und Biometrie, 

TU-Dresden, Dresden, Germany 

Tissue regeneration and maintenance are 

generally driven by a population of stem cells. 

These cells are considered to have closely 

similar functional capabilities and contribute to 

the production of mature somatic cells by 

differentiation and continuous proliferation. Clonal 

marking studies in the hematopoietic system 

revealed that the peripheral blood is generally 

composed of the ancestry of multiple 

hematopoietic stem cells at each point in time. 

However, in certain pathological situations (i.e., in 

leukemias) this balance is upset and the system 

converts to a monoclonal, often malign situation. 

Similar results are confirmed in vivo and in vitro 

using a wide range of viral markers. However, the 

general questions remain how this clonal 

dominance is generated and how it can be 

potentially detected at early stages. Using a 

range of mathematical modeling approaches we 

characterize the temporal development of 

multiple clones under different assumptions 

about the relative fitness. In particular we use 

these models to study the case of neutral 

competition for certain in vitro and in vivo settings 



| 68 

and extend our studies to the characterization of 

clonal dominance. Based on our model 

simulations we make predictions about the 

variability of clone sizes as a function of time for 

the situation of neutral and non-neutral clonal 

competition. Our results suggest that clonal 

conversion is inevitable even in the neutral 

situation and is only accelerated if dominating 

clones exist. We furthermore suggest 

experimental strategies for a quantitative 

characterization of the conversion process. 


P 52 

Efficient Zinc-Finger Nucleases-Mediated 

Gene Knockout in Pluripotent Stem Cells 

Anna Osiak 1 , Frank Radecke 2 , Eva Guhl 1 , Sarah 

Radecke 2 , Kafaitullah Khan 3 , Fabienne Lütge 3 , 

Tobias Cantz 4 , Regine Heilbronn 1 , Klaus 

Schwarz 2 , Toni Cathomen 3 

1 Institute of Virology (CBF), Charité Medical 

School, Berlin, Germany; 2 Institute for 

Transfusion Medicine, University of Ulm, Ulm, 

Germany; 3 Department of Experimental 

Hematology, Hannover Medical School, 

Hannover, Germany; 4 REBIRTH Cluster of 

Excellence, JRG Group Stem Cell Biology, 


Murine embryonic stem cells (mESCs) have been 

an excellent model system for introducing 

specific modifications into the genome to study 

gene function in vitro or to generate knockout 

mouse models. However, gene targeting in 

mESCs using standard techniques is quite 

inefficient and typically reaches frequencies of 

10-7 to 10-6. In consequence, the usage of 

complex positive/negative selection strategies to 

isolate gene targeted clones has been crucial. A 

promising strategy to introduce specific changes 

into complex genomes is based on zinc-finger 

nucleases (ZFNs). A ZFN subunit consists of a 

nonspecific endonuclease domain fused to a 

specific DNA-binding domain composed of 

engineered zinc-finger motifs that tether the 

nuclease domain to a preselected chromosomal 

site. Upon dimerization of two ZFN subunits at 

the target site, the ZFN pair specifically cleaves 

the DNA to trigger the ensuing DNA damage 

response, which can be harnessed for targeted 

genome engineering. Here, we used an EGFPpositive 

mESC line in order to explore the 

potential of ZFNs to generate a genetic knockout 

in the absence of antibiotic selection. EGFP-

69 | 

specific ZFNs carrying different previously 

described endonuclease variants were initially 

tested in an EGFP-positive human cell line and in 

EGFP + mESC to identify ZFN variants that 

combine high activity with minimal toxicity. Under 

optimized conditions, the single copy EGFP locus 

was disrupted in 9% of EGFP + mESC after 

lipofection of the ZFN expression vectors and 3% 

upon electroporation. Importantly, the percentage 

of knockout cells remained stable over time, 

suggesting minor ZFN-associated toxicity. For 

two independent clones the EGFP knockout 

status was verified on the genome level. 

Currently, these clones are being tested for their 

pluripotent status by a teratoma formation assay. 

In the advent of applying patient-derived induced 

pluripotent stem cells in regenerative medicine, 

this approach serves as a paradigm for knocking 

out a dominant mutant allele with minimal 

genomic intervention and without the need of 

introducing an antibiotic selection marker. 


P 53 

Optimizing Virus-Magnetic Nanoparticle 

Complexes for Gene Transfer in Cell Lines 

and Stem Cells 

Olga M Mykhaylyk 1 , Yolanda Sanchez- 

Antequera 1 , Markus Döblinger 2 , Stefan 

Thalhammer 3 , Per Sonne Holm 1 , Christian Plank 1 

1Institute of Experimental Oncology and Therapy 


Munich, Germany; 2 Department of Chemistry and 

Biochemistry, Ludwig-Maximilians-Universität 

München, Munich, Germany; 3 Helmholtz Zentrum 

München, Institute of Radiation Protection, 

NanoAnalytics, Munich, Germany 

The goal of this work was providing magnetic 

nanoparticles (MNPs) and their formulations for 

efficient viral genetic modification of cell lines and 

primary cells in vitro and ex vivo. MNPs of the 

core-shell type with magnetite cores of about 10 

nm stabilized and decorated with surfactants and 

charged polymers were selected from our library 

of in-house synthesized MNPs for self-assembly 

with viral particles. To achieve maximal vector 

binding magnetic vectors were optimized with 

account for the vector association and magnetic 

sedimentation with MNPs. Measuring the time 

course of the turbidity of suspensions of the 

virus-MNP complexes in defined magnetic fields 

was used for the evaluation of the 

magnetophoretic mobility. We have learned that 

for magnetic viral vectors, it is reasonable to 

express the composition in terms of iron weight 

per PHYSICAL virus particle, and NOT per 

infectious virus particle, taking into account that 

both infectious and non-infectious virus particles 

are associated with appropriate MNPs. We 

suggest a “rule” to formulate virus magnetic 

complexes with suitable MNPs, based on the 

association and magnetic sedimentation of the 

virus with MNPs as well as with account for the 

functionality of gene delivery. The optimal 

complex composition of 2.5-20 fg iron per 

physical virus particle, depending on the MNPs 

used, is applicable to both adenoviral and 

lentiviral vectors. Optimized magnetic virus 

complexes were stable in 50% FCS. Electron and 

atomic force microscopy data showed structurally 

intact viruses surrounded by multiple MNPs. 

Genetic modification of cells on a cell separation 

column modified with formulated magnetic viral 

vectors (magselectofection) is highly efficient in 

hematopoietic stem cells and mesenchymal stem 

cells from human umbilical cord, hUC-HSCs and 

hUC-MSCs, respectively. Under optimized 

transduction conditions, viral magselectofection 

of hUC-MSCs with SO-Mag2 lentivirus 

complexes at MOI as low as 0.5 pfu/cell resulted 

in 60-100% transduced cells. We have also found 

a two-fold increase in the percentage of the 

reporter gene expressing cells postmagselectofection 

of the hUC-MSCs prelabelled 

with 20 pg Fe/cell of MNPs. 


P 54 

T Cell-Based Immunotherapy for Renal Cell 

Carcinoma 

Matthias Leisegang 1 , Adriana Turqueti-Neves 2 , 

Boris Engels 1 , Thomas Blankenstein 1 , Dolores J 

Schendel 2 , Elfriede Nößner 2 , Wolfgang Uckert 1 

1 Max-Delbrück-Center for Molecular Medicine, 

Berlin, Germany; 2 Helmholtz Zentrum München, 


Adoptive therapy with genetically engineered T 

cells carrying redirected antigen specificity (TCR 

gene therapy) is a new option to treat cancer, 

especially melanoma. However, this treatment is 

not yet available for metastatic renal cell 

carcinoma (RCC), due to the scarcity of 

therapeutically useful reagents. We analyzed 

tumor-infiltrating lymphocytes (TIL) from RCC to 

identify T cells with shared tumor-specific 

recognition for the generation of T cell receptor 



(TCR)-engineered T lymphocytes for TCR gene 

therapy of RCC. We established a T cell clone 

from TIL that recognized an HLA-A2-restricted 

tumor antigen. The TCRalpha- and beta-chain 

genes (TCR53) were isolated, modified by codon 

optimization and murinization, and retrovirally 

transduced into peripheral blood lymphocytes 

(PBL). TCR53-engineered PBL recapitulated the 

specificity of the TIL and demonstrated tumorspecific, 

HLA-A2-restricted effector activites (IFNγ, 

TNF-α, IL-2, MIP-1β, cytotoxicity). TCR53engineered 

PBL of healthy donors and RCCpatients 

exhibited similar TCR expression levels, 

expansion, and polyfunctional profile. Based on 

murine B3Z cells, a TCR53-expressing indicator 

line (B3Z-TCR53) was established for the 

screening of the antigen prevalence in RCC, in 

other malignancies, and in normal cell 

counterparts. Using B3Z-TCR53 cells, 130 tumor 

and normal cells were analyzed and shared 

TCR53 peptide:MHC expression was found in 

more than 60% of RCC and 25% of tumor cell 

lines of other histology, while normal tissue cells 

were not recognized. To date, TCR53 is the only 

TCR with shared HLA-A2-restricted recognition of 

RCC. It fulfills important criteria for utilization in 

TCR gene therapy and could advance T cellbased 

immunotherapy to patients with RCC and 

other malignancies expressing the TCR-ligand. 



