3Rs in use of laboratory animals for testing and developing regenerative medicine. Frank Staal

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1 3Rs in use of laboratory animals for testing and developing regenerative medicine Frank Staal Stem Cell Biology, Dept. Immunohematology November 2016

2 Replacement in regenerative medicine 3 Rs = Replacement, Reduction, Refining Replacement in general by: -Human volunteers, tissues and cells -Mathematical and computer models -Established animal cell lines, -Invertebrates, such as Drosophila and nematode worms.

3 Regenerative medicine or severe immune disorders: Gene therapy SCID= Severe Combined Immune Deficiency. Genetic disease of the immune system Sometimes called Bubble Boy Disease Treatment: bone marrow transplantation. Or: gene therapy with own stem cells

4 The Immune system: Defense aginst invaders Macrofages Granulocytes B cells T cells

5 Development of B and T cells

6 What is gene therapy? Transfer of correct version of mutated gene with therapeutic intent How? Virus as vector Crippled virus with therapeutic gene

7 How does stem cell - gene therapy work? Blood Stem Cell from Patient Immature B Immature B B cell making antibodies Mature B plus Stem cell precursor Pre T Pre T T Mature T THYMUS Relief of developmental block BLOOD

8 Mouse studies needed as proof of principle Efficacy: specific disease models Mice that miss the same gene as human patients Heatlth cells Deficient cells Gene therapy treated cells Restoration of T cells in gene therapy treated mice

9 Successful gene therapy in patients: example from Paris Restoration of T cells, thymus tissue and immunity in the first 5 treated patients in the world NEJM, 2000

10 Severe adverse effects in SCID gene therapy trials Leukemia after gene therapy in 5 patients: 4 in the Paris trial and 1 in the London trial T-cell kinetics lymphoid blasts Hacein-Bey-Abina Science, 2003

11 Safety studies in gene therapy (and other cellular therapies!) Traditional: transplanting hundreds of mice with gene therapy treated cells. Waiting for leukemia to happen. Not predictive for human disease time and labor consuming severe stress on lab animals Required GMP testing of viral batches in suckling mice In > 10,000 mice never positive, but required Alternative: IVIM assay: in vitro immortalization assay (Hannover)

12 In vitro immortalization assay FDA approved surrogate safety assay

13 IVIM assay is a better predictor for safety than mouse experiments Data from Axel Schambach, Ute Modlich, Hannover Needs inmprovement: human cells No primary cells lymphoid cells/leukemia Urgent need for grant money

14 But mice are not humans: larger animals needed? Often proposed: studies in dogs or monkeys However, seldom the type of disease (SCID) N.B. Testing cosmetics on animals not acceptable Better: use transplantation of human cells into a limited number of mice that accept human cells xenotransplantation models

15 CD8 IgD SSC-A CD13/33 CD19 CD3 CD16/56 Xenotransplantation: human stem cells into deficient mice (NSG) Transplantation of healthy CD34+ BM Alternative for large animal studies! Myeloid B T NK hcd45 hcd45 Thymocytes hcd45+ GT GT Healthy donor Treated SCID PB B cells Healthy donor Treated SCID 3.6% 78.2% 0.50% 73.9% hcd45+/cd % 15.3% 7.50% 18.0% CD4 IgM

16 Lab animals in gene therapy applications Efficacy: use in proof of principle studies: disease models (e.g. KO mice): Needed, limited numbers (dozens) Alternative for large animals (dogs, monkeys): xenotransplantation (human stem cells into mice) Safety studies: to investigate severe adverse effects. Required by legislation, but not informative. 100s of mice needed IVIM or other in vitro assays better Better legislation: requirements for safety testing need change. There often are equivalent or better alternatives, or tests are not informative Harmonized procedures across Europe/EU: repeat of same animal studies (in various EU countries) useless

17 Acknowledgements Immunohematology and Blood Transfusion Karin Pike-Overzet Miranda Baert Marja van Eggermond Edwin de Haas Sandra Vloemans Anna-Sophia Wiekmeijer Department of Immunology, Rotterdam Mirjam van den Burg Jacques van Dongen Hannover Medical School Michael Rothe Axel Schambach Frank Staal Wim Fibbe Jaap-Jan Zwaginga Pediatrics Dagmar Berghuis Arjan Lankester Robbert Bredius Pharmacy Pauline Meij Maarten Zandvliet UCL, London Fang Zhang H. Bobby Gaspar Adrian Thrasher Necker Hospital, Paris Chantal Lagresle-Peyrou Marina Cavazzana Molecular Cell Biology Rob Hoeben