Natural gene therapy, a healing source

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Natural gene therapy, a healing source
Natural gene therapy, a healing source
Marcel Jonkman
Groningen, the Netherlands
[email protected]
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
[email protected]