P 55 

Novel Immunostimulatory Therapy by 

Adjuvant Magnetofection Prolongs Relapse- 

Free Survival of Fibrosarcoma Bearing Cats: 

A Veterinary Clinical Study 

Ulrike Schillinger 1 , Miriam Rutz 2 , Cornelia 

Fischer 2 , Anika Jahnke Jahnke 2 , Florian Walsch 2 , 

Mehrije Ferizi 1 , Veronika Benda 1 , Johannes 

Hirschberger 2 , Roberto Köstlin 3 , Bernd 

Gänsbacher 1 , Thomas Brill 1 , Christian Plank 1 

1 Institute of Experimental Oncology and Therapy 


Munich, Germany; 2 Department of Small Animal 

Medicine, Ludwig-Maximilians-University Munich, 

Munich, Germany; 3 Department of Small Animal 

Surgery and Reproduction, Ludwig-Maximilians- 

University Munich, Munich, Germany 

Feline fibrosarcoma is an everyday challenge in 

veterinary practice. Despite aggressive pre or 

post-operative treatment it has a high relapse 

rate of aprox. 75 % within 6 months after surgical 



| 70 

resection. To obtain a better therapeutic 

outcome, novel strategies are necessary. Hence, 

we established immunostimulatory therapy by 

magnetofection. Here we report preliminary 

results from a comparative clinical study where 

the genes for feline GM-CSF, IFN-γ and IL-2, 

feline GM-CSF alone or human GM-CSF were 

administered. The study design is prospective, 

randomized, placebo-controlled ( = standard 

therapy) and includes four arms: (1) standard 

therapy, i.e., surgery alone; (2) nonviral 

magnetofection of the triple-combination of feline 

GM-CSF, IFN-γ and IL-2 genes into the tumor 

before surgery or nonviral administration by 

magnetofection of feline (3) or human GM-CSF 

(4) gene alone. Preliminary clinical endpoints of 

the studies are relapse-free survival. Nonviral 

magnetofection, a procedure developed in our 

laboratory, is gene therapy by plasmids 

associated with magnetic nanoparticles under the 

influence of a magnetic field. The magnetic field 

was applied to achieve improved retention of the 

injected vector dose in the tumor. All genetherapeutic 

treatments were well tolerated and 

led to significantly prolonged relapse-free 

survival. Recent results from FACS-analysis of 

the primary tumor cells from the treated groups 

showed increased MHC-II expression compared 

to those of control cats. This is encouraging 

concerning future use in veterinary practice as 

this treatment can be easily administered. The 

results are promising with respect to their 

potential in human medicine, as they have been 

obtained in real patients instead of experimental 

tumor models. 


Therapy

71 | 

P 56 

Targeting the Epidermal Growth Factor 

Receptor (HER) Family by T Cell Receptor 

Gene-Modified T Lymphocytes 

Peter Meyerhuber 1 , Heinke Conrad 2 , Lilian 

Stärck 1 , Matthias Leisegang 1 , Dirk H. Busch 3 , 

Helga Bernhard 4 , Wolfgang Uckert 1 

1Max-Delbrück-Center for Molecular Medicine, 

Berlin, Germany; 2 Department of 

Hematology/Oncology, Technical University of 

Munich, Klinikum rechts der Isar, Munich, 

Germany; 3 Institute of Microbiology, Immunology 

and Hygiene, Technical University of Munich, 

Munich, Germany; 4 Department of 

Hematology/Oncology, Klinikum Darmstadt, 

Darmstadt, Germany 

HER2 is over-expressed in 25–40% of all breast 

cancers and in a variety of other tumors such as 

ovarian cancer and hematological malignancies. 

Due to the selective over-expression in malignant 

tissue, HER2 is considered one of the most 

attractive targets for therapeutic interventions, 

such as monoclonal antibodies, kinase inhibitors, 

and cancer vaccines. However, tumor regression 

following immunization with vaccines is rare, 

likely a consequence of HER2 being a self 

antigen and therefore inducing tolerance. To 

overcome tolerance, adoptive T cell transfer 

(ATT) strategies have been developed. However, 

clinical application of ATT is limited, because 

isolation and characterization of HER2-reactive T 

cells is laborious. Yet, a promising approach is 

the transfer of TCRαβ genes, which have been 

isolated from non-tolerant settings, into T cells. In 

this study, we isolated the TCR genes of a 

HER2-reactive, allo-HLA-A2-restricted CTL clone 

and introduced the genes into a retroviral vector. 

Efficient cell surface expression of the TCR and 

improved functional avidity of the gene-modified 

T cells were achieved after murinization 

(replacement of the human TCR constant regions 

by mouse counterparts), codon-optimization and 

application of the P2A gene linker (HER2-TCRopt). 

Thus, the TCR expression in transduced T 

cells increased from 1.5% to 41% measured by 

A2/HER2-multimer staining. The ability to secrete 

IFN-γ of HER2-TCR-opt transduced T cells was 

comparable to that of the CTL clone, which 

showed a half maximum IFN-γ secretion at 10-7 

M towards HER2 peptide-loaded T2 cells. 

Furthermore, HER2-TCR-opt-transduced T cells 

lysed HER2-expressing tumor cell lines as 

efficient as the parental clone (lysis of 58% at a 

E:T of 30:1). The TCR showed a cross-reactivity 

to HER3 and HER4 that was similar to the 

parental CTL clone. Extensive testing with 

HER2/3/4 negative cell lines did not reveal any 

further epitopes that were recognized by the 

TCR. Our results contribute to the development 

of a TCR-based approach for the treatment of 

HER2-positive breast cancer, as well as of other 

malignancies expressing HER2, HER3 and/or 

HER4. 



P 57 

Humanized Mouse Models for the Pathogenic 

Analysis of T-Cell Leukemia/Lymphoma 

Development 

Benjamin Rengstl 1 , Sebastian Newrzela 1 , Sylvia 

Hartmann 1 , Alexander Benz 1 , Tim Heinrich 1 , 

Martin-Leo Hansmann 1 , Dorothee von Laer 2 

1 

Department of Pathology, University of 

Frankfurt, Frankfurt am Main, Germany; 

2 

Department of Virology, University of Innsbruck, 


The oncogenic potential of human hematopoietic 

stem cells (HSCs) in the context of retroviral 

gene therapy, as reported in patients treated for 

X-linked severe combined immunodeficiency (X- 

SCID) or X-linked chronic granulomatous disease 

(X-CGD), is now well described. Syngeneic 

mouse models for the development of 

leukemia/lymphoma are established as well as 

the reconstitution of immunodeficient mice with 

human stem cells. The combination of both 

systems, a mouse model for the development of 

human leukemia/lymphoma is still missing. To 

address this issue, we aim to test different 

humanized mouse models. Currently, the most 

promising mouse strains available for the 

engraftment of human cells are NOD-SCID γc −/− 

(NOG) and Rag2 −/− γc −/− mice. These strains will 

be repopulated with human HSCs or mature T 

cells expressing potent (proto-)oncogenes after 

retroviral/lentiviral gene transfer. To further 

enhance the repopulation dynamics of mature T 

cells, human HSCs will be transduced with 

mouse strain-specific murine T-cell receptors 

(TCRs). After successful repopulation the strainadapted 

human, mature T cells will be isolated, in 

vitro transduced with potent (proto-)oncogenes 

and transplanted into secondary recipients. A 

second strategy to overcome the mismatch 

between the human TCR and the murine major 

histocompatibility complex (MHC) is to utilize 

huMHC-transgene mice. Due to the lack of 

functional lymph nodes in the immunodeficient 

mouse strains in a third strategy we want to use 



the so-called trimera mouse model: lethally 

irradiated and cyclophosphamid treated 

immunocompetent mice will be reconstituted with 

bone marrow of highly immunodeficient mice to 

generate an optimal environment for the 

repopulation with retrovirally/lentivirally modified 

human, mature lymphocytes. Moreover, all 

described mouse models will be used to 

investigate established human 

leukemia/lymphoma cell lines in vivo. The future 

goals of the project are on the one hand to 

compare expression profiles of healthy and 

neoplastic lymphocytes and on the other hand 

the identification of leukemic stem cells (LSCs) of 

leukemia/lymphoma cell lines. 



P 58 

Clonal Dynamics Within Mature T-Cell 

Populations in the Preleukemic Phase of T- 

Cell Leukemia/Lymphoma 

Tim Heinrich 1 , Dorothee von Laer 2 , Sebastian 

Newrzela 1 , Benjamin Rengstl 1 , Sylvia Hartmann 1 , 

Martin-Leo Hansmann 1 

1 Department of Pathology, University of 

Frankfurt, Frankfurt am Main, Germany; 2 Medical 

University of Innsbruck, Department of Virology, 


Retroviral insertional mutagenesis in 

hematopoietic progenitor cells can activate 

neighboring proto-oncogenes and thus contribute 

to leukemia development. This genotoxic activity 

of integrating viruses is highly relevant for the risk 

assessment of stem cell gene therapy and also 

has been the basis for the identification of several 

proto-oncogenes. In previous studies, we 

analyzed the susceptibility of mature T cells to 

transformation (Newrzela et al., Blood, 2008). 

