I Congreso Nacional de Anemias Raras, Barcelona, 20 -21 Set 2013

Anemia de Fanconi: el síndrome de los cromosomas de cristal

Dr. Jordi SurrallésCatedrático de Genética

Universitat Autònoma de BarcelonaGenome Instability Group: http://gig.uab.catGenome Instability Group: http://gig.uab.cat

LA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONILA ANEMIA DE FANCONI

-First described by Guido Fanconi in 1927

-Autosomal recessive (one subtype is X linked)

-A very rare disease with a frequncy of ~1/400.000

-highly hetyerogenous (genetically and clinically)

Ghido Fanconi con Andrea Lee Kuritzky Children's Hospital, Los Angeles, 1959

Disease evolution

Kutler et al., Blood, 2003

Dr.Eunike Velleuer (Dusseldorf) in action in Barcelona, March 2010

Xeroderma pigmentosum

Chromosome fragility in Fanconi anemia

Control

Fanconi

OVERLAPPING SYNDROMES

Inherited bone marrow failure syndromes: Dyskeratosis congenita, Diamond-Blackfan anemia, Shwachman-Diamond syndrome, severe congenital neutropenia, thrombocytopenia absent radii (TAR) syndrome, amegakaryocytic thrombocytopenia.amegakaryocytic thrombocytopenia.

Other overlapping syndromes:Baller-Gerold syndrome, Nijmegen breakage syndrome (MMC+) Rothmund-Thomson syndrome, Roberts syndrome (MMC+)Warsaw Breakage syndrome (MMC+)DK-phocomelia, VACTERL hydrocephalus syndrome, Wiskott-Aldrich syndrome

C. Group Gene Reference

FA-A FANCA Lo Ten Foe et al. Nature Genetics 1996

FABC Consortium. Nature Genetics 1996

FA-B FANCB Meetei et al Nature Genetics 2004

FA-C FANCC Strathdee et al. Nature, 1992

FA-D1 FANCD1/BRCA2 Howlett et al. Science, 2002

FA-D2 FANCD2 Timmers et al. Mol Cell, 2001

FA-E FANCE De Winter et al. Am. J. Hum Genet 2000

FA-F FANCF De Winter et al. Nature Genet. 2000

15 complementation Groups in 15 complementation Groups in FanconiFanconi AnemiaAnemia

FA-G FANCG De Winter et al. Nature Genet. 1998

FA-I FANCI Smogorzewska et al. Cell 2007

FA-J FANCJ/BRIP1 Levran et al. Nature Genet. 2005; Levitus et al. Nature Genet. 2005;

FA-L FANCL Ruhikanta et al. Nature Genet. 2003

FA-M FANCM Meetei et al Nature Genetics 2005

FA-N FANCN/PALB2 Reid et al Nature Genetics 2007

Xia et al Nature Genetics 2007

FA-O FANCO/RAD51C Vaz et al Nature Genetics 2010

FA-P FANCP/SLX4 Stoepker et al Nature Genetics 2011

Kim et al Nature Genetics 2011

Common phenotype in all genetic subtypes

FA-A FA-C

100

80

60

FANC-CFANC-G

FANC-FFANC-A

Rel

ativ

e su

rviv

al %

EGFP

Retrovirus-meditated genetic subtyping

1 10 100 1000

40

20

0

MMC (nM)

Rel

ativ

e su

rviv

al %

Genetic subtyping of 111 Spanish FA patients

D1

D26%

E3%

G4%

J3%

Unknown

8%

Retroviral subtyping, mutational screening, western blot

A74%

D11%

C1%

Callen et al., Blood 2005; Casado et al., J Med Genet 2007; Kalb et al Am J Hum Genet 2007; Castella et al Blood 2011

whole exome sequencing

ERCC4/XPF mutations and XPF-deficiency in Fanconi anemia patients (FANCQ)

c.2065C>A c.689T>C

c.1484_1488delCTCAA c.2371_2398dup28

Am J Hum Genet 2003

XPF cDNA genetically complements MMC sensitivity of FA104 lymphoblasts

XPF/ERCC4 mutations lead to three rare disorders: XP, XFE-progeria, and FA (FA-Q)

Xeroderma pigmentosum XFE-progeria Fanconi anemia

FANCQ alias for XPF

Interstrand crosslink repair (ICLR)

Fork stalling

Nucleotide excision repair (NER)

recognition

dual incision

UV

Unhooking

Homologous recombination

excision

DNA synthesis

ligation

NER

ICLR

NER

ICLR NER

ICLR

NER

ICLR NER ICLRNER ICLR

1 gene (XPF), 2 repair pathways, 3 syndromes

Xeroderma pigmentosumXeroderma pigmentosum Fanconi anemiaFanconi anemia ProgeriaProgeria

Bogliolo et al., Am J Hum Genet 2013

Skin cancer Leukemia and SCC Cancer?

