Massimo Zeviani, MD, PhD

Dublin April 2013

Animal models in mitochondrial disorders

der bestirnte Himmel über mir, und das moralische Gesetz in mir

Q QH2

DHODH

uridine

Q

QH2

cred

QH2

Ca2+

H+

Pyruvate

Lactate

NAD+

NADH

Alanine

Glucose

AIF CAD

Apoptosis

ADP ATP

O2

H2O

NAD+

NADH

H+

H+ Ac-CoA

TCA cycle

b-oxidn

H+

fumarate

succinate

NADH

NAD+

CO2

Apaf-1

inactive

Apaf-1

active

Activated

caspases

H+

Ca2+

e-

O2 -

O2

GPT

H2O2

Fe2+

Fe3+

OH -

H2O

GPx

GSH

GS-SG

H+

PDH

LDH

IV

IV

II

III I

I

II

III

V

III

ADP

ATP

SOD2

cox

cred

cred

61%

21% 18% mtDNA mutation

nDNA mutation

unknown

Paediatric Patients with Respiratory Chain Deficiency

0%

20%

40%

60%

80%

100%

Com

ple

x I

Com

ple

x II

Com

ple

x III

Com

ple

x IV

Com

ple

x V

Multip

le

Tota

l

Deple

tio

n

LINKAGE/HOMOZYGOSITY

Linkage analysis or homozygosity mapping

Candidate genes

Families with (autosomal recessive) mitochondrial disorders

Mutational screening

Small families and single individuals

Filtering strategies

Candidate genes

Exome sequencing Coding variants

NGS

Discovering new disease genes

? Phenotype Function

OXPHOS D2S2373

D2S2259

Locus

I II III IV V

G A A T G A G C A

Gene

Protein TTC19

C #1-1 #1-2 i.v.

In silico

Filtering genes Integrated genomics

Phylogenetic analysis

Systems analysis

Omic- analysis

Structure

Cell models

patients

MEFs

yeast

HeLa/cybrids

iPS

recombinant E. coli

In vivo

In vivo pathogenesis

Experimental therapy

Protein factory

Clinical phenotype

Organ pathophysiology

Cell biology

Systems biology and omics

Gene replacement

Cell replacement

Drugs Organ replacement

Other bioreactors

Mutant proteins

Tagged proteins

Complexes

Gene and protein interactions

Disease models

SURF1 constitutive KO

MPV17 constitutive KO

ETHE1 constitutive and conditional KO

COX15 constitutive KO (embryonic lethal) and conditionals (ACTA-COX15; GFAP-COX15)

SCO2constitutive KOKI

NDUFS1 constitutive KO

NDUFS4 constitutive KO

FASTKD2 constitutive KO

TTC19 constitutive and conditional KO

PARP1 constitutive KO

FASTKD2 constitutive and conditional KO

Transgenic mice

PGC1

TG CRE: general deleter

ALB CRE: liver specific

MYOD CRE: (muscle specific, early expressed)

ACTA CRE: (skeletal muscle specific)

TG FLPe (general deleter)

GFAP-CRE: (brain specific)

AOX (knockin in locus ROSA26)

NDI1 (knockin in locus ROSA26)

Mouse models

CNS •early-onset hypotonia. developmental delay •later spasticity, then global failure

Vascular system •acrocyanosis •petechiae, microematuria, internal bleedings

Gastrointestinal system •chronic diarrhoea

Biochemistry •COX deficiency •lactic acidosis •ethylmalonic aciduria

0

1

2

3

4

5

6

7

8

9

Ab

un

dan

ce

urea

ethylmalonate

I.S.

2-ketoglutarate

I.S.

isobutyrylglycine

isovalerylglycine

Time

Ethylmalonate Butyryl-CoA CO2 -> CH3-CH2-CH2-CO-SCoA + CH3-CH2-CH-COOH

COOH

CoASH

Ethylmalonic Encephalopathy (EE)

440del11

3G>T

113A>G 131delAG

222insA

406A>G

488G>A

487C>T

604insG

559insC 375+5G>A

505+1G>A

505+1G>T

554T>G

EX1-7del

EX4del

1 2 3 4 5 6 7

1 Kb

ETHE1 is the EE disease gene

HeLa

ETHE1 Mitotracker merge

ETHE1p

hom. sup. mitos matrix memb

Works as a homodimer

Binds 1 atom of iron/dimer

Fe++

genotypes

Lifespan

days

Behavior

Creating an ETHE1-/- mouse E4

NEO E1 E2 E3 E5 E6 E7

LOXp LOXp frt frt

0

50

100

150

200

250

300

350

g

/mg

cre

ati

nin

e

CTR KO

mouse CTR EE

human

Ethylmalonic acid

(urine)

Respiratory chain complex activities

n.s.