Surprisingly, T-cell transplanted animals showed 

no sign of leukemia/lymphoma development 

during a follow-up of more than 500 days. These 

results show that polyclonal mature T cells are 

less susceptible to transformation in vivo by 

known T-cell oncogenes than progenitor cells. In 

contrast, TCR-monoclonal T lymphocytes 

expressing oncogenes developed T-cell 

leukemia/lymphoma in recipient mice. We thus 

postulate that clonal competition may control 

leukemogenesis in mature T-cell populations. To 

analyze the mechanisms that control the 

outgrowth of malignant clones in TCR polyclonal, 

but not in TCR monoclonal mice, clonal 



| 72 

fluctuation within the T-cell population will be 

analyzed during the manifestation of mature Tcell 

leukemia/lymphoma. DNA samples from 

peripheral blood T cells will be frozen in regular 

intervals from mice transplanted with polyclonal 

or monoclonal T cells expressing specific (proto- 

)oncogenes or a control gene. As the 

gammaretroviral vector used for gene transfer 

integrates randomly into the host genome, 

individual T-cell clones are marked by their 

specific integration sites. Integration site analysis 

by LM-PCR will reveal dominant clones within the 

T-cell population. In order to visualize the clonal 

fluctuation and progressing dominance of certain 

clones with a growth advantage due to insertional 

mutagenesis, these T-cell clones will be 

investigated retrospectively by integration-site 

specific PCR. In addition, the TCR diversity in the 

polyclonal setting will be analyzed on a genomic 

level during leukemia/lymphoma 

development/suppression to correlate the 

homeostatic clonal competition and the outgrowth 

of malignant T-cell clones. 

Session: Cancer Immune Therapy and T-cell 


P 59 

A Hexon Modification in hAd5-Based Vectors 

Results in an Increased Transduction Efficacy 

of Pancreatic Cancer and Stellate Cells 

Tanja Lucas 1 , Karim Benihoud 2 , Andreas 

Wortmann 3 , Max G Bachem 4 , Stefan Kochanek 1 

1Department of Gene Therapy, University of Ulm, 

Ulm, Germany; 2 CNRS UMR 8121, Vectorology 

and Gene Transfer, Gustave Roussy Institute, 

Villejuif, France; 3 Tierforschungszentrum, 

University of Ulm, Ulm, Germany; 4 Department of 

Clinical Chemistry, University of Ulm, Ulm, 


Pancreatic cancer is the fifth leading cause of 

cancer-related death in Germany. Pancreatic 

adenocarcinomas (PDACs) are characterised by 

rapid progression, early metastasis, diagnosis at 

an advanced stage, and high resistance to 

standard therapy. Thus, patients suffering from 

PDACs have a very poor prognosis. PDACs are 

composed of infiltrating tumor cells surrounded 

by extracellular components and other nonneoplastic 

cell types. Very frequently, tumor cells 

can be acounted for significantly less than half of 

the cellular mass within the tumor. The complex 

mutual interaction between tumor cells and nonneoplastic 

human pancreatic stellate cells

73 | 

(hPSCs – the major stromal cell type present in 

pancreatic cancer) is highly regulated through 

secretion of different growth factors (e.g., TGF-β), 

strongly influencing growth and malignancy of 

PDACs. In mouse experiments coinjection of 

tumor cells and hPSCs resulted in an advanced 

tumor progression. For treatment of pancreatic 

cancer with adenoviral (Ad) vectors, it would be 

therefore of advantage to target these vectors to 

both cell types. To date the systemic 

admininstration of Ad vectors, however, is 

characterised by two major drawbacks: the low 

transduction of tumor cells and the severe 

damage of healthy tissues (e.g., liver). To 

address these obstacles a hexon-modified Ad 

vector has been developed displaying the TGF-β 

receptor binding peptide CSK17 on the 

hypervariable region 5 of hexon resulting in 

AdhCKS17. Subsequent in vitro studies have 

shown a significantly enhanced transduction of 

early tumor cells and primary hPSCs after 

infection with AdhCKS17. As a consequence the 

hexon-modification resulted in an increased burst 

size and cytotoxicity in both cell types. A 

preliminary biodistribution study indicates a 

decreased liver uptake of this hexon-modified 

vector compared to the control vector indicating 

detargeting from liver, thereby potentially 

reducing liver damage. Subsequent in vivo 

investigations are directed at analysing this 

vector within the human tumor in an ex vivo 

culture system and in murine xenotransplantation 

models bearing composite tumors consisting of 

human pancreatic cancer cells and stroma cells. 


P 60 

Controlled Removal of a Nonviral Episome 

Claudia Hagedorn 1 , Sina Rupprecht 2 , Hans J. 

Lipps 2 

1 Universität Witten/Herdecke, Institut für 

Zellbiologie, Witten/Herdecke, Germany; 2 Institut 

für Zellbiologie ZBAF – Zentrum für 

biomedizinische Ausbildung und Forschung 

Universität Witten/Herdecke, Witten/Herdecke, 


The vector pEPI is the first non-viral autonomous 

replicon that was constructed for mammalian 

cells. It represents a minimal model system to 

study the epigenetic regulation of replication and 

transcription. Its function relies on a transcription 

unit linked to an S/MAR sequence. In gene 

therapy, situations exist in which transgenes 

have to be expressed for a limited time and thus 

it would be of considerable interest not only to 

silence the expression of the transgene but to 

construct a vector which can be removed from 

the cell at any time. Therefore we followed two 

approaches: (I) We constructed the inducible 

episomal vector pEPI-Tet in which transcription 

running into the S/MAR is regulable with 

doxycycline. We found that for vector replication 

and long-term maintenance an ongoing 

transcription running into the S/MAR element is 

required. Once established, the vector is lost 

from the cell upon switching off transcription from 

the gene linked to the S/MAR. (II) Secondly, we 

used the siRNA approach: Cells in which pEPIeGFP 

has been stably established were 

transfected with a vector expressing siRNA 

directed against eGFP. As a consequence, due 

to the abrogated transcription of the transgene 

running in to the S/MAR, reduced plasmid copies 

per cell were detected using quantitative PCR. 

Furthermore, when comparing cells transfected 

with an eGFP-siRNA with those transfected with 

a nonsense-siRNA, we found that active 

chromatin markers, e.g., H3K36me or H3K4me3, 

got lost. 


P 61 

Transposition from a High-Capacity 

Adenoviral Vector: Optimization of the 

System and Mapping of Transposon 

Integration Sites in Murine Liver 

Wenli Zhang 1 , Martin Hausl 1 , Anja Ehrhardt 1 

1 Department of Virology, Max von Pettenkofer- 


High-capacity adenoviral vectors (HC-AdVs) 

devoid of all viral coding sequences offer high 

transduction efficiencies, large packaging 

capacities (up to 35 kb) and reduced toxicity. To 

further improve the persistence of transgene 

expression and the maintenance of the vector 

genome in cycling cells, we recently developed 

an adeno/transposase hybrid-vector-system 

utilizing a hyperactive Sleeping Beauty 

transposase HSB5 for somatic integration (Hausl, 

Zhang et al., Molecular Therapy, accepted). 

However, with respect to preclinical evaluation 

this hybrid-vector system needs to be 

characterized in more detail. Herein, we 

optimized the virus dose required for long-term 

effects without toxicity and we performed a vector 

fate analysis. Based on our established hybrid- 



vector system, female and male C57Bl/6 mice 

were co-injected at three dosages [1.6 × 10 8 8 × 

10 8 and 4 × 10 9 transducing units (TU) total per 

mouse] with a canine factor IX (cFIX) transposon 

donor vector and a second vector encoding the 

hyperactive SB transposase HSB5 and Flp 

recombinase (n = 5 per group). In groups which 

received the highest dose, toxicity was observed 

in mice of both genders. At the lowest dose, only 

low levels of serum cFIX were detected ( ≈ 100 

ng/ml). This was in sharp contrast to the middledose 

group in which we measured ≈ 1000 ng/ml 

serum cFIX levels without toxicity. We concluded 

that an efficient non-toxic dose for our hybridvector 

lies between 8 × 10 8 to 4 × 10 9 TUs per 

mouse. Notably, at the middle dose we observed 

a gender influence on transgene expression 

levels. In order to address issues related to 

genotoxicity after HSB5-mediated somatic 

integration, transposition events were analyzed 

by a PCR-based high-throughput sequencing 

method. For each individual we generated 4 

genomic DNA libraries using different restriction 

enzymes. Of the 73 transposition events 

identified in liver of HSB treated mice, 74% 

(54/73) were found in non-gene areas and 26% 

(19/73) in genes indicating a random integration 

pattern. Of the transposition events detected in 

genes, 17 insertion sites were in introns and only 

2 in exons. From all animals, no integration site 

was found in or near putative oncogenes. 


P 62 

Insertional Mutagenesis in Mature T Cells – A 

Mathematical Modelling Approach 

Sebastian Gerdes 1 , Sebastian Newrzela 2,3 , Tim 

Heinrich 2,3 , Dorothee von Laer 2,4 , Ingmar 

Glauche 1 , Ingo Roeder 1 

1 Institute for Medical Informatics and Biometry, 

Faculty of Medicine “Carl Gustav Carus”, 

Dresden University of Technology, Dresden, 

Germany; 2 Frankfurter Stiftung für krebskranke 

Kinder, Frankfurt am Main, Germany; 3 Pathology 

of the Goethe-University Hospital Frankfurt, 

Frankfurt am Main, Germany; 4 Innsbruck Medical 

University, Innsbruck, Austria 

Leukemia caused by insertional mutagenesis is a 

major risk of gene therapy with HSCs. In 

contrast, polyclonal mature T cells have turned 

out to be surprisingly resistant to leukemogenic 

insertional mutagenesis. Newrzela et al. showed 

that leukemia/lymphoma did not occur upon 



| 74 

transplantation of wild-type mature T cells with 

polyclonal T-cell receptors (TCR) being 

transduced with high copy numbers of 

gammaretroviral vectors encoding potent T- cell 

oncogenes into RAG1-1-deficient recipients [1]. 

