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Natural gene therapy, a healing source
Natural gene therapy, a healing source Marcel Jonkman Groningen, the Netherlands m.f.jonkman@umcg.nl umcg DEBRA International congress 2014, Paris, 19-21 September 2014 Natural gene therapy, a healing source 1. Revertant mosaicism 2. Revertant cell therapy umcg DEBRA International congress 2014, Paris, 19-21 September 2014 Revertant mosaicism (RM) What is it? What are the underlying mechanisms? Clinical incidence and revertant mutation probalility? Why does a revertant skin patch stops growing? How can I recognise revertant skin? Somatic mutations Germline: wild type Germline: mutant Forward mosaicism (nevus) Revertant mosaicism What is revertant mosaicism? Junctional EB Jonkman, Arch Dermatol 1996 What is revertant mosaicism? Junctional EB Collagen 17 deficient Jonkman, Arch Dermatol 1996 What is revertant mosaicism? Jonkman, Jonkman,Arch ArchDermatol. Dermatol1996 1996 Revertant red sectors in mutant blue petunia Wild type Mutant Faraco, Cell Reports 2013 The secret of the blue petunia Faraco, Cell Reports 2013 1996, Jefferson University (Philadelphia) Leena Pulkinnen Marcel Jonkman Jouni Uitto Found the mutation and reversion in COL17A1 Junctional EB Collagen 17 deficient COL17A: c.3781C>T / c.1706delA Jonkman, Arch Dermatol 1996 Jonkman, Cell 1997 What is the mechanism of RM? normal mitosis Jonkman, Cell 1997 What is the mechanism of RM? normal mitosis gene conversion Jonkman, Cell 1997 Homologous recombination by mutant KRT10 Ichthyosis in confetti Courtesy of dr Keith Choate, Yale Choate, Science 2010 What is the mechanism of RM? normal mitosis gene conversion homologous recombination Pasmooij, Discovery Med 2012 EB 093-01 Male 1928 COL17A: c.4424-5insC / c.3781C>T c.3781T>C Pasmooij, Am J Hum Genet 2005 What is the mechanism of RM? normal mitosis gene conversion homologous recombination back mutation Pasmooij, Discovery Med 2012 EB 026-01 2005 Pasmooij, Am J Hum Genet 2005 COL17A1 gene in revertant ankle patch Germline mutations: c.3781C>T are present Second site mutation: c.3782G>C abolishes stop codon wild type mutant CCT CGA GGG Pro Arg Gly CCT TGA GGG Pro Stop Gly revertant CCT TCA GGG Pro Ser Gly Pasmooij, Am J Hum Genet 2005 What is the mechanism of RM? normal mitosis gene conversion homologous recombination back mutation second-site mutation Pasmooij, Discovery Med 2012 Revertant mosaicism in inherited diseases Year Gene Disease Cell/tissue 1988 HPRT Lesch-Nyhan erythrocyte, fibroblast 1992 DYS Duchenne muscular dystrophy muscle fiber 1994 FAH Tyrosinaemia type I hepatocyte 1995 BLM Bloom syndrome B-lymphoid 1996 ADA IL2RG SCID T-lymphoid CMT1A Charcot-Marie-Tooth blood, nerves, hair root, buccal mucosa COL17A1 Junctional EB keratinocyte KRT14 EB simplex LAMB3 Junctional EB COL7A1 Dystrophic EB KRT10 Ichthyosis in confetti FRMT1 Kindler syndrome TERC Dyskeratosis congenita FANCA FANCC Fanconi anemia B-lymphoid RAG-1 Cd3ζ (CD247) 1996 1997 FANCD2 2001 WAS Wiskott-Aldrich syndrome T-lymphoid 2004 NEMO X-linked ectodermal dysplasia and T-lymphoid immunodeficiency 2007 CD18 Leukocyte adhesion deficiency type 1 T-lymphoid 2009 MLH1 Lynch syndrome (HNPCC) blood, skin, hair, intestines 2014 JAK3 Combined immunodeficiency lymphocyte Junctional EB EB 035-01 Male 1958 COL17A: c.2342delG / c.3781C>T, p.R1226X 2004 2007 Pasmooij, J Invest Dermatol 2012 BP180 Mutant 2342delG 07-137 07-136 Revertant 07-130 Revertant 07-055 Revertant Pasmooij, J Invest Dermatol 2012 BP180 Mutant 2342delG 07-137 07-136 Revertant second locus mutation 07-130 Revertant second locus mutation 07-055 Revertant back mutation or gene conversion Pasmooij, J Invest Dermatol 2012 What is the incidence of RM? Pasmooij, J Invest Dermatol 2012 EB 035-01 COL17A1-related junctional EB: 1. The incidence of RM is 100% 2. Multiple patches of RM Pasmooij, J Invest Dermatol 2012 COL17A1 related junctional EB: 1. The incidence of RM is 100% 2. Multiple patches of RM Pasmooij, J Invest Dermatol 2012 EB 026-01 Disseminated revertant mosaicism in Kindler syndrome FERMT1: c.456dupA FERMT1: c.676dupC slipped mispairing of duplicationn deletes one nucleotide Kiritsi, J Clin Invest 2012 What is the probability that a nucleotide is reverted? 1∙10-9 per nucleotide point mutation rate per cell division. 36∙109 – 1 number of mitosis needed to obtain total number of basal keratinocytes. 