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RNAi:
the technology to revolutionise functional genomic research
What are the limitations?
20 - 22 November 2003
Harnack-Haus,
Berlin-Dahlem
Organisers:
Thomas
F. Meyer, MPI for Infection Biology, Berlin
Thomas Rudel, MPI for Infection Biology, Berlin
Hans
Prydz, Biotechnology Centre of Oslo, Oslo
Joachim W. Engels, Institut für Organische Chemie, Goethe
Universität Frankfurt am Main
Report
Scientific
Content
The
major aim of this workshop was to bring together researchers
from different areas such as RNA chemistry, bioinformatics,
cell biology and molecular medicine in order to fathom the
impact of the new developments on the practical aspects of
the RNA interference technology.
The
workshop covered four major topics:
Topic
1. Basic mechanism and model organisms. There are indications
that differences exist in mammals and in lower organisms regarding
the components of the basic machinery and the mechanisms.
In some organisms such as Dictyostellium amplification steps
involving RNA dependent RNA polymerases are required whereas
in others both the amplification and the required enzymes
have not been identified so far. The workshop gave the opportunity
to learn about the composition and the function of the RNA
interference signalling complex which opens up the possibility
to modulate the process itself.
Topic
2. Gene suppression in mammals. The speed by which novel
systems have been developed to perform RNAi in the living
animal is breath taking. The intention in this field of applied
RNAi research is to develop new tools to knock down gene expression
in transgenic animals but also to apply synthetic RNAs to
animals as therapeutic approach. The workshop led to the conclusion
that RNAi and lentivirus-mediated transgenesis provide efficient
new approaches to study the genetics of various diseases and
that RNAi inhibitor libraries can be used to identify potential
therapeutic targets in vivo. The use of a Pol II (CMV) promoter
for the expression of shRNA in a prion disease model system
was demonstrated.
Topic
3. Therapeutic approaches. Directly related to RNAi in
the living organism is its use for therapy. The properties
of RNA interference may lead to entirely new approaches in
diseases where single or multiple knock downs of gene expression
might be required for the therapeutic success. Initial experiments
have been undertaken to investigate the possibility to interfere
with cancer, viral infections and degenerative diseases.
Topic
4. Novel approaches to RNAi. Of high priority for RNAi
research are technical developments like an innovative combination
of proteomics and RNAi, modified siRNAs, new vector systems,
technology to develop random libraries or transporters for
siRNA delivery in order to achieve an efficient knock down
of gene expression.
The
meeting offered 18 presentations by invited speakers, 1 key
note lecture and 5 additional presentations as posters related
to the sessions.
Final Programme
Opening
and Session 1 - Basic mechanism and model organisms
Thursday afternoon, 14:00-18:00 Chairperson: Joachim W. Engels
14.00-14.10 Opening Remarks. Thomas F. Meyer, MPI for Infection
Biology, Berlin
14.10-14.50 Genetic analysis of gene silencing in plants.
Alan Herr, The Sainsbury Laboratory, Norwich
14.50-15.30 RNA-RNA recognition between siRNA and its target:
a kinetic view.
Georg Sczakiel, University of Lübeck
15.30-16.10 Coffee Break
16.10-16.40 Genome-wide RNAi screening in C. elegans, Drosophila
and human cells. Christophe Echeverri, Cenix Bioscience GmbH,
Dresden
16.40-17.20
Mechanisms of antisense and RNAi mediated gene silencing in
the model organism Dictyostelium. Wolfgang Nellen, University
of Kassel
17.20-18.00 Deliberate and accidental RNAi in higher plants.
Jan Kooter, Vrieje Universiteit, Amsterdam
Dinner
19.30-20.15 Keynote Lecture…Chairperson: Hans Prydz Fritz
Eckstein, MPI for Experimental Medicine, Göttingen
Session
2 - Gene suppression in mammals
Friday
morning 9.00 - 12.00 Chairperson: Fritz Eckstein
9.00-9.40 Exploring the genetics of disease using RNAi. Luk
van Parijs, Center for Cancer Research, MIT, Cambridge
9.40-10.20
Potential control of prion propagation using RNAi. Gaëlle
Tilly, Institut National de la Recherche Agronomique, Jouy-en-Josas
10.20-11.00 Coffee break
11.00-12.00 Poster discussion
12.00-14.00 Lunch
Session 3 - Therapeutic approaches
Friday afternoon 14:00-18:00…Chairperson: Luk van
Parijs
14.00-14.40 RNAi to probe the Oxygen-sensing pathway and Tumor
Angiogenesis. Jacques Pouyssegur, CNRS UMR 6543, Nice
14.40-15.20 Biological parameters in tumor cells where HER-2/neu
expression has been down-regulated by RNAi technology. Raja
Choudhury, Karolinska Hospital, Stockholm
15.20-16.00 Coffee Break
16.00-16.40 Lentiviral mediated expression of RNAi in the
context of neurodegenerative diseases. Patrick Aebischer,
EPF Lausanne
16.40-17.20 RNA interference of HIV replication José
A. Este, Universitat Autònoma de Barcelona
17.20-18.00 Effects of siRNA and antisense RNA on tumor metastasis.
