FISHING FOR NEW THERAPEUTIC TARGETS IN THE FROG PONT

Transcription

1 FISHING FOR NEW THERAPEUTIC TARGETS IN THE FROG PONT Kris Vleminckx Ghent University Belgium DTRF2016 Patient Meeting

2 1. Why lower model organisms?

3 Most popular model organisms

4 Most popular model organisms Worm Fruit fly Fish and Frog Mouse Experimental Flexibility Relevance to humans Costs Housing & Research

5 model organisms evolutionary distance

6 model organisms evolutionary distance

7 Developmental signaling pathways hijacked in cancer (e.g. Wnt, Hedgehog, Notch)

8 2. Why the frog Xenopus?

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10 Hellsten et al., 2010 Nakayima et al., 2005

11 Xenopus main advantages - Vertebrate - tetrapod - High number offspring > 1000 eggs/spawning - embryonic development occurs entirely outside the mother animal - embryos have a large size and a high degree of transparancy during organogenesis - embryogenesis proceeds rapidly

12 Xenopus simple injection set-up

13 see Xenopus early development

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15 Xenopus injections RNA and Morpholino Injections RNA Or Morpholinos (2 8 cell stage) Phenotypic Readout

16 Example: injection Wnt RNA axis duplication

17 3. A new Era of genome editing with custom-made CRISPR/Cas9 system?

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19 Emmanuelle Charpentier and Jennifer A. Doudna Breakthrough Prize 2015 Life sciences

20 CRISPR/Cas9 mechanism Adaptation of bacterial CRISPR locus (prokaryotic immune system) Cas9 = nuclease protein binding to specific secondary RNA structure (present in tracrrna) Pennisi (2013) Guide RNA connects synthetic RNA module (complementary to target) to trac-like sequences 20

21 CRISPR/Cas9 Repair mechanisms after double strand DNA break NHEJ : Non-Homologous End Joining - insertions or deletions of reading frame

22 Xenopus injections CRISPR/Cas9 Injections Cas9 and CRISPR guide RNAs (2 8 cell stage) Phenotypic Readout

23 CRISPR/Cas9 easy and very efficient in Xenopus F0 mosaic F1 offspring Ishibashi et al., 2012

24 < 2 months ~ 6 months < 2 weeks < 2 weeks CRISPR/Cas9 Experimental Pipeline Design grna (CRISPRscan) Primer ligation + direct T7 RNA synthesis (cloning free) Inject grna + Cas9 protein Assess efficiency (NGS MiSeq) Mostly > 70% Analysis F0 mosaic (e.g. cancer models) Raise or Adjust protocol F0 F0 F1 phenotyping

25 4. Modeling human cancer syndromes in Xenopus

26 Familial Adenomatous Polyposis in Xenopus (targeting APC tumor suppressor gene) Van Nieuwenhuysen et al., 2015

27 Familial Adenomatosis Polyposis in Xenopus (targeting APC tumor suppressor gene) Extra-colonic manifestations: Congenital Hypertrophy of Retinal Pigment Epithelium (CHRPE) Desmoid tumors Brain tumors (medulloblastoma Abnormal dentition Osteomas Epidermal cysts

28 DTRF supported application 1 : Desmoid tumor preclinical compound testing BC-2059 : inhibitor of TBL1 dependent β-catenin signaling

29 DTRF supported application 2 : Desmoid tumor therapeutic target identification via grna multiplexing workflow Genome modification of APC and gene x Dissect tumor Isolate DNA Sequence Gene X Gene X disrupted In tumor? YES Gene X not important for tumorigenesis NEVER Gene X REQUIRED for tumorigenesis Gene X = therapeutic target

30 Collaborations: Center Medical Genetics Ghent Annekatrien Boel Wouter Steyaert Andy Willaert Paul Coucke Dept. Pathology David Creytens Lab of Developmental Biology Ghent University Tom Van Nieuwenhuysen Hong Thi Tran Thomas Naert Rivka Noelanders Dionysia Dimitrakopoulou Suzan Demuynck