Protein Replacement Therapy
for Mitochondrial Diseases
Integrative Biology,
Berlin, July, 2016
Prof. Haya Lorberboum-Galski
Institute for Medical Research
(IMRIC)
Faculty of Medicine, Hebrew
University
Modern medicine offers
no cure for mitochondrial
disorders….
Treatment is mostly
palliative:
Vitamins •
Co-factors •
Oxygen-radical scavengers•
The Approach:
Enzyme/Protein Replacement Therapy
• The deficient protein/enzyme - artificially manufactured and
purified with modifications.
• Given to patients regularly.
• Current treatment for lysosomal storage diseases.
(Gaucher, Fabry, MPS-1)
• Not for metabolic deficiencies with CNS involvement.
The Approach:
Cell Directed Protein Replacement Therapy (CDPRT)
To fuse the Mitochondrial protein with a delivery moiety,
leading it into cells and their mitochondria.
Patients’ cells
Delivery moiety
Delivery moiety
Delivery moiety
Patients’ mitochondria
Mitochondrial Protein
Mitochondrial Protein
Mitochondrial Protein
Patients’ mitochondria
Protein complexes
Once in the mitochondria, it will be naturally processed
and replace the mutated endogenous enzyme.
Which delivery moiety to choose?
In mitochondrial disorders:
 Several tissues involved
(with different severity).
 No need for specific
targeting.
Protein Transduction Domains (PTDs)
* a group of short peptides that serve as delivery
vectors for large molecules.
* are defined as short, water-soluble and partly
hydrophobic, and/or polybasic peptides.
* at most 30– 35 amino acids residues.
* with a net positive charge at physiological pH.
PTDs
* The main feature of PTDs is that they are able to penetrate
biological membranes at low micromolar concentrations in vitro
and in vivo without using any chiral receptors and without
causing significant membrane damage.
* Furthermore, and even more importantly, these peptides are
capable of internalizing electrostatically or covalently bound
biologically active cargoes such as drugs with high efficiency and
low toxicity.
The Delivery Moiety - TAT
• The first known Protein Transduction Domain (PTD).
• 11 amino-acid peptide of the HIV-1 virus TransActivator of
Transcription (Tat) protein.
• Arginine rich.
• Positively charged.
The Delivery Moiety - TAT
TAT-fusion proteins cross cell membranes
including mitochondrial membranes
while retaining their biological activity
both in-vitro and in-vivo!
The Problem……TAT-fusion proteins are crossing biological membranes: In-Out…!!!
The Idea: To fuse an enzyme/protein
with a delivery moiety Plus MTS, leading
it into cells and their mitochondria:
TAT-MTS (heterologous or homologous)-Mito. Protein
M
P
it.
Cell
in
te
ro
Cell
M
TS
T
TA
Mitochondrial
Protease
M
TS
Mitochondria
it.
M tein
o
Pr
Mitochondria
T
TA
TAT=TransActivator of Transcription protein
MTS= Mitochondrial Targeting Sequence
Mito. Protein= Mitochondrial Protein
Cell/Organelle-Directed Protein
Replacement Therapy for:
•
Lipoamide Dehydrogenease (LAD) (Rapoport et
al 2008, 2011)
•
C6ORF66 (NDUFAF4; ORF) (Marcus et al 2013)
•
Ornitine Transcarbomylase (OTC)
•
Frataxin (FXN) (Marcus et al, submitted)
•
Methylmalonyl CoA mutase (MCM) (Erlich et al,
in preparation)
Lipoamide Dehydrogenase (LAD):
• The E3 subunit of the three -ketoacid
dehydrogenase complexes:
 Pyruvate Dehydrogenase (PDHC).
 -Ketoglutarate Dehydrogenase (KGDHC).
 Branched-Chain Ketoacid
Dehydrogenase (BCKDHC).
• The L-protein component in
the glycine cleavage system .
Lipoamide Dehydrogenase (LAD) Deficiency:
Table I. Published Mutations in the DLD Gene and Clinical Enzyme Profiles of Patients. (DD, developmental delay;
MR, mental retardation). Taken from Cameron, J.M., et al.7
LAD Deficiency
Several different metabolic pathways affected:
Extensive metabolic
disturbances:
Biochemical abnormalities:
Massive damage caused by:
• Free radicals.
• Toxic metabolites.
• Low rate of energy production.