Hans Prydz, University of Oslo
Dinner
Round table discussion Chairperson: Thomas F. Meyer
Session 4 - Novel approaches to RNAi
Saturday morning 8:30-12:30 Chairperson José A. Este
8.30-9.10
Identification of cell death regulators by RNA interference.
Thomas Rudel, MPI for Infection Biology, Berlin
9.10-9.50 Chemical Aspects of RNA-Silencing. Joachim W. Engels,
University of Frankfurt/Mn.
9.50-10.30
Antisense and RNAi approaches against HIV-1 - finding the
right target.
Jørgen Kjems, University of Arhus Arhus
10.30-11.10 Coffee break
11.10-11.50 Modulation of gene expression by small interfering
RNA in hematopoietic cells.
Michaela Scherr, Hannover Medical School
11.50-12.30 Validation of novel transporters for siRNAs. Ute
Schepers, University of Bonn
Summary: Alan Herr
Assessment
of the results and contribution to the future direction of
the field
This
workshop has provided an excellent vision of the RNAi field
and focussed on latest developments and practical aspects
of RNAi. It provided an opportunity to exchange information,
to learn about the newest findings on the basic mechanisms
of RNAi and variations thereof found in different organisms
as well as about novel applications in therapy. Participants
not only presented their successful work but also discussed
unsuccessful approaches that are potentially helpful for the
planning of future research projects.
One
important factor for assessing the results of this event was
the lively and constructive discussion during all sessions
of the workshop and in particular during the round table discussion
on Friday evening. The discussions mainly concerned the possible
limitations and the current challenges of RNAi approaches
and led to the following conclusions:
• The basic RNAi biochemsitry such
as enzymology of RISC must be elucidated before its manipulation
can be considered.
• Non-validated inhibitors are
usually less expensive than validated ones but the use of
non-validated RNAi inhibitors is typically on the expense
of specificity. The investigation of multiple targets with
non-validated inhibitors may comprise useless efforts. Validation
of inhibitors will continue to be fundamental in order to
ascertain high specificity and reliability of results.
• Even the best design tools will
not be able to predict functional RNAi inhibitors with 100%
reliability. Standard "blast searches" are not considered
suitable for short stretches of sequence homology searches.
Rather "Smith Waterman" analysis is generally favoured.
• Maximum RNAi inhibitor stability
is not always the best choice. Sometimes a more transient
RNAi effect may be better.
• Possibilities how to draw a distinction
between off-target effect and experimental variability in
expression studies were discussed.
• Misinterpretation may arise from
doing initial RNAi studies in HeLa cells which are known to
be highly degenerated.
• The efficiency of siRNA delivery
was considered as important as its stability. This should
first be tested in cell culture systems and as well later
in animals.
• There are several applications
in which RNAi is being crucial to understanding the mechanisms
related to complex diseases or drug resistance, but the usefulness
of therapeutic RNAi approaches in severe genetic disease studies
was questioned. An open question also was if the focus of
therapeutic efforts should be on acute (infections such as
HIV) or chronic diseases (e.g. cancer).
• The current lack of basic knowledge
should not hold up human therapeutic trials. There is a lack
of knowledge about genetic side effects of conventional drugs
that have been administered for long.
• In viral disease such as HIV
sequence changes occur frequently. Using RNAi as therapeutic
tool it will be possible to quickly react to sequence changes.
A
question raised several times was which internal controls
are required for valid RNAi studies. Possible controls include
complementation with the respective cDNA, a second RNAi inhibitor
or a pharmacological inhibitor. General consent existed that
gene complementation cannot be made a basic standard for all
types of RNAi experiments because complementation often brings
up a phenotype that is different from wild types as well as
the knock down phenotype.