However, further studies demonstrated that the 

transplantation of T cells from TCR-monoclonal 

OT1 mice that were transduced with the same 

protocol resulted in leukemia/lymphoma. The 

underlying mechanisms that prevent oncogenesis 

in the polyclonal situation and permit the 

outbreak of leukemia in the monoclonal situation 

are currently unclear. Using a mathematical 

modelling approach, we challenge the arising 

hypothesis that polyclonality induces competition 

within the T-cell repertoire, which in turn 

suppresses the emergence of a leukemic clone. 

As a first step, we developed a mathematical 

model of T-cell homeostasis that is derived from 

a similar niche-based based model of 

hematopoiesis [2]. The key assumption of the 

novel model is that T-cell survival is critically 

dependant on the interaction of the clone-specific 

TCR with self-peptide-MHC-complexes and 

therefore subject to competition between different 

T-cell clones. Our model consistently reproduces 

the polyclonal pattern observed in T-cell 

homeostasis, and responds in an adequate 

manner to perturbations of the system such as 

infection. Furthermore, based on our modelling 

results, we speculate how the deregulation of Tcell 

receptor affinities supports the formation of 

dominant clones. The modelling results 

underscore the possibility that clonal competition 

can prevent the outbreak of overt mature T-cell 

leukemia/lymphoma. References: [1] Newrzela S 

et al. Resistance of mature T cells to oncogene 

transformation. Blood. 2008;112(6):2278–2286. 

[2] Roeder I., Loeffler M. A novel dynamic model 

of hematopoietic stem cell organization based on 

the concept of within-tissue plasticity. Exp. 

Hematol. 2002;30(8):853-861. 

Session: Pharmacology and Toxicology

75 | 

P 63 

Zinc-Finger Nucleases for Targeted Gene 

Repair: Studying Off-Target Activities 

Frank Radecke 1 , Sarah Radecke 2 , Toni 

Cathomen 3 , Klaus Schwarz 1,4 

1 Institute for Transfusion Medicine, University of 

Ulm, Ulm, Germany; 2 Institute for Clinical 

Transfusion Medicine and Immunogenetics Ulm, 

Ulm, Germany; 3 Department of Experimental 

Hematology, Hannover Medical School, 

Hannover, Germany; 4 Institute for Clinical 

Transfusion Medicine and Immunogenetics, 

University of Ulm, Ulm, Germany 

Targeted gene repair with single-stranded 

oligodeoxynucleotides as repair matrix often 

yields low rates of corrected cells, but these rates 

can be enhanced by the concomitant induction of 

a DNA double-strand break (DSB) close to the 

mutation. As a tool to achieve defined DSBs, 

customisable zinc-finger nucleases (ZFNs) are 

studied. They consist of a specific DNA-binding 

domain (designed zinc-finger motifs) linked to a 

nonspecific FokI-derived dimer-forming nuclease 

domain. Here, off-target activities of two ZFNs 

(GZF1 and GZF3) were investigated using three 

complementing approaches. To reduce toxicity, 

both ZFNs already harbour a variant dimer 

interface to favour heterodimerisation. For in vitro 

cleavage assays, the ZFN proteins were 

incubated with target locus DNA. Off-target 

activity was detected at two positions due to 

unwanted GZF1 homodimerisation, where one 

ZFN bound its perfect 9-mer site while the 

partner functionally attached to a mismatched 

sequence. To examine whether this might 

happen in a 293 cell line with a transgenic singlecopy 

target locus, one expression vector (for 

GZF1 or GZF3) was transfected to then analyse 

target locus DNAs for signatures of DSB repair 

(e.g., nonhomologous end joining (NHEJ)). At the 

regular GZF1/GZF3 site, one NHEJ-derived 

modification per ≈ 15,000 alleles was observed 

when only GZF1 was present. Since similar 

binding site variants were found also for GZF3 

homodimers, related off-target activity—not yet 

detected—might also occur. In silico analyses 

(Ensembl BLASTN on haploid genome) predicted 

28,100 and 12,695 additional perfect sites for 

GZF1 and GZF3, respectively. Based on 

sequence analyses from the in vitro and in cellula 

assays, ≈ 6% of these sites were classified as 

potential off-targets with canonical 5–7 base-pair 

spacers and second binding sites (9-mers) with 

6–8 matches. At half of these sites, the favoured 

heterodimers might form. In conclusion, our 

multifaceted platform consisting of genome-wide 

in silico predictions combined with in vitro and in 

cellula assays represents a potent tool to 

evaluate ZFN specificities and can be used to 

estimate biological activities of any ZFN with 

known DNA binding parameters. 


P 64 

Control Mechanisms in Mature T Cell 

Leukemia/Lymphoma 

Sebastian Newrzela 1 , Nabil Al-Ghaili 2 , Tim 

Heinrich 1 , Mina Petkova 2 , Kerstin Cornils 3 , Sylvia 

Hartmann 1 , Martin-Leo Hansmann 1 , Boris 

Fehse 3 , Sebastian Gerdes 4 , Ingo Roeder 4 , 

Dorothee von Laer 5 

1Pathology of the Goethe-University Frankfurt, 

Frankfurt am Main, Germany; 2 Frankfurter 

Stiftung für krebskranke Kinder, Frankfurt am 

Main, Germany; 3 University Medical Center 

Hamburg-Eppendorf, Hamburg, Germany; 

4 Institute for Medical Informatics and Biometry, 

Faculty of Medicine “Carl Gustav Carus”, 

Dresden University of Technology, Dresden, 

Germany; 5 Innsbruck Medical University, 


In safety studies in a mouse model, we found that 

in hematopoietic stem cells high copy numbers of 

gammaretroviral vectors expressing T-cell 

oncogenes readily induced T-cell 

leukemia/lymphoma in recipient mice. However, 

mature polyclonal T cells could not be 

transformed (Newrzela et al., Blood 2008). Yet, 

and quite surprisingly, T-cell receptor (TCR) 

monoclonal T lymphocytes derived from OT-I 

mice were transformed with the same kinetics 

and efficiency as stem cells. This propensity of 

monoclonal T-cell populations to develop 

malignancies is highly relevant for evaluating the 

safety of therapeutic gene transfer into mature T 

cells, especially the adoptive transfer of 

therapeutic TCRs. On the other hand, these 

observations imply that polyclonal T-cell 

populations can control the outgrowth of 

malignant T-cell clones. The population-dynamics 

of T cells will be analyzed with respect to the 

diversity of the T-cell repertoire. The generated 

data on T-cell dynamics will furthermore help to 

validate mathematical models on the normal and 

malignant T-cell homeostasis, which will be 

developed in the group of Ingo Roeder, Dresden. 

In addition, the molecular mechanisms that 

control T-cell malignancy in the polyclonal, but 

enhance leukemogenesis in the monoclonal 

setting will be identified. The mechanisms 



involved in the pathogenesis of mature T-cell 

malignancies are addressed in this study. 


P 65 

Characterization of rAAV Genomes After 

Delivery of ssAAV or scAAV in Rats by LAM- 

PCR and Deep Sequencing 

Christine Kaeppel 1 , Ali Nowrouzi 1 , Uwe Appelt 1 , 

Anne Arens 1 , Christian Weber 1 , Maude Flageul 2 , 

Paula Miranda 3 , Nicolas Ferry 2 , Piter Bosma 3 , 

Christof von Kalle 1 , Manfred Schmidt 1 

1National Center for Tumor Diseases Heidelberg, 

Department of Translational Oncology, German 

Cancer Research Center, Heidelberg, Germany; 

2 INSERM UMR 948, Nantes, France; 3 Academic 

Medical Center Liver Center, Amsterdam, The 

Netherlands 

Long-term gene expression can be achieved 

following recombinant adeno-associated virus 

(rAAV) vector delivery in vivo. Previously, we 

have shown that rAAV8 delivery to newborn rat 

liver results in low frequent integrated rAAV 

genomes in a minority of cells. However, even 

the rare integration frequency of the rAAV vector 

raises concerns about its clinical safety. To 

evaluate the safety of different AAV-serotypes 

and vector doses in preclinical settings we 

estimated rAAV integration characteristics in rat 

liver after single-stranded (ss)AAV or selfcomplementary 

(sc)AAV injection of different 

vector doses. The persistence of ssAAV and 

scAAV was analysed in transduced rat liver 14 

months and 13 months post-rAAV injection. In 

rats transduced with scAAV partial hepatectomy 

(HP) was performed 13 months post-injection 

and the integration frequency was evaluated 3 

months post HP. LAM-PCR was performed on 

250-500 ng of total DNA derived from 12 ssAAV1, 

6 scAAV1 and 6 scAAV8 transduced rats and 

combined with deep sequencing allowing 

simultaneous and efficient identification of 

complex rAAV concatemeric forms and 

proviruses. Sequencing of > 68.000 LAMamplicons 

revealed 67 unique insertion sites (IS), 

in ssAAV transduced rats up to seven per sample 

and in scAAV transduced ones up to eight per 

sample. The averaged number of IS was higher 

in rat liver transduced with 1.2 × 10 12 TU/kg 

ssAAV1 compared to a vector dose of 8 × 10 12 

TU/kg (4.6 IS vs. 0.5 IS). Semi-quantitative 

measurements of rAAV concatemers showed 

that in rat liver transduced with 8 × 10 12 TU/kg 



| 76 

ssAAV1 up to 100% of the analysed sequences 

were due to concatemers while in rat liver 

transduced with 1.2 × 10 12 TU/kg up to 3/4 of all 

analysed sequences could be referred to IS. In 

addition, sequencing of rAAV LAM-amplicons 

derived from ssAAV and scAAV transduced rat 

liver was used to show the feasibility of complete 

ITR sequencing in vivo. In partial ITR no 

preferential ITR breakpoints were detected in 

ssAAV transduced rat liver while in scAAV 

transduced ones preferential ITR breakpoints 

were detected. These insights are of clinical 

relevance for potential vector genotoxicity of 

different rAAV serotypes and doses. 


