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