Cicle de Conferències en Malalties Rares, VHIR, Barcelona, 16 Des 2014

Rare diseases as platform to develop

novel therapeutic strategies: the

example of Fanconi anemia

Dr. Jordi Surrallés

Catedràtic de Genética

Professor ICREA-Acadèmia

Universitat Autònoma de Barcelona

Genome Instability Group: http://gig.uab.cat

Fanconi anemia

http://www.malaltiaamagada.com

A tale: Arnau has a hidden disease

Garaycoechea and Patel (2014) Blood 123:26-34

Bone marrow failure in Fanconi anemia

Disease evolution in Fanconi Anemia

Kutler et al., Blood, 2003

Chromosome fragility in Fanconi anemia

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

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

15+1 complementation Groups in Fanconi Anemia

Interstrand crosslink repair (ICLR)

Fork stalling

Unhooking

Translesion sythesis

Recombination

1 10 100 1000

100

80

60

40

20

0

FANC-C

FANC-G

FANC-F

FANC-A

MMC (nM)

Re

lati

ve

su

rviv

al

%

Empty

Retroviral subtyping of FA

Genetic subtyping of 143 Spanish FA patients

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

Unassigned

whole exome sequencing

501500

498 499

FA 287

Mutations in the XPF nuclease lead to three

rare disorders: XP, XFE-progeria and FA

Xeroderma pigmentosum XFE-progeria Fanconi anemia

FANCQ alias for XPF in HUGO

Bogliolo et al., 2013, Am J Hum Genet

Interstrand crosslink

repair (ICLR)

Fork stalling

Unhooking

Homologous recombination

Nucleotide excision

repair (NER)

recognition

dual incision

excision

DNA synthesis

ligation

UV

NER

ICLR

Xeroderma pigmentosum

NER

ICLR

Xeroderma pigmentosum

NER

ICLR

Fanconi anemia

NER

ICLR

Fanconi anemia

NER ICLR

Progeria

NER ICLR

Progeria

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

Bogliolo et al., Am J Hum Genet (2013)

UV skin sensitivity anemia Anemia + UV skin

sensitivity

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

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

16 complementation Groups in Fanconi Anemia

DNA damage response

Ataxia Telang.

Nijmegen

Fanconi

Bloom

Seckle

Breast cancer risk in BRCA1/2 mutation carriers

April 2005

Synthetic lethality in cancer therapeutics

Synthetic lethality in cancer treatment

Lord and Ashworth BMC Biology 2010 8:38 doi:10.1186/1741-7007-8-38

1 10 100 1000

100

80

60

40

20

0

FA disrupted

tumor cell

Tumor cell

Cisplatin concentration

Re

lati

ve

su

rviv

al

%

Chemo-sensitizing tumor cells through

disruption of the FA/BRCA pathway

Lyakhovich and Surralles, 2009

From genes to therapy

-Bone marrow or umbilicar cord blood

transplant

-Preimplantational embryo selection

-Gene therapy?

-Stem cell based regenerative medicine???

Dr. Eliane Gluckman and Matthew Farrow

1988

1993

Dr. Pablo Rubinstein and Mitch Santa

Leukemia

Metabolic

syndromes

Saviour babies to cure FA

Molly Nash

Barcelona, August 15th 2006

Trujillo and Surralles, in press

Savior babies and Fanconi anemia: low success

rates after 38 trials in 7 families

Gene therapy of severe immunodeficiencies

Fancostem/Fancolen team

C. Díaz de Heredia

Vall Hebron

J. Surrallés

UAB

J. Bueren

CIEMAT

J. Sevilla

Niño Jesús

Phase I/II Gene therapy trial of Fanconi anemia

patients with a new Orphan Drug consisting of a

lentiviral vector carrying the FANCA gene

Coordinator: Juan Bueren (Madrid)

http://www.eurofancolen.eu

EuroFancoLen Project

WP1 WP2 WP3 WP4

J. Surralles A. Thrasher M. CavazzanaA. Galy and F. Mavilio

Transduction with therapeutic

lentiviral vectors

blood mobilization

Plerixafor+filgrastim

CD34+ cells

Selection

Infusion of transduced graft

Gene therapy: Genetic Correction of

Hematopoietic Stem Cells (CD34+)

cPPT Wpre*

GA

U3 R U5

PGK

SD SA

RRE

yFANCA

CMV R U5

PGK-FANCA.Wpre* LV

Orphan Medicinal Product Designation: EU 3/10/822

Lentiviral vector containing the Fanconi anemia A (FANCA) gene

Lentiviral vector: better and safer

Figure 3

Navarro et al., 2008, Blood; Rio et al., 2009, Blood

It works in mice

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

“Natural” vs “medical” gene therapy

Mutation Reverse mutation (mosaics)

After gene therapy

Mosaicism often results in fast clinical improvement

hemoglobin

platelets

Des 2008

T=G

Oct 2013

T>>>G

Feb 2011

T>G

c.3335T>G

Future

FA fibro

IPS

cytokines Blood

Haematopoietic

progenitors

3-4 transcription

factors* (retro)

*KLF4, cMYC, OCT4, SOX2

FA gene (lenti)

Raya et al. Nature, 2009

Disease-corrected haematopoietic progenitors from

Fanconi anemia induced pluripotent stem (iPS) cells

Disease-free cardiomyocytes from Fanconi anemia skin

Nature 460:53-59

Making blood cells from IPS cells

Liu et al., Nature Communications, 2014

Modeling Fanconi Anemia

therapeutics using patients iPSCs

Liu et al., Nature Communications, 2014

DSB

DSB

mutation repair (genome editing or

homologous recombination)

save integration (save harbour)

Nucleases

(ZFN, TALEN, CRISPR)

Fixing rare diseases by genome editing

Spanish FANCA mutational spectrum

Point mutationSplicing mutation

Microdeletion

MicroinsertionDeletion

Callén et al., 2005; Blood; Castella et al., Blood 2011

C>T missense mutation

Genome editing to correct a FANCA mutation

by homologous recombination

(helper dependent adenoviral vectors targeting FANCA)

FA Crt FA-CFA Crt FA-C

Liu et al., Nature Communications, 2014

Save harbour: the AAVS1 locus and ZFN

Rio et al., EMBO Mol Med, 2014

Harvest of

CD34+ cells

Infusion of

genetically-

corrected Cells

PATIENT

Gene therapy in the future

Freezing of

CD34+ cells

Genome

editing

Why research on rare genetic diseases?

• First umbilical cord blood transplant

• First savior baby

• Among the first gene therapy trials

• First generated disease-free IPS

• Model to understand fundamental biological processes (genome stability, cancer, ageing, pluripotency…)

• Tool to find new cancer drugs and new cancer genes

Surrallés’ lab members, BarcelonaDr. M. Bogliolo

Dr. L. Mina

Dr. J. Minguillón

Dr. R. Pujol

Dr. MJ Ramírez

Dr. J. Surrallés

Dr. G. Hernández

Dr. M. Aza-Carmona

A. Molina

M. Marin

H. Montanuy

M. Peiró

Genome Instability Group: www.gig.uab.cat