P 66 

Improved Drug-Selectable Fluorescent 

Proteins as Bifunctional Marker Genes 

Kristoffer Weber 1 , Ellen Preuß 1,2 , Lisa 

Weingarten 1 , Boris Fehse 1 

1 Universitätsklinikum Hamburg-Eppendorf, 

Hamburg, Germany; 2 Frankfurter Stiftung für 

krebskranke Kinder, Hamburg, Germany 

The use of fluorescent proteins (FPs) as markers 

expressed by gene transfer vectors facilitates fast 

and unambiguous identification of gene-modified 

cells. However, to select cells based on their 

expression of FPs, fluorescence-activated cell 

sorting would have to be used. This complex and 

expensive technique is not available in all 

laboratories, bears a certain contamination risk 

and might be too stressful for some delicate cell 

types. To overcome these limitations we have 

recently developed ten fusion proteins as 

bifunctional markers composed of a variety of 

fluorescent proteins (Cerulean, eGFP, Venus, 

dTomato, mCherry) fused to drug resistance 

proteins (NeomycinR, ZeocinR, PuromycinR, 

HygromycinR, BlasticidinR). Now we present the 

second generation of bifunctional markers which 

are greatly improved in brightness by inserting a 

2A peptide in between the fluorescent protein 

and the resistance protein. In fact, the brightness 

of the so far dimmest marker (eGFP fused to 

PuromycinR) was increased by a factor of seven, 

now reaching 90% of the brightness of unfused 

eGFP. All bifunctional markers have been tested 

in lentiviral vectors (www.LentiGO-Vectors.de) 

and were cloned as “building blocks” to easily 

interchange the resistances in a single cloning 

step. Importantly, working with the new 

bifunctional markers revealed that the efficacy of

77 | 

cell selection could be overestimated with 

standard antibiotic-resistance genes. In fact, in 

different adherent cell lines we found by flow 

cytometry that antibiotic-mediated selection of 

cells was by far not complete, even though all 

control cells without the respective antibiotic 

resistance were dead. This data not only has 

important implications for assays with antibioticsselected, 

apparently pure cell populations, but 

also indicates some so far unknown bystander 

protection of nontransduced cells in adherent 

cultures. In conclusion we suppose the novel 

bifunctional markers are well suited for numerous 

applications and different vector systems. 


P 67 

From Insertion Site Profiles to Clonal 

Contributions 

J. Uwe Appelt, Frank A. Giordano, Anne Arens, 

Cynthia C Bartholomae, Manfred Schmidt, 

Christof von Kalle, Stephanie Laufs 

Department of Translational Oncology, National 

Center for Tumor Diseases and German Cancer 

Research Center, Heidelberg, Germany 

Initially, our goals were to test if viral insertion is 

nonrandom and, specifically, to clarify whether 

host and disease-specific integration patterns 

exist. We developed the bioinformatic 

applications IntegrationSeq, IntegrationMap and 

QuickMap, and others (manuscript in revision) 

which allow high throughput analyses (next 

generation sequencing) of insertion sites of viral 

vectors (QuickMap available at 

http://www.gtsg.org). Furthermore, we designed a 

first version of a database, which to date contains 

more than 77,334 different vector insertion sites 

derived from previous analyses on different 

vectors (ASLV, FIV, MMTV, HTLV, HIV, EIAV, 

FV, MLV, and SIV) and different host cells. We 

subjected all insertion sites stored in this 

database to our analysis pipeline and 

characterized insertions with regard to 

localization on chromosomes, in or next to a 

genes, cancer genes, fragile sites, transcription 

factor binding sites, CpGs, or repetitive elements 

(SINE, LINE, LTR). A random insertion site set 

(10 6 sequences) reliably reflects random 

distribution and serves as our reference for all 

analyses of experimental data. We saw that the 

distinct insertion profiles of the different 

retroviruses resemble genomic finger prints, 

which allow distinguishing between different 

retroviruses. Further, a high-resolution metaanalysis 

HIV vector insertions revealed that HIV 

significantly spare a region of 1 kb+ / − to 

transcription start sites (Giordano et al., AIDS 

2009). Using the insertion site profiles and the 

information on redundancy of recovered 

sequences (read counts), both provided by 

QuickMap and our other integration site 

sequence data mining tools, we could reliably 

estimate clonal compositions of complex samples 

(preclinical and clinical studies), thus allowing 

monitoring of transduced cells and assessment 

for the safety of gene transfer. We are now 

building up a database that will allow the dynamic 

storage of integration site sequences in the Terra 

range and comprehensively fulfill the needs for (i) 

more specialized features of vectors (e.g., IDLV, 

AAV), (ii) genomic and transcriptomic features 

near the integration site and (iii) new powerful 

sequencing technologies. 


P 68 

Next Generation Sequencing Data 

Management for Integration Site Analyses 

Anne Arens, Derek Gustafson, Cynthia C 

Bartholomae, Uwe Appelt, Frank Giordano, 

Annette Deichmann, Hanno Glimm, Stephanie 

Laufs, Christof von Kalle, Manfred Schmidt 

Department of Translational Oncology, National 

Center for Tumor Diseases (NCT) and German 

Cancer Research Center (DKFZ), Heidelberg, 


Integration site analyses have become a valuable 

and powerful tool to detect clonal repopulation 

kinetics in clinical gene therapy, to address the 

biosafety of a specific gene transfer approach or 

to uncover vector-induced side effects. With a 

combinatorial approach of standard or 

nonrestrictive (nr) linear amplification-mediated 

(LAM) PCR followed by next generation 

sequencing, time- and cost-efficiency of 

integration site sequence retrieval can be 

improved by several logs compared to standard 

Sanger sequencing technologies. Using our 

implemented analyses tools, such as Quickmap 

(www.gtsg.org) and others (Paruzynski A et al., 

Nature Protocols 2010), sequencing data output 

provides relevant integration site information 

such as chromosome, chromosomal locus, 

distance and orientation to the next RefSeq 

genes and retrieval frequency of individual 

integration sites. However, integration site data 



mining nowadays not only requires setting up 

certain software that allow an automated and 

standardized readout which can be handled by 

non-bioinformaticians. The vast amount of 

different vector types used in various areas of 

gene transfer approaches (e.g., high frequent 

integrating oncoretro- and lentiviral vectors, lowfrequent 

integrating integrase deficient lentiviral 

and AAV vectors as well as transposons such as 

Sleeping Beauties) for different cell types and 

species also requires a hitherto unknown 

flexibility of relevant downstream programs to 

answer the specific scientific questions beyond. 

Here, we will present some aspects of our 

‘downstream’ bioinformatical data analysis 

system that meet these requirements, including 

among others (i) therapeutically relevant 

informations on clonal constitution and dynamics 

of gene-modified cell populations in preclinical 

and clinical gene transfer settings 

(‘pharmacokinetics'), (ii) precision of vector 

integration and (iii) sequence homology 

comparison and others. 


P 69 

Investigation of the Localisation and 

Interactions of Adenoviral Vectors with 

Immune Cell Populations in the Spleen 

Following Intravascular Delivery 

Lynda Coughlan 1 , Angela C. Bradshaw 1 , Raoul 

Alba 1 , Robert A. McDonald 1 , Nico van Rooijen 2 , 

James M. Brewer 3 , Paul Garside 3 , Stuart A. 

Nicklin 1 , Andrew H. Baker 1 

1 BHF GCRC, University of Glasgow, Glasgow, 

UK; 2 Department of Molecular Cell Biology, Vrije 

Universiteit Medical Centre (VUMC), Amsterdam, 

The Netherlands; 3 Division of Immunology, 

Infection and Inflammation, GBRC, University of 

Glasgow, Glasgow, UK 

The spleen is an important site for the induction 

of immune responses to adenoviral (Ads) 

vectors, especially following intravascular 

delivery. Ads have been shown to interact with 

macrophage populations and dendritic cells in the 

spleen, and high level splenic uptake has been 

associated with vector-induced toxicity in vivo. 

