MITOCHONDRIAL DNA DEPLETION SYNDROME: TWO GENOMES TALKING

CRG-VHIR ENCOUNTER ON RARE DISEASESBarcelona, January 18th, 2012

Ramon MartíLaboratori de patologia neuromuscular i mitocondrial

Vall d’Hebron Institut de Recerca

Intergenomic communication

mtDNA replicationenzyme machinery and

substrates

Molecular phenotype on mtDNA-Depletion

-Multiple deletions

-mtDNA somatic point mutations

OXPHOS dysfunction

gene protein function disease reference

TYMP TP dNTP metabolism MNGIE Nishino et al. 1999

ANT1 ANT1 ADP/ATP carrier PEO Kaukonen et al. 2000

DGUOK dGK dNTP metabolism MDS Mandel et al. 2001

TK2 TK2 dNTP metabolism MDS Saada et al. 2001

POLG pol ɣ(catalitic subunit) polymerase PEO, Alpers Van Goethen et al. 2001

C10orf2 Twinkle helicase PEO Spelbrink et al. 2001

SUCLA2 SCS-A(β subunit) dNTP metabolism MDS Elpeleg et at. 2005

POLG2 pol ɣ(ancillary subunit) polymerase PEO Longley et al. 2006

MPV17 MPV17 unknown MDS Spinazzola et al. 2006

RRM2B p53R2 dNTP metabolism MDS Bourdon et al. 2007

SUCLG1 SCS-A(α subunit) dNTP metabolism MDS Ostegaard et al. 2007

Genes involved in mtDNA depletion / deletions syndromes

Mutations in TYMP

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE)

-OMIM: #603041; MITOCHONDRIAL DNA DEPLETION SYNDROME 1 (MNGIE TYPE); MTDPS1.-Orphanet: ORPHA298

Thymidine phosphorylase catalizes the first step of thymidine and deoxyuridine catabolism

Thymidine

Deoxyuridine

Pi

TP

2-deoxyribose1-phosphate thymine

uracil

Homodimer TP

Thymidine and deoxyuridine in plasmaHPLC assay

control

MNGIEpatient

Martí et al, Biochem Biophys Res Commun 2003

dThddCMPdTMP dTDP dTTP

dCDP dCTPdCtd

dAdodGMPdAMP dADP dATP

dGDP dGTPdGuo

dThd dTMP dTDP dTTPThyTP

DEPLETION MULTIPLE DELETIONS SOMATIC SITE-SPECIFIC POINT MUTATIONS

• MITOCHONDRIAL DYSFUNCTION

• Gastrointestinal dysmotility and cachexia

• Ptosis, ophthalmoparesis, or both

• Peripheral neuropathy

CLINICAL PHENOTYPE

1-Studies on biochemical pathomechanisms accounting for mtDNA depletion in MNGIE.

(mtDNA depletion is the most relevant molecular defect associated to this disorder)

2-Preclinical studies on gene therapy using a lentiviral vector

1-Studies on biochemical pathomechanisms accounting for mtDNA depletion in MNGIE.

(mtDNA depletion is the most relevant molecular defect associated to this disorder)

2-Preclinical studies on gene therapy using a lentiviral vector

González-Vioque et al, PloS Genet 2011, 7: e1002035

González-Vioque et al, PloS Genet 2011, 7: e1002035

González-Vioque et al, PloS Genet 2011, 7: e1002035

dThddCMPdTMP dTDP dTTP

dCDP dCTPdCtd

dAdodGMPdAMP dADP dATP

dGDP dGTPdGuo

dThd dTMP dTDP dTTPThyTP

González-Vioque et al, PloS Genet 2011, 7: e1002035

González-Vioque et al, PloS Genet 2011, 7: e1002035

Deoxycitydine supply prevents mtDNA copy-number decay induced by thymidine excess in quiescent human cultured cells

Limited dCTP availability, secondary to thymidine overload, accounts for mtDNA depletion in MNGIE

González-Vioque et al,PloS Genet 2011, 7: e1002035

CONCLUDING REMARKS (I)

1-Studies on biochemical pathomechanisms involved accounting for mtDNA depletion in MNGIE.

