View
213
Download
0
Embed Size (px)
Citation preview
www.genoway.com
Transgenic models :
Increasing predictability of in vivo studies:
Alexandre Fraichard, CSO & CEO
www.genoway.com
• Transgenic animals : overexpression of transgene
• KO mice : inactivation of a gene
Gene function / Gene validation
Genetically modified models
• Genetically modified models are research tools
• They help to obtain a better answer
=> Tools for a more efficient research
www.genoway.com
Two majors axes
• Improve the read out of the experiment– Increased sensitivity– Earlier read out– Higher throughput
• Increase the predictability of the response obtained through “Humanization”
– Human protein
– Humanized regulation
– Humanized pathway
Genetically modified models
www.genoway.com
Develop the most predictable and reliable animal model
• Consulting: Each model is different, fully adapt the model to the project
expectations and constraints.
• Innovation: New technologies for better models.
• Reliability: a GM models is always important, it must rely on the most robust
and validated technological plate form.
genOway’s mission
www.genoway.com
A focused tool provider
• Focused: Design and develop animal models.
– Animal facility is subcontracted to professional breeders
– No phenotyping activity but a network of European biotechs
• Customized services: Flexible offers with the most adapted technologies
• Quality: A fully dedicated laboratory and experienced scientists.
– SOP
– Quality plan
A track record of satisfied customers … … with more and more repeat business
genOway’s mission
www.genoway.com
A few figures
• Created in 1999
• Staff: 38
• Customers in 15 countries
• Export > 75%
• A fully dedicated facility of 1 300 m2 in Lyon
• The European leader in the “Transgenic business”
genOway’s mission
www.genoway.com
Genetically modified models: tools for a better research
• Transgenesis: key technologies
• Case studies
– Read out and monitoring of physiological response
– Humanization
• Next step
Agenda
www.genoway.com
DNA pronuclear injection
DNA microinjection
1- Ovocyte re-implantation
2- Birth
Founders breeding
3- ScreeningPCR Then Southern Blot
Transgene
PCR Southern Blot
Ovocytes
www.genoway.com
DNA pronuclear injection
CONS– Expression pattern– Several trangenic lines required– Comparative studies not feasible
PROS– Molecular biology work is simple– Development time: 6 to 9 months
The insertion locus is unknown: random integration
www.genoway.com
Endogenous locus
3 ’5 ’Exon 1
Homologous recombination
Targeting vector
Homologous recombination
PGK Neo -Tk
3 ’5 ’PGK Neo -Tk
Targeted locus
www.genoway.com
Model development
Electroporation
Screening
Selection
BlastocystsChimeric mice
HeterozygousHomozygous
animals
PGK
Neo
www.genoway.com
Endogenous locus
3 ’5 ’Exon 1
Targeting vector
Homologous recombination
PGK Neo -Tk
3 ’5 ’PGK Neo -Tk
Inactivated locus
Targeted insertion: Knock out
www.genoway.com
Targeted insertion: Knock out
PROS– gene function analysis: broad spectrum– source of -/- cells
CONS– Deletion occurs at the ovocyte stage– Artefactual genetic compensation– Development time: 12 months
=> « Rapid Kin Technology »TM developed by genOway
NB: siRNA
www.genoway.com
Targeted insertion: Knock-in
Endogenous locusTarget gene
Human gene
Targeted locus Human gene
www.genoway.com
Targeted insertion: Knock-in
PROS
• Expression pattern mastered
• One transgenic line per transgene
• Relevant comparative studies
CONS
• Development time: 12 months
=> « Quick Kin Technology »TM developed by genOway
www.genoway.com
Targeted insertion: 3’end Knock-in
Reporter gene expression mirrors the biomarker expression
XFPIRES
PGKNeo Recombined locus Bio marker
Locus of interest
Bio marker
Homologous recombination
XFPIRES
PGKNeo
www.genoway.com
Applications
• Case study 1: Knockout
• Case study 2: Monitoring of cell response
• Case study 3: Humanization by BAC
• Case study 4: Humanization by Kin
www.genoway.com
Case study 1: MDR1 knock-out
• Deletion of the Mdr1a gene Schinkel et al, Cell 1994
– Elevated drug levels in many tissues (brain …)
– Decreased drug elimination
• Deletion of both Mdr1a+b genes Schinkel et al, PNAS 1997
– No major phenotype
– Genetic compensation: normal or not?
• Interaction Mdr1 - CYP3A Schuetz et al,Mol. Pharm. 2000
– Up-regulation of CYP3A in Mdr -/- mice
– Environment can suppress this result
• A knockout is physiologically very complex to understand
www.genoway.com
Case study 2
Monitoring of gene response - cellular response
www.genoway.com
Case study 2: classical analysis
.
BiochemicalAnalysis
0
10
20
30
40
50
60
70
80
90
1 2 3 4
Intracellular Staining
In vitro steps
CK
CK
Detection
www.genoway.com
Case study 2: 3’end Knock-in
XFPIRES
PGKNeo recombined locus Bio marker
Locus of interest
Bio marker
Homologous recombination
XFPIRES
PGKNeo
www.genoway.com
Case study 2: Principle
Gene 1 R1
Gene 2 R2Stimuli Detection
R2
Protéine 2 +
R1
Protéine 1 +
www.genoway.com
Case study 2: How is the model valuable
Usual analysis
.