LAD deficiency - Clinical Course
• Variable clinical course (type of mutation, homozygosity/compound),
Ranging from:
Infantile neurodegenerative disease:
• Severe psychomotor retardation in infancy.
• Lactic acidemia.
• Death by early childhood.
Episodes of liver failure:
• Recurrent vomiting and abdominal pain.
• Encephalopathy.
• Elevated liver transaminases and prolonged prothrombin time.
• Lactic and ketoacidemia.
LAD-based proteins:
Schematic Structure
TAT-LAD
(58.1 kDa)
TAT
MTS
TAT
MTS
LAD
LAD
LAD-based proteins
Schematic Structure
TAT
MTS
MTS
LAD
TAT-LAD (58.1 kDa)
MTS
LAD
LAD (56.7 kDa)
TAT
LAD
TAT--LAD (54.2 kDa)
LAD-based proteins
Expression
and Purification
:
TAT-LAD, LAD and TAT--LAD purification
Rapoport et al. Molecular Therapy, 2008
LAD-based proteins
In-Vitro activity assay
LAD-based proteins: In-Vitro
LAD enzymatic activity within the
treated cells did not
reach a plateau thus suggesting
activity
assay
that we did not reach a saturating concentration (Fig 7B).
7. LAD activity in G229C/Y35X cells after 2 hrs incubation with different concentration
LAD activityFigure
in G229C/Y35X
cells after 2 hrs incubation with different concentration
of TAT-LAD. (A) LAD activity in patients' cells treated with increasing concentrations of TATof TAT-LAD.
(A)
activity
patients'
cells
treated with
increasing
concentrations
LAD.
TheLAD
enzymatic
activityin
values
are presented
as nmol/min/mg
protein.
(B) Data from
(A)
presented
as
fold
increase.
of TAT- LAD. The enzymatic activity values are presented as nmol/min/mg protein.
(B) Data from (A) presented as fold increase.
5.4
TAT-LAD is delivered into mitochondria of patients’ cultured cells, increasing
Delivery of TAT-LAD into
patients’ fibroblasts
G229C/Y35X patient cells
E375K patient cells
Delivery of TAT-LAD into the
mitochondria of patients’ fibroblasts
G229C/Y35X patient cells
Delivery of TAT-LAD into the mitochondria of
patients’ fibroblasts - CS and LAD/CS ratio
1.3
Normal range
LAD deficiency range
G229C/Y35X patient cells
Delivery of TAT-LAD into the mitochondria
Import and processing assay
In-vitro translation
Inside mitochondria
TAT-LAD is processed to it’s mature form inside the
mitochondria
Pyruvate Dehydrogenase Complex (PDHC)
• 9.5x106Da “Machine”
 Core of 60 subunits of E2.
 30 molecules of E1 (heterodimer of
2a;2b subunits).
 6 molecules of the homodimeric E3.
 12 molecules of the E3 binding protein.
• Similar structure to all -ketoacide dehydrogenase
complexes.
Co-Localization of FITC-TAT-LAD with PDHC
D479V
patient cells
PDHC activity in treated patients’ fibroblasts
Mg2+, TPP
Pyruvate + CoA + NAD+
Acetyl-CoA + NADH + CO2
PDHC activity - measuring 14CO2 production from [1-14C] pyruvate
75%
69%
50%
30%
9%
5%
Time of incubation (hrs)
Summary - Patients’ fibroblasts
TAT-LAD successfully competes with the endogenous mutated LAD:
Dimerization and PDHC integration.
Replacement of ONE mutated component is sufficient to restore
the activity of a HUGE ENZYMATIC COMPLEX.
Rapoport et al. Mol Ther. 2008
The mouse model of LAD deficiency (E3 Mice)
• Heterozygotes to a recessive loss-of-function mutation affecting the
Dld gene.
• Homozygous mice (Dld-/-) stop developing and die in-utero at early
gastrulation.
LAD activity: ~50% of WT
-ketoacid dehydrogenase complexes: ~50% of WT
Heterozygous E3 mice are phenotipically normal
(humans carriers of LAD deficiency present no
clinical symptoms).
Experimental Design
Time (hrs)
At different time points Organs removal and analysis
(4-12 mice at each time point)
A single injection (i.v.)
of TAT-LAD or LAD
(10 units)
LAD activity in mice plasma
Rapoport, M. et al. Journal Molecular Medicine, 2011,
LAD activity in treated E3 mice tissues
Liver
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.
LAD activity in treated E3 mice tissues
Heart
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.