The
participants recommended that EURIT, the European RNAi network
representing several hundred active RNAi researchers (www.eurit-network.org),
should help to standardize required controls in RNAi applications
to facilitate the peer review procedure of publications.
The
multidisciplinary character of the audience was especially
enriching and promoted the exchange of experiences between
researchers with different backgrounds. The opinion and advice
of the experts working for years in the field of RNA technologies
was particularly important and was well received by the participants
working for a short time with RNAi.
The
workshop has supported the consolidation of existing national
and international collaborations by providing feedback and
reviewing advances. It also represented an opportunity for
discussing potential standardization projects, and the advancement
of the RNAi technology through EURIT. Furthermore, it allowed
some of the participants to establish new collaborations involving
exchange of technical expertise, share of information resources
and participation in existing projects. Some of the participants
would be interested in implementing activities such as research
visits and exchanges.
List of participants
Berra, Edurne CNRS-UMR 6543 33 av. Valambrose
06189 Nice, France berra@unice.fr2
Bruland, Torunn NTNV Dep. Cancer Research and
Mol. Medicine MTFS Olav Kyrresgt. 3, 5th floor N-7489 Trondheim,
Norway Torunn.Bruland@medisin.ntnv.no
Choudhury, Raja Karolinska Department of Oncology-Pathology,
Karolinska Hospital, SE-171 76 Stockholm rajcho@mbox.ki.se
Dong, Wubei IPK-Gersleben Corrensstr. 3 Gatersleben,
Germany dong@ipk-gatersleben.de
Douchkov, Dimitar IPK-Gersleben Corrensstr.
3 Gatersleben, Germany douchkov@ipk-gatersleben.de
Duong, Chi Vinh MPI für Hirnforschung
Deutschordenstraße 46 60528 Frankfurt, Germany Duong@mpih-frankfurt.mpg.de
Eckstein, Fritz MPI für experimentelle
Medizin Hermann-Rein-Str. 3 37075 Göttingen, Germany
eckstein@em.mpg.de
Engels, Joachim Institut für Organische
Chemie, Johann Wolfgang Goethe-Universität, Marie Curie
Str. 11,
60439 Frankfurt, Germany joachim.engels@chemie.uni-frankfurt.de
Este, Jose A. Retrovirology Laboratory
IrsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat
Autònoma de Barcelona 08916 Badalona, Spain jaeste@ns.hugtip.scs.es
Fetzer, Christian Ruhr-Universität Bochum,
Molekulare Neurobiochemie Universtätsstraße 150
44780 Bochum, Germany christian.fetzer@rub.de
Fiedler, Wolfgang Martin-Luther-University
Halle-Wittenberg, KIM 1 - Molecular Gastro. Oncology, BIOZENTRUM
Weinbergweg 22 D-O6120 Halle, Germany wolfgang.fiedler@medizin.uni-halle.de
Giordano, Tiziana San Raffaele Science Institute
Neuroscience Dept.-DIBIT via Olgettina 58 20132 Milan IT giordano.tiziana@hsr.it
Guerra, Marisol University of La Laguna Delgado
Barreto 38207 La Laguna, Tenerife mguerra@ull.es
Hejnar, Jiri IMG Prague Flemingoro.nam.2 Prague
CZ jhejnar@ing.cas.cz
Herr, Alan The Sainsbury Laboratory, John Innes
Centre Norwich, NR4 7UH UK alan.herr@sainsbury-laboratory.ac.uk
Heß, Simone Inst. F. Medizin. Mikrobiologie,
MHH Hannover Carl-Neuberg-Str. 1 30625 Hannover, Germany Hess.Simone@mh-hannover.de
Johanssen, Tim Eisai Research London Gower
Street London UK Timothy.Johanssen@eisai.net
Jungnickel, Berit Institut für Klinische
Molekularbiologie und Tumorgenetik
GSF-Forschungszentrum für Umwelt und Gesundheit Marchioninistrasse
25 81377 München DE jungnickel@gsf.de
Karlas, Alexander RKI Robert Koch Institute