Biochemical studiesSurface stainingIntracellular staining…
1 Stimuli
2 Cell recovery
3 Detection
4 Cells are dead
No In vitro step
Viable cells
genOway’s model
Direct identification of fluorescence
www.genoway.com
Case study 3: Human “Acetylases”
• Project
– 210 Kb BAC project: 2 Human genes of interest
• Outcome of classical transgenesis- Not enough founders for a reliable scientific analysis
www.genoway.com
Outcome
- 8 founders positive for the 4 PCRs: 5 ’ end, hu gene1 , hu gene2 and 3 ’ end- 11 founders positives for all PCRs except 3 ’ or 5’ end (# 3B)- 6 other situations- Integrity of the BAC is controlled- Copy number: from 1 to 5 - 10
Case study 3: Human “Acetylases”
Safe Large fragment TransgenesisTM
11 19 24
11 19 24
Hu gene1 + + +
Hu gene2 + +
#4A #8A #3B4A 8A 3B5 ’ + +
3 ’ + + +
www.genoway.com
Case study 3: Human “Acetylases”
Experiments performed
• In vivo analysis
v In progress
• In vitro experiments based on primary culture
v Molecular mechanisms studies
v « Screening »
www.genoway.com
Case study 4
Humanized xenobiotic response in miceNature, vol 406, pp435 - 439
Regulators of CYP3A: SXR versus PXR• Deletion of PXR (mouse gene)• Addition of SXR (Human protein)
www.genoway.com
PXR null mice
• KO mice loose the CYP3A induction by PCN and DEX• CYP1A2 ... not altered
www.genoway.com
SXR transgenic mice
Induction« Human »
« Human » properties are added to the mouse
RIF induction• Reversible• Dose dependent
Induction« Mouse »
www.genoway.com
SXR Tg / PXR -/-
Induction« Human» No more induction
« Mouse»
« Humanized » mouse, no more mouse response
www.genoway.com
Conclusion 1
The model is
• PXR -/-
• SXR Tg
The model could be used to screen molecules for SXR neutrality
• Higher predictability of the results obtained in vivo
www.genoway.com
SXR Tg analysis
The model: PXR -/- & SXR Tg
The model could be used to screen molecules for SXR neutrality
=> A higher predictability of the results is obtained in vivo
CONS–Expression pattern: Albumin promoter–Level of expression–Copy number
PROS–Nice results …
A Knock-in approach should have been selected
www.genoway.com
Targeted insertion: Knock-inGene substitution approaches
Reccombined locusSXR
IRES
Endogenous locusPXR gene
• PXR is inactivated: PXR -/-• SXR: one copy, PXR promoter and regulatory sequences• Genetic background, breeding ...
www.genoway.com
Conclusion 2
Targeted insertion provides• A clear genetic understanding of the model
• Copy number• Insertion site• Exact sequence inserted
• Controlled characteristics• Expression level • Expression pattern
• Practical advantages• Breeding constraints• Opportunities for model evolution
Humanisation SXR-PXR is in development
www.genoway.com
Applications
• Case study 1: Knockout
• Case study 2: Monitoring of cell response
• Case study 3: Humanization by BAC
• Case study 4: Humanization by Kin
www.genoway.com
Rat is a key animal model for the biomedical research• Its physiology is closer to Human than most other species• Huge amount of data has been accumulated in the past decades• 25% of animal models are rat models
• More publications are done with rat than with mice
Rat cloning
www.genoway.com
Genetically modified rat Genetically modified cloned
animal
Rat cloning
Foster mother
Genetic modification
Oocyte Nuclear transfer Activation
Cloned embryo
Nucleus donorcell
Cloned animal
CompetitorsCompetitors
www.genoway.com
Oocyte Nuclear Transfer Activation Cloned embryo
Main steps:
DevelopmentDevelopmentabnormally abnormally
startedstarted
DeathDeathControlledControlleddevelopmentdevelopment
1er cloned embryos
New York, 2001
Rat cloning
www.genoway.com
Cloned pups Male cloned rats Female cloned rats
Rat cloning
www.genoway.com
Rat cloning & applications
• Development of Humanized rat models
v CNS
v Cardiovascular
v ADME - Toxicology
• Rat consortium in develoment
v One animal breeder: genetic & health status
v Pharmaceutical partners
v Academic laboratories.
www.genoway.com
Conclusion
• Genetically modified modelsv Gene validationv Monitoring of physiological responsev Humanization for physiological studies
• Service provider: Customized models v Innovative technologiesv Flexibility v Reliability.
• genOway is developing a range of fully humanized models
for « metabolism » studies A consortium will be developed with pharmaceutical companies to validate and use these models.
www.genoway.com
Customer portfolio
Biopharmaceutical companies
Altana Pharma, Aventis Pharma, Bayer, CellTech , Galderma, GSK, Johnson & Johnson, Laboratoires Pierre Fabre, Laboratoires Servier, Merck Sharp & Dohme, Novartis, Novusphrama, Pfizer, SangStat, sanofi-synthelabo, Schering, Schering-Plough, Syn-X Pharmaceuticals …
Non profit organisations
Advanced Biotechnology Center (Italy), Centro de Biologia Molecular (Spain), CNRS (France), DIBIT (Italy), Genethon (France), Imperial College of London (UK), Inserm (France), Institut Gustave Roussy (France), Institute for cancer Research and Treatment (Italy), Kimmel Cancer Center (USA), King's College of London (UK), Ludwig Institute For Cancer Research (Sweden), Mario Negri Institute for Pharmacological Research (Italy), Mount Sinai (USA), Pasteur Institute (France), University College London (UK), University of Antwerp (Belgium), University of Barcelona (Spain), University of Cambridge (UK), University of Frankfurt am Main (Germany), University of Geneva (Switzerland), University of Konstanz (Germany), University of Lausanne (Switzerland), University of Oxford (UK), University of Tampere (Finland), University of Wageningen (The Netherlands) …