LAD activity in treated E3 mice tissues
Brain
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.
TAT-LAD is Delivered to Mice
Tissues
Heart
Liver
Brain
TAT-LAD is Delivered to Mice Tissues
Figure
15. Delivery of
TAT-LAD
into E3-treated mice
tissues.
FITC-labeled
TAT-LAD
and
FITC-labeled
LAD
were injected into E3 mice. After 2 hrs
FITC-labeled TAT-LAD and FITC-labeled LAD were injected into E3 mice. After 2 hrs
mice were sacrificed
and and
brain
liver
removed.
Frozen
sections
were prepared
mice were sacrificed
brainand
and liver
werewere
removed.
Frozen sections
were prepared
and
for FITCsignal
signal (green).
The sections
stained also
with DAPI
to mark
cells with DAPI to mark
and analyzedanalyzed
for FITC
(green).
The were
sections
were
stained
also
nuclei with in the tissues (blue). Original magnifications #40.
cells nuclei with in the tissues (blue). Original magnifications ×40.
PDHC activity in treated E3 mice tissues
Liver
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.
PDHC activity in treated E3 mice tissues
Heart
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.
PDHC activity in treated E3 mice tissues
Brain
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.
LAD vs PDHC activity in treated E3 mice at 24
hrs
Summary in-vivo
* TAT-LAD is delivered into the liver, heart and brain of injected E3 mice.
* A single injection of TAT-LAD restores LAD and PDHC enzymatic activity
in the liver, heart and brain of E3 mice.
* This effect lasts for several hours (up to 24-48 Hr’).
Matan Rapoport et al. Journal Molecular Medicine, 2011, 89:161-70
Cell/Organelle-Directed Protein
Replacement Therapy for:
•
Lipoamide Dehydrogenease (LAD) (Rapoport et
al 2008, 2011)
•
C6ORF66 (NDUFAF4; ORF) (Marcus et al 2013)
•
Ornitine Transcarbomylase (OTC)
•
Frataxin (FXN) (Marcus et al, submitted)
•
Methylmalonyl CoA mutase (MCM) (Erlich et al,
in preparation)
Advantages of TAT-fusion proteins in
treating mitochondrial disorders
1. The ability to be delivered into all cells and tissues •
No need for specific targeting.
•
Delivery into high-energy demanding tissues - Liver, muscles, CNS.
2. TAT-fusion proteins cross the blood-brain barrier
•
3.
An advantage in metabolic disorders involving the CNS.
No need to restore enzymatic activity back to 100%
•
Raise it above a certain energetic threshold (can differ between
patients).
The Future…
This approach could be applied to the many other known
mitochondrial and metabolic disorders
Revolutionize the management of these types of disorders
in modern medicine
TAT open the Door……
Acknowledgements…..
Lab members:
Dr. Matan Rapoport
Lina Salman
Dr. Michal Lichtenstein
Prof. Orly Elpeleg
Dana Marcus
Prof. Ann Saada
Natali Cohen
Metabolic Disease Unit,
Hadassah University
Hospital, Jerusalem, Israel
Dr. Tal Erlich-Hadad
Rita Hadad
Department of Biochemistry and Molecular Biology
IMRIC, Faculty of Medicine, Hebrew University
BioBlast - Pharma Ltd.
Prof. Patel MS
Department of Biochemistry, School
of Medicine, State University of
New York, Buffalo, USA
Point of max % increase - LAD VS PDHC
Delivery of TAT-LAD into the mitochondria of patients’ fibroblasts
Frataxin-based fusion proteins
Schematic Structure
Hi
s
H TAT MTSfra
FXN
27 kDa
H TAT MTScs
FXN
20.7 kDa
H TAT MTSorf
FXN
22.1 kDa
H TAT MTSlad
FXN
22.2 kDa
H=His; FRA=Frataxin; MTS=mitochondrial translocation sequence;
cs=Citrate synthase; orf=C6ORF66; lad=LAD
Rescue of FA patients’ cells from oxidative stress
30
Increase in cell survival (%)
25
20
15
BSO 1 mM
10
BSO 25 mM
5
0
-5
-10
-15
-20
MTS-FXN
MTS-CS
MTS-ORF
MTS-LAD
TAT-MTS-FXN increases ATP levels in
Patients’ cells
TAT-MTScs-FXN fusion protein restores
Aconitase Activity in Patients’ cells
Patient #L850
400%
C- Cytosol; M- Mitochondria