However, the precise immune effectors which 

determine the release of characteristic 

inflammatory cytokines in response to Ad remain 

to be fully identified. Using a panel of viruses, 

including Ad5, Ad48 and hexon-modified Ad5, we 

assessed their interactions with cell markers in 



| 78 

the spleen (CD11b, CD11c, SIGNR1, MARCO, 

CD169 and F4/80) at low and high doses of Ad, 

and at early and late time-points post-injection. In 

order to fully analyse these interactions and their 

contribution to inflammatory responses, we 

administered a cohort of mice with clodronateencapsulated 

liposomes and assessed the 

effects on macrophage and other immune cell 

populations in the spleen. We quantified serum 

cytokines 6 hr post-injection using a 20-plex 

luminex assay, for clodronate treated and 

untreated animals. Fluorescently labeled Ads 

(Ad5, Ad48 and hexon-modified Ad5) were 

detected by immunohistochemistry in colocalisation 

with MARCO + and Moma-1 + cells, 

within the marginal zone surrounding the white 

pulp of the spleen, and with F4/80 + red pulp 

macrophages (1 h). These macrophage 

populations were almost completely absent in 

animals treated with clodronate liposomes and 

subsequently the spatial distribution of virus 

within the spleen was altered. The defining 

splenic cell markers for virus transgeneexpressing 

cell types at later time-points (48 h) 

were not the same as those detected at early 

time-points. These currently remain to be 

identified. Further studies will be required to fully 

characterise the time-course of adenoviral uptake 

in the spleen and the contribution of the various 

cellular effectors to the induction of inflammation. 