** **

**

**p<0,0005

Ethe1-/-

brain

muscle

colon

liver

Ethe1+/+

Phylogenetic search

Bradyrhizobium japonicum 27376118 9

Legionella pneumophila 54293763 9

Legionella pneumophila 52841017 9 Danio rerio 47086181 9

Caenorhabditis elegans 17538952 9 Apis mellifera 48140497 9

Drosophila melanogaster 24652740 9

Drosophila melanogaster 27819799 9 Anopheles gambiae 31210533 9 Anopheles gambiae 55240363 9

Oryza sativa 56202167 9 Oryza sativa 34910930 9

Arabidopsis thaliana 12324040 9 Arabidopsis thaliana 1644427 9 Arabidopsis thaliana 22655048 9

Xenopus laevis 27371293 9

Pan troglodytes 55649315 9 Homo sapiens ETHE1

Mus musculus 53236965 9 Rattus norvegicus 27676450 9

Burkholderia cepacia 46322841 9 F

Burkholderia cepacia 46317848 9 F Ralstonia eutropha 45515657 9

Ralstonia metallidurans 48771425 9 Nostoc punctiforme 23128919 9 F

Synechococcus elongatus 56750614 9

Nostoc sp. 17231396 9 Anabaena variabilis 45507470 9

Synechocystis sp. 16330445 9

Thermosynechococcus elongatus

Polaromonas sp. 54032671 9 F

??

Fusion events Operon

?? soxY soxA ?? soxB soxZ soxX

??

??

??

Ethe1-related domain

Rhodanese-related sulfur-transferase domain

Domain of unknown function

Predicted permease Glutathione S-transferase

Sulfur oxidase gene cluster

ETHE1-like proteins are present in operons containing Rhodanese-like proteins or

as ETHE1/Rhodanese chimaeric proteins

The Sörbo assay

Sulfur metabolism

Szabo, Nat Rev Drug Discov 2007

Thiosulfate in urines

EE-Patients

nm

ol/m

g c

reatin

ine

0

500

1000

1500

2000

2500

CTR 0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

nm

ol/m

g c

reatin

ine

CTR KO mice

0

40

80

120

160

200

+/+ +/+ +/+ +/+ +/+ -/- -/- -/- -/- -/-

M

and tissues

LIVER

0,0

500,0

1000,0

1500,0

2000,0

2500,0

3000,0

3500,0

MUSCLE

0,0

500,0

1000,0

1500,0

2000,0

2500,0

CTR KO CTR KO

p<0.0007 p=0.00007

BRAIN

0,0

10,0

20,0

30,0

40,0

50,0

60,0

70,0

80,0

90,0

CTR KO

p<0.0016

H2S concentration in tissues of ETHE1

knockout mice

H2S

(nM

)

H2S is a metabolically active compound

Szabo, Nat Rev Drug Discov 2007

H2S inhibits COX and SCAD activities

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0 1 2 0 0

0

2 0

4 0

6 0

8 0

1 0 0

1 2 0

N a S H ( µ M )

resi

du

al a

ctiv

ity SCAD in liver COX/CS

NaSH (µM)

res

idu

al a

cti

vit

y

120

0 1 2 3 4 5 6 7 8 9 10 11 12 13

0

20

40

60

80

100

NaCN

MUSCLE

BRAIN

LIVER

0 2 4 6 8 10 12

0

20

40

60

80

100

120

NaSH (µM)

resid

ua

l acti

vit

y

CI and CS in muscle

CI/CS

CS

Tiranti et al, Nat. Med. 2009

A mitochondrial pathway for H2S detoxification

Jorns MS et al, 2012

0

2

4

6

8

10

12

14

16

µm

ol O

2 m

in-1

mg

-1

ETHE1His

nm

ol O

2 m

in-1

mg

-1

0

1

2

3

4

5

ETHE1

SDH

Liver

-/- +/+

SDH

ETHE1

Hela HelaETHE1-HA

0

1

2

Fm

ol O

2 m

in-1

cell

-1

HelaETHE1-HA Hela

Liver homogenate

HeLa cells

Purified ETHE1

25kDa

A mitochondrial pathway for H2S detoxification

Jorns MS et al, 2012

H2S production: multiple sources

Exogenous:

