1. 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

2. Modern medicine offers no cure for mitochondrial disorders….

3. Treatment is mostly palliative: Oxygen-radical scavengers
Vitamins
Co-factors
Oxygen-radical scavengers

4. Enzyme/Protein Replacement Therapy
The Approach:
Enzyme/Protein Replacement Therapy
Current treatment for lysosomal storage diseases. (Gaucher, Fabry, MPS-1)
The deficient protein/enzyme - artificially manufactured and purified with modifications.
Given to patients regularly.
Not for metabolic deficiencies with CNS involvement.

5. Patients’ mitochondria Mitochondrial Protein
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
Patients’ mitochondria
Delivery moiety
Mitochondrial Protein

6. Patients’ mitochondria
Protein complexes
Once in the mitochondria, it will be naturally processed and replace the mutated endogenous enzyme.

7. Which delivery moiety to choose?
In mitochondrial disorders:
Several tissues involved (with different severity).
No need for specific targeting.

8. 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.

9. 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.

10. 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.

11. 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…!!!

12. The Idea: To fuse an enzyme/protein with a delivery moiety Plus MTS, leading it into cells and their mitochondria:
TAT=TransActivator of Transcription protein
MTS= Mitochondrial Targeting Sequence
Mito. Protein= Mitochondrial Protein
TAT=TransActivator of Transcription protein
MTS= Mitochondrial Targeting Sequence
Mito. Protein= LAD, C6ORF66, Frataxin
The Idea: To fuse an enzyme/protein with a delivery moiety, leading it into cells and their mitochondria:
The Idea: To fuse an enzyme/protein with a delivery moiety, leading it into cells and their mitochondria:
TAT=TransActivator of Transcription protein
TAT=TransActivator of Transcription protein
MTS= Mitochondrial Targeting Sequence
MTS= Mitochondrial Targeting Sequence
Mito. Protein= LAD, C6ORF66, Frataxin
Mito. Protein= LAD, C6ORF66, Frataxin

13. 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)

14. The L-protein component in the glycine cleavage system .
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 .

15. 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

16. LAD Deficiency Extensive metabolic disturbances:
Several different metabolic pathways affected:
Extensive metabolic disturbances:
Biochemical abnormalities:
Massive damage caused by:
Free radicals.
Toxic metabolites.
Low rate of energy production.

17. 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.

18. LAD-based proteins: Schematic Structure
TAT-LAD
(58.1 kDa)
TAT
LAD
MTS
TAT
MTS
LAD

19. LAD-based proteins Schematic Structure
MTS
TAT
LAD
TAT-LAD (58.1 kDa)
LAD
MTS
LAD (56.7 kDa)
LAD
TAT
TAT--LAD (54.2 kDa)

20. : LAD-based proteins Expression and Purification
TAT-LAD, LAD and TAT--LAD purification
Rapoport et al. Molecular Therapy, 2008

21. LAD-based proteins In-Vitro activity assay

22. LAD-based proteins: In-Vitro activity assay
LAD activity 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 TAT- LAD. The enzymatic activity values are presented as nmol/min/mg protein.
(B) Data from (A) presented as fold increase.

23. Delivery of TAT-LAD into patients’ fibroblasts
G229C/Y35X patient cells
E375K patient cells

24. Delivery of TAT-LAD into the mitochondria of patients’ fibroblasts
G229C/Y35X patient cells

25. 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

26. 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

27. 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.

28. Co-Localization of FITC-TAT-LAD with PDHC
D479V
patient cells

29. PDHC activity in treated patients’ fibroblasts
Pyruvate + CoA + NAD+
Acetyl-CoA + NADH + CO2
Mg2+, TPP
PDHC activity - measuring 14CO2 production from [1-14C] pyruvate 9%
69%
50%
75%
30% 5%
Time of incubation (hrs)

30. 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

31. 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).

32. Experimental Design Time (hrs) At different time points -
A single injection (i.v.)
of TAT-LAD or LAD
(10 units)
At different time points -
Organs removal and analysis
(4-12 mice at each time point)

33. LAD activity in mice plasma
Rapoport, M. et al. Journal Molecular Medicine, 2011,

34. LAD activity in treated E3 mice tissues
Liver
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.

35. LAD activity in treated E3 mice tissues
Heart
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.

36. LAD activity in treated E3 mice tissues
Brain
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.

37. TAT-LAD is Delivered to Mice Tissues
Heart
Liver
Brain

38. TAT-LAD is Delivered to Mice Tissues
FITC-labeled TAT-LAD and FITC-labeled LAD were injected into E3 mice. After 2 hrs
mice were sacrificed and brain and liver were removed. Frozen sections were prepared
and analyzed for FITC signal (green). The sections were stained also with DAPI to mark
cells nuclei with in the tissues (blue). Original magnifications ×40.

39. PDHC activity in treated E3 mice tissues
Liver
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.

40. PDHC activity in treated E3 mice tissues
Heart
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.

41. PDHC activity in treated E3 mice tissues
Brain
* P<0.05 of non treated mice vs treated mice with TAT-LAD or LAD.

42. LAD vs PDHC activity in treated E3 mice at 24 hrs

43. Matan Rapoport et al. Journal Molecular Medicine, 2011, 89:161-70
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 Hr’).
Matan Rapoport et al. Journal Molecular Medicine, 2011, 89:161-70

44. 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)

45. Advantages of TAT-fusion proteins in treating mitochondrial disorders
The ability to be delivered into all cells and tissues -
No need for specific targeting.
Delivery into high-energy demanding tissues - Liver, muscles, CNS.
TAT-fusion proteins cross the blood-brain barrier
An advantage in metabolic disorders involving the CNS.
3. No need to restore enzymatic activity back to 100%
Raise it above a certain energetic threshold (can differ between patients).

46. The Future… TAT open the Door……
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……

47. Dr. Michal Lichtenstein
Acknowledgements…..
Lab members:
Dr. Matan Rapoport
Lina Salman
Dr. Michal Lichtenstein
Dana Marcus
Natali Cohen
Dr. Tal Erlich-Hadad
Rita Hadad
Department of Biochemistry and Molecular Biology IMRIC, Faculty of Medicine, Hebrew University
Prof. Orly Elpeleg
Prof. Ann Saada
Metabolic Disease Unit, Hadassah University Hospital, Jerusalem, Israel
Prof. Patel MS
Department of Biochemistry, School of Medicine, State University of New York, Buffalo, USA
BioBlast - Pharma Ltd.

49. Point of max % increase - LAD VS PDHC

50. Delivery of TAT-LAD into the mitochondria of patients’ fibroblasts

51. Frataxin-based fusion proteins Schematic Structure
His
TAT
MTSfra
FXN
27 kDa H
TAT
MTScs
FXN
20.7 kDa H
TAT
MTSorf
FXN
22.1 kDa H
TAT
FXN
MTSlad
22.2 kDa
H=His; FRA=Frataxin; MTS=mitochondrial translocation sequence;
cs=Citrate synthase; orf=C6ORF66; lad=LAD

52. Rescue of FA patients’ cells from oxidative stress

53. TAT-MTS-FXN increases ATP levels in Patients’ cells

54. TAT-MTScs-FXN fusion protein restores Aconitase Activity in Patients’ cells
Patient #L850
400%
C- Cytosol; M- Mitochondria