Session: Pharmacology and Toxicology

79 | 

DGGT 2010 Abstract author index 

Abken, Hinrich, Inv 22 

Acosta-Sanchez, Abel, Inv 12 

Al-Ghaili, Nabil, P 64 

Alba, Raoul, P 38, P 69 

Albarenque, Stella, P 48 

Ali, Robin R, Inv 14, P 30 

Almarza, David Gomez, Or 2 

Aloysius, Mark, Inv 23 

Altomonte, Jennifer, P 14, P 44, P 45 

Anton, Martina, Inv 2, Or 18, P 41, P 46, P 50 

Antoniou, Michael, Or 9 

Apfel, Sibylle, P 14 

Appelt, Uwe, P 65, P 67, P 68 

Arens, Anne, Or 7, P 65, P 67, P 68 

Argyros, Orestis, P 23, P 24 

Armeanu, Sorin, P 42 

Arzumanov, Andrey A, Inv 11 

Bachem, Max G, P 59 

Bahadur, Dhirendra, P 15 

Bai, Lin, Or 6 

Baier, Ruth, P 18, P 19 

Baiker, Armin, P 21 

Bainbridge, JWB, Inv 14 

Baker, Andrew H, Inv 10, Or 21, P 38, P 69 

Bakshi, Rakesh, P 26 

Ball, Claudia R, Or 7, P 37 

Ballmaier, Matthias, Or 10 

Banerjee, Rinti, P 15 

Banfi, Andrea, P 28 

Bartholomae, Cynthia C, P 67, P 68 

Bauer, Ralf, P 29 

Baum, Christopher, Or 10, Or 15, Or 19 

Becirovic, Elvir, Or 6 

Beck, Susanne C, Or 6 

Bednarski, Christien, P 17, P 22 

Belay, Eyayu, Inv 12 

Bell, John C, Inv 18, Inv 19 

Benda, Veronika, P 55 

Bendle, Gavin M, Or 17 

Benihoud, Karim, P 59 

Benz, Alexander, P 57 

Bergemann, Christian, P 8 

Bernhard, Helga, P 56 

Bhalla, Joti, Inv 23 

Bhattacharya, SS, Inv 14 

Biel, Martin, Or 6 

Bies, Laura, Or 17 

Bitzer, Michael, P 42 

Blaesen, Markus, P 18, P 19 

Blankenstein, Thomas, P 54 

Blasczyk, Rainer, Or 7 

Bock-Marquette, Ildiko, Or 8, P 27, P 36 

Bode, Juergen, P 12 

Boekstegers, Peter, P 27, P 28 

Böhm, Marie, Or 7 

Böning, Guido, Or 14 

Bosma, Piter, P 65 

Boztug, Kaan, Or 7 

Bradke, Frank, P 14 

Bradshaw, Angela C, Or 21, P 69 

Braren, Rickmer, P 45 

Bräuchle, Christoph, Or 5, P 3 

Breitbach, Caroline, Inv 18 

Brewer, James M., P 69 

Brill, Thomas, P 55 

Brugman, Martijn, Or 10, Or 15 

Buch, Prateek, P 30 

Buchholz, Christian J, P 42 

Buck, Andreas K, P 45 

Buckley, Suzanne MK, Inv 13 

Büning, Hildegard, Or 2, Or 6, P 32 

Busch, Dirk H, P 56 

Büsche, Guntram, Or 10 

Caizergues, Didier, Inv 24 

Cantz, Tobias, Or 15, P 52, P 52 

Carpentier, A, Inv 15 

Cathomen, Toni, Or 3, P 11, P 17, P 22, P 52, P 52, P 

63 

Cattaneo, Roberto, Inv 17, P 42 

Cengizeroglu, Arzu, Or 18, P 41 

CGD Consortium, Inv 16 

Chan, Jerry, Inv 13 

Chan, Lucas, Inv 23 

Charrier, Sabine, Inv 24 

Chick, Helen, P 38 

Chuah, Marinee KL, Inv 12 

Cichutek, Klaus, Inv 25, P 42 

Cim, Abdullah, Or 9 

Collins, Louise, Or 9 

Conrad, Heinke, P 56 

Cooper, Jonathan D, Inv 13 

Corjon, Stephanie, Or 1 

Cornils, Kerstin, P 64 

Cornu, Tatjana I, P 17 

Coughlan, Lynda, P 69 

Coutelle, Charles, P 23, P 24 

Cowled, Christopher, Inv 23 

Daenthanasanmak, Anusara, P 26 

Dannemann, Nadine, P 22 

de Bruin, Karla, P 3 

Deedigan, Laura, P 48 

Deichmann, Annette, P 68 

Deindl, Elisabeth, Or 8 

Deutsch, Daniela, P 41 

Diaz, Margarita, Or 20 

Diepolder, Helmut, P 26 

DiMaio, Michael, P 27 

Döblinger, Markus, P 53 

Dohmen, Christian, P 5 

Dorer, Dominik E, P 16, P 39 

Draganovici, Dan, Or 14 

Dreyer, Anne-Kathrin, P 22 

Duffy, Margaret R, Or 21 

Dumler, Katja, P 46, P 50 

Ebert, Oliver, P 14, P 44, P 45 

Edinger, Daniel, P 7 

Egana, Tomas J, P 34 

Egerer, Lisa M, P 31, P 32 



Ehrhardt, Anja, Or 3, P 9, P 10, P 20, P 21, P 35, P 61 

El-Aouni, Chiraz, P 28 

Ellwart, Joachim, Or 18 

Emmrich, Stephan, P 43 

Engels, Boris, P 54 

Engler, Tatjana, Or 1 

Eremin, Oleg, Inv 23 

Espenlaub, Sigrid, Or 1, P 13 

Fabre, John, Or 9 

Fahl, Edda, Or 6 

Farkasova, Katarina, Or 18, P 7, P 41 

Farzaneh, Farzin, Inv 21, Inv 23 

Fedonidis, Constantinos, P 23 

Fehse, Boris, P 64, P 66 

Fella, Carolin, Or 1 

Ferizi, Mehrije, P 55 

Ferna´ndez Ulibarri, Ine´s, P 16 

Ferry, Nicolas, P 65 

Filipczyk, Adam, Or 15 

Fischer, Cornelia, P 55 

Fischer, M Dominik, Or 6 

Fitzke, FW, Inv 14 

Flageul, Maude, P 65 

Foralosso, Ruggero, P 5 

Frankenberger, Bernhard, Or 16 

Frenz, Jessica, P 25 

Frieb, Wolfgang, P 1 

Fröhlich, Thomas, P 5, P 6 

Fulda, Simone, P 48 

Gait, Michael J, Inv 11 

Galla, Melanie, Or 15 

Galy, Anne, Inv 24 

Gänsbacher, Bernd, P 33, P 46, P 50, P 55 

Garritsen, Henk, Inv 21 

Garside, Paul, P 69 

Gaudet, D, Inv 15 

Gellhaus, Katharina, P 11, P 17 

Geraerts, Martine, Inv 12 

Gerdes, Sebastian, P 51, P 62, P 64 

Gildehaus, Franz Josef, Or 14, P 47 

Giordano, Frank A, P 67, P 68 

Giri, Jyotsnendu, P 15 

Giroglou, Tsanan, Or 11 

Glauche, Ingmar, P 51, P 62 

Glimm, Hanno, Or 7, Or 17, Or 20, P 37, P 68 

Globisch, Franziska, P 28, P 36 

Göke, Burkhard, Or 13, Or 14, P 47 

Gollisch, Tim, Or 6 

Gottlieb, Elena, P 36 

Greentree, S, Inv 15 

Grez, Manuel, Inv 16, Or 20 

Grünwald, Geoffrey K, Or 13, P 47 

Guhl, Eva, P 52, P 52 

Gustafson, Derek, P 68 

Gutzmer, Ralf, Inv 21 

Haase, Rudolf, Or 18, P 4, P 21, P 41 

Habib, Nagy, Inv 23 

Hagedorn, Claudia, P 60 

Hallek, Michael, Or 2 

Händel, Eva M, P 17 

Hanschmann, Kay-Martin O, P 42 

Hansmann, Martin-Leo, P 57, P 58, P 64 

Harbottle, Richard, P 23, P 24 

Hardwick, Nicola, Inv 23 

Harms, Nina, Or 3, P 35 

Hartmann, Marianne, P 31 

Hartmann, Sylvia, P 57, P 58, P 64 

Hartog, Christoph, P 25 

Hatzopoulos, Antonis K, P 27 

Hausl, Martin, P 10, P 20, P 21, P 35, P 61 

Heckl, Dirk, Or 10 

Heilbronn, Regine, P 11, P 17, P 52, P 52 

Heinrich, Tim, P 57, P 58, P 62, P 64 

Heinz, Niels, Or 19 

Hensel, Karin, P 8 

Herbst, Friederike, P 37 

Hermann, Felix G, P 31 

Hinkel, Rabea, Inv 3,Or 8, P 27, P 28, P 36 

Hirschberger, Johannes, P 55 

Hoeffer, Klemens, Inv 13 

Holder, GE, Inv 14 

Holm, Per Sonne, Or 13, P 13, P 46, P 49, P 53 

Hopfner, Ursula, P 34 

Horstkotte, Jan C, P 27 

Howe, Steven, P 23, P 24 

Huber, Gesine, Or 6 

Imhoff, Andreas, P 50 

Ingram, Wendy, Inv 23 

Itaka, Keiji, Or 4 

Ivics, Zoltán, Inv 12, P 35 

Izsvák, Zsuzsanna, Inv 12, P 35 

Jahnke, Anika, P 55 

Janicki, Hanna, P 32 

John, Katja, P 43 

Jones, Peter, Or 9 

Jun, Tye Gee, Inv 23 

Kaeppel, Christine, P 65 

Kahle, Joerg, P 31 

Kampik, Daniel, P 30 

Kane, NM, P 38 

Kasper, Julia C, P 1 

Kataoka, Kazunori, Or 4 

Kathöfer, Astrid, P 25 

Katus, Hugo A, P 29 

Kay, Mark A, Inv 8 

Khan, Kafaitullah, P 17, P 52, P 52 

Kimpel, Janine, P 31, P 32 

Kirn, David H, Inv 18 

Klein, Christoph, Or 7 

Klibanov, Alexander L, P 8 

Kloz, Ulrich, P 37 

Klump, Hannes, Or 15 

Klutz, Kathrin, Or 13, Or 14, P 47 

Kneiske, Inna, Or 11 

Knoop, Kerstin, Or 14 

Koch, Christian, P 15, P 33, P 34 

Koch, Susanne, Or 6 

Kochanek, Stefan, Or 1, P 59 

Koenig, Frauke M, Or 5 

Kolk, Andreas, P 33 

Kolokythas, Marie, Or 14 

Kondratenko, Irina, Or 7 

Kontermann, Roland, P 39 

Köstlin, Roberto, P 55 

Kostova, Youlia, P 46 

Kreppel, Florian, Or 1, P 13 

Krishnamoorthy, Vidhyasankar, Or 6 

Kritz, AB, P 38 



| 80

81 | 

Kuehle, Johannes, P 12 

Kühlcke, Klaus, Or 7 

Kühle, Johannes, Or 15 

Kuhs, Sandra, Inv 21, P 26 

Kupatt, Christian, Inv 3, Or 8, P 27, P 28, P 36 

Larcher, Fernando Laguzzi, Or 2 

Larkin, Daniel F.P., P 30 

Lauer, Ullrich, P 42 

Laufs, Stephanie, P 67, P 68 

Lebherz, Corinna, Or 8, P 27, P 28, P 36 

Leisegang, Matthias, Or 16, P 54, P 56 

Li, Wenzhong, P 43 

Linnemann, Carsten, Or 17 

Lipps, Hans J., Or 9, P 60 

Loew, Rainer, Or 19 

Lucas, Tanja, P 59 

Luhmann, Ulrich FO, P 30 

Lulay, Christina, Or 7 

Lütge, Fabienne, P 52, P 52 

Lyko, Frank, P 37 

Ma, Ling, Inv 12 

Machens, Hans G, P 25, P 34 

Magnusson, Terese, P 4 

Mailänder, Peter, P 25 

Maitland, Norman J, Inv 20 

Mantwill, Klaus, P 46, P 49 

Maródi, Laszló, Or 7 

Marozin, Sabrina, P 44 

Mátés, Lajos, Inv 12, P 35 

Ma´ trai, Janka, Inv 12 

Mattar, Citra, Inv 13 

Mätzig, Tobias, Or 15 

Mayr, Juliane, P 2 

McDonald, Robert A, P 38, P 69 

McVey, JH, Or 21 

Merten, Otto-Wilhelm, Inv 24 

Meyer, Johann, Or 10 

Meyerhuber, Peter, P 56 

Michalakis, Stylianos, Or 6 

Milosevic, Slavoljub, Or 16 

Miranda, Paula, P 65 

Modlich, Ute, Or 10 

Mohr, Andrea, P 48 

Momma, Stefan, Or 11 

Montazami-Astaneh, Kaweh, P 11 

Moore, AT, Inv 14 

Muehlebach, Michael D, P 42 

Mufti, Ghulam, Inv 23 

Mühlfriedel, Regine, Or 6 

Muik, Alexander, Or 11 

Müller, Oliver J, P 29 

Müller-Lerch, Felix, P 17 

Müther, Nadine, P 35 

Mykhaylyk, Olga M, P 8, P 15, P 53 

Naumann, Ulrike, P 49 

Naundorf, Sonja, Or 7 

Nelson, Peter J, Or 14 

Nettelbeck, Dirk M, P 16, P 39 

Newrzela, Sebastian, P 31, P 57, P 58, 

P 62, P 64 

Nicklin, Stuart A, Or 21, P 69 

