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Transgenic models : Increasing predictability of in vivo studies:

Transgenic models : Increasing predictability of in vivo studies:. Alexandre Fraichard , CSO & CEO. Genetically modified models. Genetically modified models are research tools They help to obtain a better answer => Tools for a more efficient research.

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Transgenic models : Increasing predictability of in vivo studies:

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  1. Transgenic models : Increasing predictability of in vivo studies: Alexandre Fraichard, CSO & CEO

  2. Genetically modified models • Genetically modified models are research tools • They help to obtain a better answer • => Tools for a more efficient research Transgenic animals : overexpression of transgene KO mice : inactivation of a gene Gene function / Gene validation

  3. Genetically modified models 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

  4. genOway’s mission 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.

  5. genOway’s mission 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

  6. genOway’s mission 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”

  7. Agenda Genetically modified models: tools for a better research Transgenesis: key technologies Case studies Read out and monitoring of physiological response Humanization Next step

  8. Transgene DNA pronuclear injection Ovocytes DNA microinjection 1- Ovocyte re-implantation 2- Birth 3- Screening PCR Then Southern Blot PCR Southern Blot Founders breeding

  9. DNA pronuclear injection The insertion locus is unknown: random integration CONS • Expression pattern • Several trangenic lines required • Comparative studies not feasible • PROS • Molecular biology work is simple • Development time: 6 to 9 months

  10. Endogenous locus Exon 1 5 ’ 3 ’ Homologous recombination Targeting vector Targeted locus 5 ’ 3 ’ PGK PGK Neo -Tk Neo -Tk Homologous recombination

  11. Neo PGK Screening Blastocysts Selection Electroporation Heterozygous Homozygous animals Chimeric mice Model development

  12. Endogenous locus Exon 1 5 ’ 3 ’ Homologous recombination Targeting vector Inactivated locus 5 ’ 3 ’ PGK PGK Neo -Tk Neo -Tk Targeted insertion: Knock out

  13. PROS gene function analysis: broad spectrum source of -/- cells Targeted insertion: Knock out • CONS • Deletion occurs at the ovocyte stage • Artefactual genetic compensation • Development time: 12 months • => « Rapid Kin Technology »TM developed by genOway • NB: siRNA

  14. Target gene Endogenous locus Human gene Targeted locus Human gene Targeted insertion: Knock-in

  15. PROS Expression pattern mastered One transgenic line per transgene Relevant comparative studies Targeted insertion: Knock-in CONS • Development time: 12 months => « Quick Kin Technology »TM developed by genOway

  16. Locus of interest Bio marker IRES IRES XFP XFP Neo Neo PGK PGK Recombined locus Bio marker Targeted insertion: 3’end Knock-in Reporter gene expression mirrors the biomarker expression Homologous recombination

  17. Case study 1: Knockout Case study 2: Monitoring of cell response Case study 3: Humanization by BAC Case study 4: Humanization by Kin Applications

  18. 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 Case study 1: MDR1 knock-out

  19. Case study 2 Monitoring of gene response - cellular response

  20. CK CK Case study 2: classical analysis Biochemical Analysis . In vitro steps Intracellular Staining Detection

  21. Locus of interest Bio marker IRES IRES XFP XFP Neo Neo PGK PGK Homologous recombination Bio marker recombined locus Case study 2: 3’end Knock-in

  22. Protéine 1 + R1 R2 Protéine 2 + Case study 2: Principle Gene 1 R1 Stimuli Detection Gene 2 R2

  23. genOway’s model . 1 Stimuli 2 Cell recovery Direct identification of fluorescence 3 Detection No In vitro step Viable cells 4 Cells are dead Case study 2: How is the model valuable Usual analysis Biochemical studies Surface staining Intracellular staining …

  24. Project 210 Kb BAC project: 2 Human genes of interest Outcome of classical transgenesis Not enough founders for a reliable scientific analysis Case study 3: Human “Acetylases”

  25. #4A #8A #3B 5 ’ + + 3 ’ + + + 11 19 24 4A 8A 3B Hu gene1 + + + Hu gene2 + + 11 19 24 Case study 3: Human “Acetylases” Safe Large fragment TransgenesisTM • 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

  26. Case study 3: Human “Acetylases” • Experiments performed • In vivo analysis • In progress • In vitro experiments based on primary culture • Molecular mechanisms studies • « Screening »

  27. Case study 4 • Humanized xenobiotic response in mice • Nature, vol 406, pp435 - 439 • Regulators of CYP3A: SXR versus PXR • Deletion of PXR (mouse gene) • Addition of SXR (Human protein)

  28. PXR null mice • KO mice loose the CYP3A induction by PCN and DEX • CYP1A2 ... not altered

  29. Induction « Human » Induction « Mouse » SXR transgenic mice • RIF induction • Reversible • Dose dependent « Human » properties are added to the mouse

  30. Induction « Human» No more induction « Mouse» SXR Tg / PXR -/- « Humanized » mouse, no more mouse response

  31. 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

  32. 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

  33. PXR gene Endogenous locus Targeted insertion: Knock-inGene substitution approaches IRES Reccombined locus SXR • PXR is inactivated: PXR -/- • SXR: one copy, PXR promoter and regulatory sequences • Genetic background, breeding ...

  34. 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

  35. Case study 1: Knockout Case study 2: Monitoring of cell response Case study 3: Humanization by BAC Case study 4: Humanization by Kin Applications

  36. More publications are done with rat than with mice Rat cloning 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

  37. Activation Oocyte Nuclear transfer Foster mother Competitors Cloned embryo Genetic modification Nucleus donor cell Genetically modified cloned animal Cloned animal Genetically modified rat Rat cloning

  38. Nuclear Transfer Activation Cloned embryo Oocyte Death Development abnormally started 1er cloned embryos New York, 2001 Controlled development Rat cloning Main steps:

  39. Cloned pups Male cloned rats Female cloned rats Rat cloning

  40. Rat cloning & applications • Development of Humanized rat models • CNS • Cardiovascular • ADME - Toxicology • Rat consortium in develoment • One animal breeder: genetic & health status • Pharmaceutical partners • Academic laboratories.

  41. Conclusion • Genetically modified models • Gene validation • Monitoring of physiological response • Humanization for physiological studies • Service provider: Customized models • Innovative technologies • Flexibility • 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.

  42. 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 (TheNetherlands) …

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