C. Group Gene Reference

FA-A FANCA Lo Ten Foe et al. Nature Genetics 1996

FABC Consortium. Nature Genetics 1996

FA-B FANCB Meetei et al Nature Genetics 2004

FA-C FANCC Strathdee et al. Nature, 1992

FA-D1 FANCD1/BRCA2 Howlett et al. Science, 2002

FA-D2 FANCD2 Timmers et al. Mol Cell, 2001

FA-E FANCE De Winter et al. Am. J. Hum Genet 2000

FA-F FANCF De Winter et al. Nature Genet. 2000

FA-G FANCG De Winter et al. Nature Genet. 1998

16 complementation Groups in 16 complementation Groups in FanconiFanconi AnemiaAnemia

FA-G FANCG De Winter et al. Nature Genet. 1998

FA-I FANCI Smogorzewska et al. Cell 2007

FA-J FANCJ/BRIP1 Levran et al. Nature Genet. 2005; Levitus et al. Nature Genet. 2005;

FA-L FANCL Ruhikanta et al. Nature Genet. 2003

FA-M FANCM Meetei et al Nature Genetics 2005

FA-N FANCN/PALB2 Reid et al Nature Genetics 2007

Xia et al Nature Genetics 2007

FA-O FANCO/RAD51C Vaz et al Nature Genetics 2010

FA-P FANCP/SLX4 Stoepker et al Nature Genetics 2011

Kim et al Nature Genetics 2011

FA-Q FANCQ/ERCC4 Bogliolo et al Am J Hum Genet 2013

Spanish FANCA mutational spectrum

Point mutationPoint mutationSplicing mutationMicrodeletionMicroinsertionDeletion

130 mutations, 52 different, 20 novelCallén et al., 2005; Blood; Kalb et al, Am J Hum Genet 2007;

Castella et al., Blood 2011

HN-SCC Cancer

Bowel Ca.Colorectal Ca

Colorectal Ca.56 y.o

Lung Ca58 y.o.

Lung + Breast Ca, 74 y.o.

Cancer pedigree due to mutations in BRIP1/FANCJ found by exome sequencing

Cervical 33 y.o

Protate Ca. 60 y.o.

Prostate Ca58 y.o.

Kindey Ca

Brain Ca55 y.o

Dead at birth Down Synd.

Lymphoma70 y.o.

Fanconi anemiaIntrauterine growth retardation

Dead at birth Congenital malformations

Saviour babies: embryo selection for HLA matched

Molly Nash

Preimplantational genetic diagnosis with HLA-matching selection: savior babies

(38 cycles, 7 families)

524 oocytes

299 embryos299 embryos

75 healthy26 HLA compatible

16 tranferred to uterus

5 implanted (pregnancy)1 born

Barcelona, August 15th 2006

Bone marrow transplant increases cancer risck (SCC)

J Natl Cancer Institute, 2008

Blood CD34+ cells Selection

Gene therapy: Genetic Correction of Hematopoietic Stem Gene therapy: Genetic Correction of Hematopoietic Stem Cells from Patients with Fanconi anemiaCells from Patients with Fanconi anemia

Transduction with therapeutic lentiviral vectors

Infusion of transduced graft

“Natural” vs “medical” gene therapy

Mutation Back mutation (mosaics)Gene therapy

Mosaic FA patient

FA patientFA patient

Mosaicism

DEB test: ?DEB test: positive

DEB test: positive

Back mutation

Clonal expansion

Mosaicism often results in clinical improvement:

“natural” gene therapy

RE

LAT

IVE

VA

LUE

(%

)

Age (months)

Figure 3 Gene therapy of FANCD1 KO mice (BRCA2-/-)B

120

3 Gy

FA-D1Males

FA-D1Females

+ EGFP-LVs

+ BRCA2- LVs

3 Gy

0

20

40

60

80

100D

onor

Chi

mer

ism

(%) # 3

# 1# 4# 6# 5

# 7

# 9

# 8

1 2 3 4 5 6

Months after Bone Marrow Transplantation

0 10 30 MMC (nM)

CF

Cs

Sur

viva

l ( %

)# 1

# 3

# 4

# 5# 6

# 8# 7

# 90

20

40

60

80

100

120

MMC (nM)0 10 30

# 1

# 3

# 9# 7

# 8

# 6

# 4

# 5

2 months after BMT

0

20

40

60

80

100

120

CF

Cs

Sur

viva

l ( %

)

B

6 months after BMTA

Gene therapy of FANCD1 KO mice (BRCA2-/-)

FA-D1+ EGFP-LV FA-D1+ BRCA2-LVWT

- MMC

+ MMC

B

- +

WT

MMC

% A

berr

ant

Cel

ls

0

20

40

60

80

100

- +

FA-D1 + EGFP-LV

# 9

FA-D1 + BRCA2-LV

- +

# 4

Phase I/II Gene therapy trial of Fanconi anemia patients with a new Orphan Drug consisting of a lentiviral vector carrying the FANCA gene: A

Coordinated International ActionCoordinated International Action

Coordinator: Juan Bueren (Madrid)

� Fanconi anemia diagnosis.

