TRANSGENIC RAT Knock- out for CFTR

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Fabienne Lafleur
il y a 8 ans
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1 TRANSGENIC RAT Knock- out for CFTR In silico design and valida+on of sgrna func+onality in cellulo (rat C6 cells) U6 sgrna CAG Human op$mized cas 9 bghpa FLAG Nls 3X SV40 Nls SV40 T7 endonuclease I (T7EI) muta+on detec+on assay % coupure C6 NT CFTR1.1 CFTR1.2 CFTR1.3 CFTR

2 TRANSGENIC RAT Knock- out for CFTR In vitro transcrip+on of sgrna Gene invalida+on by KI NHEJ KO by NHEJ ODN 60 nt Xba1, TGA Embryo micro- injec+on : Cas9+sgRNA+ ODN Xba1, TGA KO by KI FO: 11 (+) out 32 animals (34%) (sgrna: 13% ac+vité in vitro)

3 TRANSGENIC RAT : Gene marking Design of the strategy Endogenous promoteur Endogenous 3 UNT In vitro valida+on of sgrna candidates in C6 (TE71 assays) Embryo micro- injec+on : Cas9+sgRNA+ donor Fo: 2 (+) out 40 (5%) (sgrna: 55% ac+vité in vitro) % indel F1: wai+ng for pups

4 CRISPR/Cas: stratégie antivirale anti-hcmv Réplication Gancyclovir, Cidofovir, Foscarnet, Fomivirsen, CMX001, Maribavir, cyclopopavir, Artesunate Ciblent des molécules similaires Emergence de souches résistantes Fixation Fusion IVIg, CF102 Différents récepteurs en fonction de la cible Education immunitaire Vaccin anti-gb+mf59 DNA vaccin (pp65 and gb) DLI LT (CD4-CD8) anti-cmv Pas d action sur les pools latents Invalidation du gène IE Stratégie novatrice et complémentaire Nouvelle cible : l ADN viral Action sur des virus réplicatifs et LATENTS Fabienne Haspot UMRS 1064

5 CRISPR/CAS anti-hcmv : Block hcmv reac$va$on and replica$on Preliminary results D0: 1 st transfec$on with grna/ cas9 plasmid D1: 2 nd transfec$on with prc/cmv target gene Plasmid extrac$on PCR T7 assay Mismatches digested in the sample from anti-hcmv grna/cas9 treated cells and not in the irrelevant or Cas9 alone treated sample Selected grna/cas9 are func$onal

6 human ES, induced Pluripotent Stem cells (ips) Source for almost all cells of the body (ips princeps : Yamanaka et al., cell 2006)

7 Personalized regenera+ve medecine using autologous ips Treatment of metabolic liver diseases CN1 (UGT1A1 -/- ) Crigler-Najjar 1 (CN) Patients 1) skin biopsy Fibroblastes 2) ips Reprogramming factors: SOX2, OCT4, KLF4, c-myc ips UGT1A1 + ips 3) Targeted gene correction (genome editing using artificial nuclease) UGT1A1 + hepatocytes 4) Differentiation into hepatocytes 5) Cell transplantation UTG1A1-deficient Rat

8 Personalized regenera+ve medecine using autologous ips Gene edi+ng strategy: Integra+on of a therapeu+c cassepe in «safe harbor locus» AAVS1

9 Personalized regenera+ve medecine using autologous ips Preliminary results ZFNs, TALEN : no puromycin resistant clones

10 Maladies cardio- vasculaires héréditaires : l ère des ips spécifiques Etude d une arythmie cardiaque ventriculaire Dysplasie arythmogène du VD Syndrome LEOPARD Syndrome d Hamamy Syndrome de Brugada Enjeux : Evalua+on pré- thérapeu+que Mise en évidence de nouveaux acteurs thérapie cellulaire Mercola et al., Circ Res. 2013;112(3): N. Gaborit, UMR1087

11 Genome edigng grâce à CRISPR/Cas: Apports à l étude d une arythmie cardiaque ventriculaire Prélèvements contrôle Reprogramma+on Pa$ent aeeint de pathologie cardiaque Complexe SgARN/Cas9 : U6 sgrna NLS Cas9 ips Plasmide donneur: Edi+on Bras 5 GFP PGK puro Bras 3 ips- GFP Différencia+on Tri cellulaire (FACS ) ips- CM- GFP ventriculaires Contrôle ips- CM- GFP Phenotype SB? MYL2 Exon Bras 5 Codon stop GFP PGK puro Bras 3 Inser+on du gène de la GFP sous contrôle du promoteur de MYL2, d expression ventriculaire.

12 Premiers résultats µm sgrna MYL2- GFP- puro Caractérisa$on avant tri cellulaire: prédominance de cardiomyocytes auriculaires Transfec$on ips Mlc2v Mlc2a Diﬀérencia$on d ips éditées en cardiomyocytes 50 µm

13 Summary CRISR/Cas gene edi+ng Very easy to program (guide RNA: 20 nt) Very fast to generate (cloning of the guide RNA) Very cost- effec+ve Highly- efficient Works in almost all species Broad applica+ons Animal models : New models, (cys+c fibrosis, immunology) In vitro cell models : KO/KI for analysis of gene, primary cells, ips An+- viral therapie: CMV (cell therapy), PERVs (pig KO for xenotransplanta+on) Cell and Gene therapy: Gene correc+on, phenotype correc+on, IPS Biotechnology : Producer cell lines (transgenes introduced by KI) Imaging: gene marking (luciferase, GFP, ) Target disease : Cardiopathy, Crigler- Najjar, Hun+ngton, An+- bacterial therapy : phage therapy

14 Future Direc+on Delivery is an enormous challenge - Transgenesis: direct injec$on of sgrna, cas 9 mrna, Donor DNA into embryo - Cell lines : not all transfectable - Primary cells, animals: transfec$on is inefficient Specificity is s+ll something to work - Predic$on of on- target and off- target ac$vity (sgrna design, type of cell, delivery) - modified Cas9 : Cas 9 nickase - truncated sgrna (17/18 nt long sgrna) - Controlling cas 9 enzyma$c reac$on : sgrna/cas9 concentra$on dura$on of the ac$vity New applica+on of CRISPR/Cas9 CRISPR/Cas transcrip$on ac$vator CRISPR/Cas transcrip$on inhibitor Epigenome edi$ng.

15 Acknowledgement Inserm U1064, Team 2 hipsc liver, CFTR Tuan Huy Nguyen Angélique Fourrier Claire Poiron Jessica Bellec Frederic Delbos Transgenesis plateform Rat KO/KI Ignacio Anegon Laurent Tesson Séverine Ménoret Séverine Rémy Claire Usal Inserm U1064, Team 1 An+- CMV Fabienne Haspot Franck Halary Flora Coulon ips pla{orm, SFR Bonamy hipsc L.David P. Lemarchand A. Derevier A. Gaignerie INSERM U565 TALEN, CRISPR JP Concordet C Giovannangeli Laboratory of Clinical Inves+ga+on III, Geneva CFTR Marc Chanson Inserm UMR 1087 / CNRS UMR 6291 and collaborators hipsc heart N. Gaborit P. Lemarchand X. Latypova Others Marta CRISPO (Ins+tut Pasteur de Montevideo, Uruguay): Merino Sheep Castro Maria (University of Michigan) : adenoviral vector CRISPR cas Jeremy BELLIEN (CHU Rouen): rat, introduc+on of a speciﬁc single muta+on in endogenous gene Laure Delestre (crc, Jussieu): rat, introduc+on of 2 speciﬁc muta+ons in endogenous gene Cedric Louvet (U1064): mice, immunology

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