Fernando Larcher Laguzzi-Enfermedades raras de la piel
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Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
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Fernando Larcher Laguzzi-Enfermedades raras de la piel
- 1. Advances in Dystrophic Epidermolysis Bullosa treatment Dr. Fernando Larcher Simposio Internacional “Enfermedades raras de la piel: de la clínica al gen y viceversa” Madrid, 20-21 de Octubre de 2016
- 2. * Collagen VII immunofluorescence Healthy skin RDEB skin
- 3. Collagen VII CURRENT THERAPEUTIC APPROACHES FOR RDEB
- 4. CURRENT THERAPEUTIC APPROACHES FOR RDEB
- 5. CURRENT THERAPEUTIC APPROACHES FOR RDEB Clinical trials: • Allogeneic Bone Marrow • Allogeneic MSC
- 7. Bone Marrow Transplantation* 30 patients treated “A durable improvement in mucocutaneous integrity was demonstrated in 17 of 18 patients alive and engrafted” (77% survival)
- 8. CURRENT THERAPEUTIC APPROACHES FOR RDEB Fibroblasts Keratinocytes iPSC Other cell types
- 9. Autologous fibroblasts Genetically modified (C7) Autologous fibroblasts Genetically modified (C7) Combined cell (suspension) and gene therapy trials US UK
- 10. Ex vivo gene therapy (grafting) trials for RDEB
- 11. Phase I/II ex vivo gene therapy clinical trial for recessive dystrophic epidermolysis bullosa using skin equivalent grafts genetically corrected with a COL7A1 Project website: http://www.genegraft.eu/ A. Hovnanian Ex vivo gene therapy (grafting) trials for RDEB
- 12. Retroviral construct: pLZRS-ColVII 1 2 3 217 kD 123 kD gf e RDEB-HK Corrected RDEB-HK 5’LTR Col VII cDNA 3’LTRIres Zeo WB ColVII Fibroblastos Grafting HK-RDEBcol7 HK-RDEB Col 7 Col 7 DAPI DAPI Biengineered skin
- 13. GMP production of the genetically modified skin equivalent
- 14. Collagen VII CURRENT THERAPEUTIC APPROACHES FOR RDEB Clinical trial: Drugs that induce readthrough of nonsense COL7A1 mutations (e.g. gentamicin)
- 15. DNA Stop (PTC) U DNA nonsense mutations
- 16. Gentamicin
- 17. 8179 Collagen VII* 81791-77 78 82-118 mARN pre-ARNm 8178 79 801 11880 MUTATION ADN / pre-mRNA 80 81791-77 78 82-(PTC)80 mARN No Collagen VII AON
- 18. Other ex vivo gene therapy approaches involving gene addition in experimental stage RNA Trans-splicing COL7 COL7 COL7 ͠ 15% of RDEB keratinocytes transposed Transposon-transposase
- 19. > 600 different COL7A1 mutations – AD or AR Uitto et al., 2002. J Investig Dermatol Symp Proc COL7A1 MUTATIONS IN DEB
- 20. COL7A1 1 118 c.6527insC (PTC) > 40 patients up to 2015
- 21. Exons 1-78 Exons 82-118Exon 79 mutant exon 80 Exon 81 No Collagen VII Mutant COL7A1 c.6527insC in exon 80: frame-shift lmutationleading to a PTC Recurrent mutation Targeted therapy
- 22. CRISPR/Cas9 Clustered regularly interspaced short palindromic repeats Gene editing by targeted nucleases
- 23. TALENs design for COL7A1 exon 80 editing 12827F 12874R DAPI GFPAdTALEN
- 24. Correction of RDEB-E67A6 cell line by HDR. HDR RDEB-E67A6 TALEN Ad.12827F TALEN Ad.12874R Template DNA AAV-PGK-NEO (p35) 20 x (p100) pass Selection of resistant clones + G418 (15 days) Ring cloning of colonies and cell expansion (p60)
- 25. Gene editing (NHEJ) by targeted nucleases
- 26. Exons 1-78 Exon 79 mutant exon 80 Exon 81 Exons 82-118 Exons 1-78 Exon 79 edited exon 80 Exon 81 Exons 82-118 No Collagen VII Collagen VII Gene editing strategies using TALEN or Crispr/Cas9 nucleases Mutant COL7A1 edited COL7A1 (re-framed or skipped)
- 27. Clones analyzed: 113 Clones containing indels: 30 (27 %) Cell line RDEB-E67A6 Ad.12827F Ad.12874R Cloning by límit dilution p60 Gene editing in RDEB-E67A6 cell line by TALEN.mediated NHEJ NHEJ
- 28. ACTIN COL7A1 NHEJ-Indels leading to COL7A1 frame restoration COL7A1 expression NHEJ gene-edited keratinocytes NHEJ clone 8 clone 124 clone 27 clone 51 C7 IF
- 29. Grafting Fibroblastos SKIN-HUMANIZED MOUSE RDEB Fibroblasts Bioengineered skin COL7A1-edited Keratinocyte clones Ad TALEN A Ad TALEN B In vivo analysis
- 31. Exons 1-78 Exon 79 mutant exon 80 Exon 81 Exons 82-118 Exons 1-78 Exon 79 edited exon 80 Exon 81 Exons 82-118 No Collagen VII Collagen VII Gene editing strategies using TALEN or Crispr/Cas9 nucleases Mutant COL7A1 edited COL7A1 (re-framed or skipped)
- 32. Exons 1-78 Exons 82-118 Exons 1-78 Exon 79 Exon 81 Exons 82-118 Exon 79 Mutant exon 80 Exon 81 Gene editing strategies using TALEN or Crispr/Cas9 nucleases No Collagen VII Collagen VII* Mutant COL7A1 edited COL7A1 (exon 80 excised)
- 33. ColVII K14 DAPI Collagen VII IF COL7A1-edited polyclonal primary RDEB culture Preliminary results: NHEJ correction of patient c.6527insC RDEB keratinocytes 2 clones isolated ΔG Hmz ΔG Htz
- 34. www.moleculartherapy.org vol. 22 no. 11 november 2014 vol.15 no. 9 september 2007
- 35. Collagen VII CURRENT THERAPEUTIC APPROACHES FOR RDEB Changes in ECM and fibroblasts: Fibrosis, inflammation, cancer TGFβ, ??
- 37. MUCHAS GRACIAS