� Diagnosis of the pathogenic mutations.

� Early diagnosis of myelodysplastic syndromes orleukemia .

WP1: To determine the genetic and hematopoietic characteristics of FA patients

leukemia .

� Diagnosis of mosaic patients with revertant mutationsaccounting for spontaneous hematological recovery.

� Subtyping of Fanconi anemia patients.

� Prediction of the hematopoietic reserve of thepatients.

AIM: To collect 4x10 6 CD34+ cells / kg of weight projected to 5 years.

WP2: To assess the safety and efficacy of an improv ed mobilization and HSC collection method based on a new mobilizatio n regimen

for FA patients with plerixafor and filgrastim.

• HSC mobilization: Filgrastim (10-12 µg / kg every 12 hours) for up to 7 days and plerifaxor (240 µg / kg) up to 4

days, 6 to 11 hours before starting apheresis

1 2 3 4 5 6 7 8 Days:

Apheresis

Mozobil

Filgrastim

WP3: To validate the safety and efficacy of the the rapeutic clinical-grade lentiviral vector

Antecedents:

cPPT Wpre*

GA

∆∆∆∆U3 R U5

PGK

SD SA

RRE

ψψψψFANCA

CMV R U5

PGK-FANCA.Wpre* LV

Orphan Medicinal Product Designation: EU 3/10/822

Lentiviral vector containing the Fanconi anemia A (FANCA) gene

WP3: To validate the safety and efficacy of the the rapeutic clinical-grade lentiviral vector

AIMs:

-Production of the therapeutic vector under GMP conditions.

- Manufacturing of the medicinal product, genetically

- Validation of the safety of the medicinal product.

- Manufacturing of the medicinal product, genetically modified FA-A CD34 + cells, under GMP conditions.

WP4: To assess the safety and efficacy of the infus ion of CD34 +

cells in FA patients, after transduction with the t herapeutic lentiviral vector

AIMTo demonstrate the safety and obtain the first evid ences of clinical efficacy associated to the infusion of the medicinal

product: Genetically modified autologous CD34 + cells.

� Patients complementation group: FA -A

Inclusion Criteria

Exclusion Criteria

� Patients with a HLA-identical related donor� Nº of cryopreserved or fresh CD34 + cells: <10 5 CD34+/kg weight

� Evidence of CD34 + cells transformation� Evidence of somatic mosaicism in HSCs associated wi th

hematological improvement

� Patients complementation group: FA -A� Moderate to severe aplasia

FutureFuture

IPScytokines Blood

3-4 transcription

FA gene (lenti)

Disease-corrected haematopoietic progenitors from Fanconi anemia induced pluripotent stem (iPS) cells

FA fibro Haematopoietic progenitors

3-4 transcription factors* (retro)

*KLF4, cMYC, OCT4, SOX2

Raya et al. Nature, 2009

DSB

mutation repair

DSB

save integration (save harbour)

acknowledgements:Juan Bueren (CIEMAT-Madrid)

Red Española de Anemia de Fanconi

Juan Carlos Izpisua-Belmonte (CMRB-Barcelona)

Javier Benítez (CNIO-Madrid)

Sheila Zúñiga (Sistemas Genómicos, Valencia)Sheila Zúñiga (Sistemas Genómicos, Valencia)

Arleen Auerbach (RU-New York)

Ruud Brakenhoff (VUMC-Amsterdam)

Detlev Schindler (Uni. Wursburg)

Johan de Winter (VUMC-Amsterdam)

Orlando Scharer (SBU-New York)

Koos Jaspers (EU-Rotterdam)

Surrallés’ lab, Barcelona, SpainDr. M. Bogliolo

Dr. L. Mina

Dr. J. Minguillón

Dr. R. Pujol

Dr. MJ Ramírez

Dr. J. Surrallés

Dr. G. HernándezDr. G. Hernández

Dr. M. Aza-Carmona

A. Molina

J.P. Trujillo

M. Marin

H. Montanuy

S. Sánchez

Genome Instability Group: www.gig.uab.cat