a by-product of anaerobes in the

large intestine

Endogenous:

a “gasotransmitter”produced in trace

by many organs

Median= 72 days

Metronidazole+NAC Metronidazole

Median=27 days Median= 55 days

METRONIDAZOLE, A BACTERICIDAL AGENT

Median=49 days

NAC

N-ACETYL CYSTEINE: A GSH PRECURSOR

* PRE

* POST

Ethe1-/-

mice

Ethe1-/-

children

Liver-targeted AAV-mediated gene replacement

AAV

NAC+Metro

NAC

naïve

p<0.0001

SDH COX SDH COX COX+SDH COX

SDH COX SDH COX COX+SDH COX

Genetic classification of OXPHOS disorders

Defects of Mitochondrial DNA

•Protein synthesis genes (rRNAs, tRNAs)

•Protein-encoding OXPHOS subunit genes

•Large deletions

Nuclear DNA mutations

•OXPHOS subunits

•OXPHOS assembly factors

•mtDNA maintenance and transcription

•Mitochondrial protein synthesis

•Biosynthesis of phospholipids or cofactors

•Mitodynamics, mitophagy, apoptosis

•Mitoprotein quality control

•Mitobiogenesis

•Mitochondrial detoxification mechanisms

6.2 5.1 7.6

3.6

10.4

0.8 2.7 3.0

16.8 14.1

7.9 10.2

0

10

20

Brain

cortex

White

matter

Liver Kidney Small

intestine

Skeletal

muscle

Mean r

atio

MNGIE Control

Real-time quantitative PCR to estimate mtDNA depletion

mtDNA multiple deletions in muscle of MNGIE patients detected

by Southern blot hybridization

MN1-1 MN2-1 MN2-2 MN3-1 MN4-2 MN7-2 Cont.

P S U P S U P S U P S U P S U P S U P U

WT D5-kb D7.7-kb D8.1-kb D10.3-kb

-3 -3 -4 -2

-<2 -<2 -4 -<2

-<2 -<2 -2

-6 -11 -3 -2

-3 -4 -2 -2

Why is mtDNA affected in MNGIE?

thymidine

deoxyuridine deoxycytidine

dTMP

dUMP

dCMP mtDNA

thymidine

deoxyuridine

dTMP nDNA

TK2

TK1

de novo

synthesis thymine

uracil

TP

mitochondria

dNT2

Vector: TBG-TYMP AAV2/8. Two-month old mice were treated at different doses: 2·1011 gc/kg (N=8) 1012 gc/kg (N=8) 2·1012 gc/kg (N=6) 1013 gc/kg (N=2)

Follow-up during 8 months after treatment and killed after 8 months. Endpoints:

Monitoring of plasma nucleosides concentration Monitoring of plasma ALT levels TP activity in liver and other tissues (8 months) Nucleosides in plasma, liver and small intestine (8 months)

To be determined:

Mitochondrial dNTP pools in liver and brain (8 months) Histological analysis of liver tissue (8 months)

By today, the following mice reached 8 months of life:

2·1011 gc/kg (N=5) 1012 gc/kg (N=4) 2·1012 gc/kg (N=4) 1013 gc/kg (N=2)

Plasma Thymidine

Liver TP activity

Nucleosides 8 months after treatment (intracardiac puncture)

Scatter plots: liver TP activity vs. plasma nucleosides 8 months after treatment

Liver TP activity vs. plasma nucleosides after

8 months of treatment

AAV-mediated gene replacement

Liver targeting of suitable Ethe1-expressing recombinant

AAV vectors is specific

Ethe1p liver-specific expression restores SDO activity

Ethe1-/- mice treated with >1010 AAV/gm of body weight show significant prolongation of the lifespan; the oldest treated animal is now 180 days old and well

Liver-targeted, AAV-based gene therapy is potentially transferable to patients

Suitable mitodisorders include conditions due to accumulation of toxic compounds such as Ethylmalonic Encephalopathy and MNGIE, as well as liver-specific mtDNA depletion

12

GMP manufacturing of AAV

Clinical and molecular characterization of specific patients

GLP Safety/Tox studies GMP vector

AAV8-ARSB GMP

certified

Pre-inquiry IMPD/IND (ISS: Italian Institute of Health)

Protocol assistance (EMA)

IMPD (ISS) IRB

0 24 6

Months

Clinical Trial (5 years)

Orphan Drug Designation

Preclinical studies

Path Towards Clinic Translation: Timeframe