(mtDNA depletion is the most relevant molecular defect associated to this disorder)

2-Preclinical studies on gene therapy using a lentiviral vector

Gene therapyAlloHSCT

1Kb EcoRV XbaI EcoRV UncutDNA +XbaIladder

10Kb 10Kb 8,5Kb

2,2Kb

TPhPGK

TPIRES

0

p305-TP

1Kb EcoRI BamHI UncutDNAladder

6Kb

1,9Kb

0,55Kb

8,5Kb hPGKIRES

BamHI

0

p305-Sham

GENERATION OF LENTIVIRAL VECTORS

Expression of TYMP in transduced B-LCL

Torres-Torronteras et al, Gene Ther 2011, 18:795-806

Function of the gene product

Torres-Torronteras et al,Gene Ther 2011, 18:795-806

Tymp-/- Upp1-/- Tymp-/- Upp1-/-

Isolation of progenitor hematopoietic cells

Myelosuppression (irradiation)

Transduction with lentiviral vector carrying TYMP

Infusion of gene-corrected cells

Study of the phenotype

TP function in vivo

Torres-Torronteras et al, Gene Ther 2011, 18:795-806

TP-treated sham-treatedTP-treated

wt ko ko-sham ko-tpGroup

0

5

10

15

Thd

(µM

)

PLASMA

wt ko ko-sham ko-tpGroup

0

20

40

60

80

100

Thd

(pm

oles

/mg

prot

)

LIVER

wt ko ko-sham ko-tpGroup

0

200

400

600

800

1000

Thd

(pm

oles

/mg

prot

)

SPLEEN

wt ko ko-sham ko-tpGroup

0

50

100

150

200

250

300

Thd

(pm

oles

/mg

prot

)

SKELETAL MUSCLE

wt ko ko-sham ko-tpGroup

0

100

200

300

400

500

600

Thd

(pm

oles

/mg

prot

)

SMALL INTESTINE

wt ko ko-sham ko-tpGroup

0

50

100

150

200

250

300

Thd

(pm

oles

/mg

prot

)

BRAIN

Thymidine levels 6 months after gene therapy (N= 10 for all groups)

TP activity

 (nmol/h/m

g prot)

Group

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

30

20

10

0

Group

TP activity

 (nmol/h/m

g prot)

200

100

0

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

TP ACTIVITY 6 MONTHS AFTER TRANSPLANTATION

Blood cells Bone marrow

TP activity

 (nmol/h/m

g prot)

Group

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

200

150

100

0

Group

TP activity

 (nmol/h/m

g prot)

50

25

0

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

TP ACTIVITY 6 MONTHS AFTER TRANSPLANTATION

Spleen Liver

75

100

TP activity

 (nmol/h/m

g prot)

Group

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

600

400

200

0

Group

TP activity

 (nmol/h/m

g prot)

10

5

0

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

TP ACTIVITY 6 MONTHS AFTER TRANSPLANTATION

Small intestine Brain15

TP activity

 (nmol/h/m

g prot)

Group

WT             KO         SHAM       TPn=10         n=10       n=10       n=10 

2

1

0.5

0

TP ACTIVITY 6 MONTHS AFTER TRANSPLANTATION

Skeletal muscle (gastrocnemius)

1.5

TP activity

 (nmol/h/m

g prot)

Group

WT          KO            SHAM       TPn=9        n=10            n=7        n=5 

75

50

25

0

18 MONTHS AFTER TRANSPLANTATION (AGE 76‐89 weeks)

Group

WT          KO            SHAM       TPn=9        n=10            n=7        n=6 

TP activity

 (nmol/h/m

g prot)

75

50

25

0

Blood cells Bone marrow

18 MONTHS AFTER TRANSPLANTATION (AGE 76 – 89 weeks)

Group

Plasma Thd (µM)

WT            KO          SHAM       TPn=9          n=10         n=7        n=5 

15

10

5

0

20

Group

WT          KO            SHAM       TPn=9        n=10            n=7        n=5 

Plasma dU

rd (µ

M) 15

10

5

0

20

1 - We have generated a lentiviral vector capable of introducing a fully functional version of the TYMP gene in vivo.

2 - The allotopic overexpression of TYMP did not produce cytotoxicity, and restored the correct balanced dThd and dUrd concentrations at low levels of chimerism.

3 - The expression and function of TP is stable.

4 - These results constitute the proof of concept that treatment of MNGIE with gene therapy is feasible.

CONCLUDING REMARKS (II)

Laboratori de patologia neuromuscular i mitocondrialVall d’Hebron Institut de Recerca

Emiliano González VioqueJavier Torres Torronteras

Research group on Cell and Gene Therapy

Jordi BarquineroAlba GómezHerena Eixarch