Noble, Alistair, Inv 23 

Noske, Nadja, Or 3 

Nowrouzi, Ali, Or 7, P 65 

Nößner, Elfriede, P 54 

Ogris, Manfred, Or 1, Or 5, Or 13, Or 18, P 1, P 4, P 

13, P 40, P 41, P 47 

Oppenheim, David, Inv 23 

Osiak, Anna, P 52 

Ott, Michael, P 26 

Pachler, Karin, P 2 

Paquet-Durand, Franc¸ois, Or 6 

Parker, A.L., Or 21 

Paruzynski, Anna, Or 7 

Perabo, Luca, Or 2 

Petkova, Mina, P 64 

Petry, Harald, Inv 15 

Pfosser, Achim, Or 8, P 28, P 36 

Philipp, Alexander, P 6, P 7 

Plank, Christian, P 8, P 15, P 33, P 34, P 53, P 55 

Pradhan, Pallab, P 8, P 15 

Preuß, Ellen, P 66 

Pützer, Brigitte M, P 43 

Quattrocelli, Mattia, Inv 12 

Radecke, Frank, P 52, P 63 

Radecke, Sarah, P 52, P 63 

Rahim, Ahad A, Inv 13 

Rahman, Shamim H, P 11 

Rauschhuber, Christina, Inv 1, P 9, P 10, P 20 

Reckhenrich, Ann K, P 34 

Reidy, Mairead, P 48 

Rengstl, Benjamin, P 57, P 58 

Robins, Adrian, Inv 23 

Rödl, Wolfgang, P 13, P 47 

Roeder, Ingo, P 51, P 62, P 64 

Rosenecker, Josef, Inv 7 

Rubin, GS, Inv 14 

Rudolph, Carsten, Inv 4 

Ruggiero, Eliana, Or 17 

Rupprecht, Sina, P 48, P 60 

Ruthardt, Nadia, Or 5, P 3 

Rutz, Miriam, P 55 

Ruzsics, Zsolt, P 20 

Rub, Verena, P 6 

Saleh, Amer F, Inv 11 

Salguero, Gustavo, P 26 

Sallach, Jessica, Or 2 

Samara-Kuko, Ermira, Inv 12 

Sampaolesi, Maurillio, Inv 12 

Sanchez-Antequera, Yolanda, P 53 

Sancho-Bru, Pau, Inv 12 

Sawyer, Greta, Or 9 

Schaffert, David, P 1, P 47 

Schambach, Axel, Or 10, Or 15, Or 20, P 12 

Schaser, Thomas, P 42 

Schendel, Dolores J, Or 16, P 54 

Scheu, Christina, P 13 

Schiedlmeier, Bernhard, Or 19 

Schillinger, Ulrike, P 55 

Schinkel, Stefanie, P 29 

Schleef, Martin, P 18, P 19 

Schmeer, Marco, P 18, P 19 

Schmidt, Manfred, Or 7, Or 17, Or 20, P 37, P 65, P 

67, P 68 

Schmitz, Georg, P 8 

Schöler, Hans, Or 15 



Scholz, Simone J, Or 20 

Schroeder, Timm, Or 15 

Schumacher, Ton NM, Or 17 

Schwarz, Klaus, P 52, P 52, P 63 

Seeliger, Mathias W, Or 6 

Senekowitsch-Schmidtke, Reingard, Or 13, P 47 

Sessa, WC, P 38 

Seymour, Len W, Inv 6, Inv 9 

Seznec, Janina, P 49 

Slobodianski, Alex, P 25 

Smith, Alexander J, Inv 14, P 30 

Spitzweg, Christine, Inv 5, Or 13, Or 14, P 13, P 47 

Spranger, Stefani, Or 16 

Stachel, Georg, Or 8, P 36 

Stärck, Lilian, P 56 

Stein, Stefan, Or 20 

Steingoetter, Andreas, P 15 

Stiess, Michael, P 14 

Stockman, A, Inv 14 

Stripecke, Renata, Inv 21, P 26 

Stroes, E, Inv 15 

Su, Baowei, P 40 

Su, Haibin, Or 9 

Sultana, Shahana, Or 8, P 36 

Sundarasetty, Bala Sai, P 26 

Tanimoto, Naoyuki, Or 6 

Thalhammer, Stefan, P 53 

Thielecke, Lars, P 51 

Thormann, Michael, P 28 

Thrasher, AJ, P 38 

Tolmachov, Oleg, P 23 

Trenkwalder, Teresa, Or 8, P 36 

Tuorto, Francesca, P 37 

Turan, Sören, P 12 

Turqueti-Neves, Adriana, P 54 

Twisk, J, Inv 15 

Uckert, Wolfgang, Or 16, P 54, P 56 

van den Born, I, Inv 14 

van der Hoeven, Franciscus, P 37 

van Rooijen, Nico, P 69 

VandenDriessche, Thierry, Inv 12 

Verfaillie, Catherine, Inv 12 

Vermeesch, Joris, Inv 12 

Vetter, Alexandra, P 13 

Vigh, Balasz, P 33 

Virdi, Kulpreet Singh, P 13 

Vlasak, Reinhard, P 2 

Vlaskou, Dialechti, P 8 

Vogt, Stephan, P 50 

Voigtlander, Richard, P 20, P 21 

Volk, Andreas, P 31, P 32 

Volz, Christian, P 29 

von Kalle, Christof, Or 7, Or 17, Or 20, P 37, P 65, P 

67, P 68 

von Laer, Dorothee, Or 11, P 31, P 32, P 57, P 58, P 

62, P 64 

Waddington, Simon N, Inv 13, P 23 

Wagner, Ernst, Or 1, Or 5, Or 14, Or 18, P 1, P 3, P 4, 

P 5, P 6, P 7, P 13, P 40, P 41, P 47 

Walsch, Florian, P 55 

Wang, Wei, P 37 

Wang, Weiwei, P 43 

Warlich, Eva, Or 15 

Weber, Christian, P 65 

Weber, Kristoffer, P 66 

Wedemeyer, Heiner, P 26 

Weingarten, Lisa, P 66 

Wells, James, Inv 23 

Wester, Hans-Ju¨ rgen, P 46 

Wexel, Gabriele, P 50 

Wicke, Daniel, Or 10 

Wilde, Susanne, Or 16 

Willhauck, Michael J, Or 13, Or 14, P 47 

Wolf, Anja, P 46 

Wolf, Nicola M, P 35 

Wong, Andrew MS, Inv 13 

Wong, Suet-Ping, P 23, P 24 

Wood, Matthew, Inv 11 

Wortmann, Andreas, P 59 

Wübbenhorst, Daniela, P 50 

Wuchrer, Alexander, P 27, P 28 

Wunderlich, Nathalie, Or 13, Or 14, P 47 

Yan, Bing, Inv 12 

Yin, Haifang, Inv 11 

Yu, Rui, P 48 

Yzer, S, Inv 14 

Zach, Christian, Or 14, P 47 

Zhang, Wenli, P 35, P 61 

Zhang, Xiahong, Or 9 

Zhang, Ziyang, P 25 

Zimmer, Gert, Or 11 

Zimmermann, Martina, P 42 

Zong, Xiangang, Or 6 

Zwacka, Ralf, P 48 



| 82

Schnellbahnnetz 

S8 

Flughafen München 

Munich Airport 

Flughafen Besucherpark 

S1 S1 

Freising 

Pulling 

S2 

Petershausen 

R 

R 

Neufahrn 

Eching 

Lohhof 

Unterschleißheim 

Partner im 

Vierkirchen- 

Esterhofen 

Hallbergmoos 

Oberschleißheim 

Röhrmoos 

Altomünster 

Kleinberghofen 

S2 

Hebertshausen 

R 

Feldmoching 

Erdweg 

Erding 

Ismaning 

Garching- 

Forschungszentrum 

Garching 

Garching-Hochbrück 

Fröttmaning 

Arnbach 

S3 

Mammendorf 

R 

Altenerding 

Markt Indersdorf 

Unterföhring 

Aufhausen 

Fasanerie 

Niederroth 

Malching 

St. Koloman 

R Moosach 

Dachau 

Karlsfeld 

Allach 

Schwabhausen 

Ottenhofen 

Johanneskirchen 

Kieferngarten 

Freimann 

Studentenstadt 

Alte Heide 

Nordfriedhof 

Dietlindenstr. 

Münchner Freiheit 

Hasenbergl Dülferstr. Harthof Am Hart 

Frankfurter Ring 

Milbertshofen 

Bonner Platz 

OberwiesenfeldOlympiazentrumPetuelring 

OEZ 

Moosacher 

St.-Martins- 

Platz 

Arabellapark 

Scheidplatz 

Olympiapark 

in Bau bis 

vsl. Ende 2010 

Bachern Dachau Stadt 

R 

Maisach 

Hohenzollernplatz 

Markt Schwaben 

R 

Richard-Strauss-Str. 

Georg-Brauchle- 

Ring 

Westfriedhof 

Gernlinden 

Poing 

Josephsplatz 

Böhmerwaldplatz 

Chinesischer 

Turm 

Giselastr. 

Englschalking 

Untermenzing 

Obermenzing 

Esting 

Grub 

Prinzregentenplatz 

Olching 

Heimstetten 

Universität 

Odeonsplatz Lehel Max-Weber-Pl. 

Theresienstr. 

MaillingerStiglmaierstr.platz Gern 

Rotkreuzplatz 

Gröbenzell 

Feldkirchen 

Riem 

Daglfing 

Königsplatz 

Messestadt 

Ost 

Rosenheimer S1 

Platz Ostbahnhof 

Marienplatz 

City Center Isartor 

Karlsplatz 

(Stachus) 

S27 

Hackerbrücke 

Hauptbahnhof 

Central Station 

Donnersbergerbrücke 

Messestadt 

West 

Hirschgarten 

Laim 

Lochhausen 

Langwied Pasing 

Berg am Laim 

Moosfeld 

R R 

R 

R 

Messe München 

International / ICM 

Leuchtenbergring 

Trudering 

Kreillerstr. 

St.-Martin-Str. Josephsburg 

Karl-Preis-Platz 

S20 

Westkreuz 

Leienfelsstr. 

Aubing 

Puchheim 

Deutsches 

Museum 

Gronsdorf 

Rathaus 

Theresienwiese 

Haar 

Heimeranplatz 

Neuaubing 

Harthaus 

Eichenau 

Untersbergstr. 

Silberhornstr. 

Kolumbusplatz 

Sendlinger 

Tor Fraunhoferstr. 

Schwanthalerhöhe 

Laimer Platz Friedenheimer 

Str. 

Giesing 

Lochham 

Germering- 

Unterpfaffenhofen 

Fürstenfeldbruck 

Vaterstetten 

Innsbrucker Ring 

Michaelibad 

Candidplatz 

Goetheplatz 

I s a r 

R 

Harras 

Westendstr. 

Partnachplatz 

Gräfelfing 

Geisenbrunn 

Buchenau 

Quiddestraße 

Baldham 

S6 

Zorneding 

Poccistr. 

Implerstr. 

Brudermühlstr. 

Perlach 

Mittersendling 

Obersendling 

Westpark 

Holzapfelkreuth 

Haderner 

Planegg 

Gilching- 

Argelsried 

Stern 

Schöngeising 

Stockdorf 

Neugilching 

R 

Siemenswerke 

Grafrath 

Eglharting 

Neuperlach Zentrum 

Therese-Giehse-Allee 

Neuperlach Süd 

Neubiberg 

Ottobrunn 

Hohenbrunn 

Wettersteinplatz 

Aidenbachstr. 

Machtlfinger 

St.-Quirin-Platz 

Thalkirchen (Tierpark) 

Fasangarten 

Mangfallplatz 

Groß- 

Str. 

hadern Klinikum 

Großhadern 

Solln 

Gauting 

Weßling 

Fasanenpark 

Türkenfeld 

Forstenrieder 

Allee 

Kirchseeon 

Geltendorf 

R 

R 

Starnberg 

R 

Possenhofen 

S4 

Grafing 

Bahnhof 

Wächterhof 

Höhenkirchen- 

Siegertsbrunn 

Dürrnhaar 

Grafing 

Stadt 

Aying 

Herrsching 

S8 

Ammersee 

Ebersberg 

Peiß 

R 

S4 

Großhelfendorf 

Kreuzstraße 

Starnberger 

See 

Tutzing 

S6 

R 

R 

Großhesselohe Isartalbf. 

Unterhaching 

Pullach 

Taufkirchen 

Höllriegelskreuth 

Furth 

Buchenhain 

S20 S27 

Baierbrunn 

Deisenhofen 

Hohenschäftlarn 

Innenraum 

Sauerlach 

Ebenhausen-Schäftlarn 

München XXL 

Icking 

Otterfing 

Wolfratshausen 

Außenraum 

Holzkirchen 

S7 S3 R 

S7 

Starnberg 

Nord 

Steinebach 

Basler Str. 

Fürstenried 

West 

Seefeld- 

Hechendorf 

Feldafing 

Tarifzonen 

Regionalzughalt 

Fernzughalt 

R 

MVV/Stand: Dezember 2009

Munich - 17th Annual Meeting German Society for Gene Therapy ...

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