Nordufer 20 Berlin , Germany KarlasA@rki.de
Kjems, Jorgen Universität Arhus,
Department of Molecular Biology C.F. Mollers Alle, Build.130
DK-8000 Arhus C, Denmark kjems@biobase.dk
Klos, Andreas Med. Microbiology, MHH
Carl-HeubergStr. 1 Hannover, Germany Klos.Andreas@mh-hannover.de
Köhler, Rolf MPI for I0 Schumannstr. 21/22
Berlin, Germany koehler@mpiib-berlin.mpg.de
Krol, Jacek IBCh Noskovsiziegok Poznan
PL jkrol@lose.man.poznan.pl24 Kurreck Jens FU Berlin Thielalle
63 14195 Berlin, Germany jkurreck@chemie.fu-berlin.de
Lord, Christopher The Breakthrough Toby Robins
Breast Cancer Research Centre, Institute of Cancer Research
Mary-Jean Mitchell Green Building, Chester Beatty Laboratories
Fulham Rd. London SW3 6JB UK lordc@ic.ac.uk
Machuy, Nikolaus MPI for Infection Biology
Schumannstr. 21/22 Berlin, Germany machuy@mpiib-berlin.mpg.de
Maciel, Patricia Institute for Research
in Life and Health Sciences, School of Health Sciences, University
of Minho
Campus de Gualtar 4710-057 BRAGA, Portugal pmaciel@ecsande.uminha.pt
Maly, Petr Institute of Molecular Genetics
Videnska 1083 Prague CZ pemal@biomed.cas.cz
Marwan, Wolfgang University of Hertfordshire
College Lane Hatfield UK w.marwan@hertr.ac.uk
Meyer ,Thomas F. MPI for Infection Biology
Schumannstr. 21/22 Berlin, Germany meyer@mpiib-berlin.mpg.de
Montanini, Luisa University of Parma
Via delle Science II/a Parma, Italy luisa.montanini@hotmail.com
Nellen, Wolfgang Universität Kassel, Fachbereich
19 Biologie/Chemie, Department of Genetics Heinrich-Plett-Str.
40 34109 Kassel, Germany nellen@uni-kassel.de
Polesskaya, Anna CNRS 7 rue G. Moguet Villejuif,
Francea poles@vjt.cnrs.fr
Pouyssegur, Jacques CNRS 33 Av. Valombrose
Nice FR pouysseg@unice.fr
Prydz, Hans Biotechnology Centre, University
of Oslo Gaustadalleen 21 0349 Oslo, Norway hans.prydz@biotek.uio.no
Pscherer, Armin DKFZ INF 580 Heidelberg,
Germany apscherer@dkfz.de3
Rimmele, Martina RiNA GmbH Takustraße
3 14195 Berlin, Germany rimmele@rna-network.com
Rodriguez, Antonio University of La
Laguna Delgado Barreto 38207 La Laguna, Tenerife acastia@ull.es
Rudel, Thomas MPI for Infection Biology
Schumannstr. 21/22 Berlin, Germany rudel@mpiib-berlin.mpg.de
Scherr, Michaela MHH Carl-Neuberg-Str. 1 Hannover,
Germany, m.scherr@t-online.de
Sczakiel, Georg Uni Lübeck Ratzeburger
Allee 160 Lübeck, Germany sczakiel@imm.uni-luebeck.de
Seltsam, Axel Institut für Transfusionsmedizin,
MHH Carl-Neuberg-Str. 1 30625 Hannover, Germany seltsam.axel@mh-hannover.de
Skopkova, Zuzana Institute of Molecular Genetics
Videnska 1083 Prague CZ zusko@biomed.cas.cz
Tao, Jiong MPI for Molecular Genetics Ihnestraße
73 Berlin, Germany jiong@molgen.mpg.de
Thommesen, Liv Norwegian University of Science
& Technology MTFS, DMS, 7489 Trondheim, Norway liv.thommesen@medisin.ntnu.no
Thompson, James D. Transgenomics The Quadrangle,
Crewe Hall, Weston Road Crewe, Cheshire, UK CW1 6UZ UK jthompson@transgenomics.com
Tilly,
Gaelle Institut National de la Recherche Agronomique,
Domaine de Vilvert 78352 Jouy-en-Josas cede, France tilly@inra.jouy.fr
Toufik, Abbas-Terki EPFL EPFL 1015 Lausanne,
France toufik.abbas-terki@epfl.ch
Trülzsch, Barbara Dept of Human Anatomy
and Genetics South Parks Rd Oxford OX1 3QX UK Barbara.Trulzsch@anat.ox.ac.uk
van Parijs, Luk MIT Mass. Ave. Cambridge, MA
lukvp@mit.edu
Wood, Matthew Oxford University South Parks
Rd. Oxford UK matthew.wood@anat.ox.ac.uk
Zaborowska, Zaneta Free University Berlin Thielalle
63 Berlin, Germany zaneta@chemie.fu-berlin.de
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