100% probability for revertant mutation in a skin stem cell. Van den Akker, Thesis 2013 Selection advantage determines expansion Youssoufian. Nat Rev Genet 2002 The revertant patches are not confined to the lines of epidermal development, i.e. the lines of Blaschko (Blaschko, 1901; Happle, 1984). Reverse mutations occur when epidermal formation from Blaschko-lines is completed, i.e. after the end of the 4th week (Loomis et al., 2008). Van den Akker, Thesis 2013 Timing and visibility No advantage Selection advantage Conception Generation 30 Probability for revertant mutation ≈ 1 Birth Generation 41 Revertant patch not visible Revertant patch visible Van den Akker, Thesis 2013 RM patch fading up in infancy 2002 2009 2012 Revertant patches stop growing after infancy 2 years 8 years 19 years Ichthyosis in confetti Courtesy of dr Keith Choate EB 026-01 No clear expansion in adulthood 1981 1994 2005 Pasmooij, Am J Hum Genet 2005 EB 208-01 No clear expansion in adulthood Mar 2008 Mar 2008 Nov 2008 Jan 2011 Jonkman & Pasmooij, N Eng J Med 2009 How can I recognise revertant skin? Ask the patient COL17A1 Preserved skin structure COL7A1 Wearing juwelry COL17A1 Pigmentation COL17A1 Preserved skin structure Van den Akker, JAMA Dermatol 2012 How can I recognise revertant skin? Ask the patient COL17A1 Wearing juwelry COL17A1 Preserved skin structure Rub COL7A1 COL7A1 Pigmentation COL17A1 Ballpoint rub test Kiritsi, J Invest Dermatol 2014 How can I recognise revertant skin? Ask the patient COL17A1 Wearing juwelry COL17A1 Preserved skin structure Rub COL7A1 COL7A1 Pigmentation COL17A1 Not identifiable KRT14 Not all revertant mutations are helpful Some open Pandora’s Box Keratitis-ichthyosis- deafness syndrome Ogawa, Plos Genet 2014 Revertant cell therapy: using natural gene therapy as source for healing Autologous revertant cell therapy 1. Non-cultured skin graft transplantation 2. Cultured epidermal sheet 3. Induced Pluripotent Stem Cell technology (iPS cells) Minigrafting of revertant skin in junctional EB (LAMB3) Gostyński, J Am Acad Dermatol 2014 Expansion of cells in culture 1. Non-cultured skin graft transplantation 2. Cultured epidermal sheet 3. Induced Pluripotent Stem Cell technology (iPS cells) Gostyński, Br J Dermatol 2009 Day 0: 0.1 mm deep superficial incision 2003 Day 0: epidermal stripping of acceptor site Gostyński, Br J Dermatol 2009 Day 0: looking at the nude lamina densa Gostyński, Br J Dermatol 2009 Day 0: grafts in situ Gostyński, Br J Dermatol 2009 Day 5: grafts on carrier gauze in situ Gostyński, Br J Dermatol 2009 Day 14: removed gauze Gostyński, Br J Dermatol 2009 4 months: completely healed Gostyński, Br J Dermatol 2009 4 months: adhesive strip test fragile skin < 3% of the graft was revertant Gostyński, Br J Dermatol 2009 Loss of revertant cells after culturing % of revertant cells: 40 % 25 % 3% 0% Gostyński, Br J Dermatol 2009 Revertant human epithelial graft in mouse model Col17 Mouse Human Col17 Gostyński, J Invest Dermatol 2013 It is normal for all humans to deplete the stem cell population with age The blood of a 115-year-old Dutch woman was derived from only two related stem cells. Hendrikje van Andel Holstege, Genome Res 2014 Fig. 1 A Stem cells can be induced B 1. Non-cultured skin graft transplantation 2. Cultured epidermal sheet 3. Induced Pluripotent Stem Cell technology (iPS cells) Col17 DAPI C Minnesota trial: allogenic bone marrow transplantation in EB After HCT pre Day 200 60x Before After HCT Day 360 Courtesy: Jakub Tolar Before Tolar, N Eng J Med 2010 Patient: XX Female Donor: XY Male Donor CD45-/CD31 cells. Donor CD45-/CD31 cells. Donor skin cells. Chimeric Skin Tolar, Lancet 2013 First HCT for EB in Europe Centre for Blistering Diseases, Groningen Pediatric Blood and Bone Marrow Program, Utrecht International Expert Advisory Panel Christine Bodemer Jaap Jan Boelens Marcel Jonkman Anna Martinez John McGrath Jakub Tolar From skin via blood to skin Autologous revertant iPSC hematopoietic stem cell transplantation mutant revertant Revertant keratinocytes iPSC Revertant hematopoietic stem cells hematopoietic reprogramming Acknowledgements Groningen, NL Madrid, Spain Marjon Pasmooij Maria Jose Escamez Antoni Gostyński Marta Garcia Kasia Gostyńska Fernando Larcher Peter van den Akker Marcela del Rio Utrecht, NL New York, USA Jaap Jan Boelens Noriko Umegaki-Arao Angela Chirstiano Freiburg, Germany Dimitra Kiritsi Philadelphia, USA Cristina Has Leena Pulkinnen Leena Bruckner-Tuderman Jouni Uitto Contact Marcel Jonkman m.f.jonkman@umcg.nl
