METHODS AND COMPOSITIONS FOR TREATMENT OF MITOCHONDRIAL DISORDERS

Abstract

The present invention concerns in general novel fusion proteins comprising a membrane transferring moiety and an enzymatic moiety. The present invention further concerns a method of treating disease using said fusion proteins.

Claims

1. A method of treatment of a mitochondrial disorder in a subject, comprising administering to the subject a fusion protein, said fusion protein comprising a protein transduction domain (PTD) fused to a functional component of a mitochondrial enzyme and a mitochondria targeting sequence (MTS), wherein said MTS is situated between said PTD and said functional component and wherein said MTS is an MTS of another mitochondrial enzyme that is encoded by a nuclear gene, wherein said mitochondrial enzyme is an enzyme of a mitochondrial multi-component enzyme complex and wherein said protein transduction domain is a TAT peptide.

2. The method of claim 1, wherein the portion of said fusion protein that is C-terminal to said MTS consists of said functional component of an enzyme.

3. The method of claim 2, wherein there are no residues heterologous to the enzyme present C-terminal to the MTS and cleavage of the said MTS generates an enzyme with the native sequence, which is readily integrated into a conformationally-sensitive multi-component enzyme complex.

4. The method of claim 1, wherein said enzyme is Lipoamide Dehydrogenase (LAD).

5. The method of claim 1, wherein said enzyme is selected from the group consisting of 2-oxoisovalerate dehydrogenase alpha subunit (Branched-Chain Keto Acid Dehydrogenase E1α), 2-oxoisovalerate debydrogenase beta subunit (Branched-Chain Keto Acid Dehydrogenase E1β), Acyl-CoA dehydrogenase, medium-chain specific, Acyl-CoA dehydrogenase, very-long-chain specific, TrifUNctional enzyme alpha subunit (Long-chain 3 hydroxyacyl CoA Dehydrogenase or LCHAD) (HADHA), Trifunctional enzyme beta subunit (Hydroxyacyl-CoA Dehydrogenase/3-Ketoacyl-CoA Thiolase/Enoyl-CoA Hydratase (HADHB]), Pyruvate dehydrogenase El component beta subunit, and Pyruvate dehydrogenase El component alpha subunit.

6. The method of claim 1, wherein said multi-component enzyme complex is selected from the group consisting of pyruvate dehydrogenase complex (PDHC), [alpha]-ketoglutarate dehydrogenase complex (KGDHC), and branched-chain keto-acid dehydrogenase complex (BCKDHC).

7. The method of claim 1, wherein said multi-component enzyme complex is selected from the group consisting of complex I (NADH-ubiquinone oxidoreductase), complex II (succinateubiquinone oxidoreductase), complex III (ubiquinol-ferricytochrome c oxidoreductase), complex IV (cytochrome c oxidoreductase), and complex V (FIFO ATPase).

8. The method of claim 1, in which said mitochondrial disorder is caused by a missense mutation in said enzyme.

9. The method of claim 1, in which said mitochondrial disorder is selected from the group consisting of LAD deficiency and isolated Complex I deficiency.

10. The method of claim 1, in which said mitochondrial disorder is a neurodegenerative disease.

11. The method of claim 1, in which said mitochondrial disorder is selected from the group consisting of encephalopathy and liver failure that is accompanied by stormy lactic acidosis, hyperammonemia and coagulopathy, Alpers Disease, Barth syndrome, Beta-oxidation Defects, Camitine-Acyl-Camitine Deficiency, Camitine Deficiency, Co-Enzyme Q1 O Deficiency, Complex I Deficiency, Complex II Deficiency, Complex III Deficiency, Complex IV Deficiency, Complex V Deficiency, COX Deficiency, CPEO, KSS, LCHAD, Leigh Disease or Syndrome, LHON, LIC (Lethal Infantile Cardiomyopathy), Luft Disease, MELAS, MERRF, Mitochondrial Cytopathy, Mitochondrial Myopathy, MNGIE, NARP, and Pyruvate Dehydrogenase Deficiency.

12. The method of claim 1, in which said mitochondrial disorder is selected from the group consisting of Ornithine Transcarbamylase deficiency (hyperammonemia) (OTCD), Carnitine Opalmitoyltransferase II deficiency (CPT2), Fumarase deficiency, Cytochrome c oxidase deficiency associated with Leigh syndrome, Maple Syrup Urine Disease (MSUD), Medium-Chain Acyl-CoA Dehydrogenase deficiency (MCAD), Acyl-CoA Dehydrogenase Very Long-Chain deficiency (LOAD), Trifunctional Protein deficiency, Progressive External Ophthalmoplegia with Mitochondrial DNA Deletions (POLG), DGUOK, TK2, Pyruvate Decarboxylase deficiency, MMC-Maternal Myopathy and Cardiomyopathy; Ataxia, Retinitis Pigmentosa; FICP-Fatal Infantile Cardiomyopathy Plus, a MELAS-associated cardiomyopathy; MELAS-Mitochondrial Encephalomyopathy with Lactic Acidosis and Strokelike episodes; LDYT-Leber's hereditary optic neuropathy and Dystonia; MHCM-Maternally inherited Hypertrophic CardioMyopathy; OM-Diabetes Mellitus; DMDF Diabetes Mellitus+Deafness; CIPO-Chronic Intestinal Pseudoobstruction with myopathy and Ophthalmoplegia; DEAF-Maternally inherited DEAFness; PEM-Progressive encephalopathy; SNHL-SensoriNeural Hearing Loss; Encephalomyopathy; DEM CHO-Dementia and Chorea; AMDF-Ataxia, Myoclonus; ESOC Epilepsy;Optic atrophy; FBSN Familial Bilateral Striatal Necrosis; FSGS Focal Segmental Glomerulosclerosis; LIMM Lethal Infantile Mitochondrial Myopathy; MOM Myopathy and Diabetes Mellitus; MEPR Myoclonic Epilepsy and Psychomotor Regression; MERME MERRF/MELAS overlap disease; MHCM Maternally Inherited Hypertrophic CardioMyopathy; MICM Maternally Inherited Cardiomyopathy; MILS Maternally Inherited Leigh Syndrome; Mitochondrial Encephalocardio myopathy; Multisystem Mitochondrial Disorder (myopathy, encephalopathy, blindness, hearing loss, peripheral neuropathy); NAION Nonarteritic Anterior lschemic Optic Neuropathy; PEM Progressive Encephalopathy; PME Progressive Myoclonus Epilepsy; RTI Rett Syndrome; SIDS Sudden Infant Death Syndrome; and MILD Maternally Inherited Diabetes and Deafness.

13. The method of claim 1, wherein said treatment is a continuous prolonged treatment for a chronic disease or comprises a single or few time administrations for treatment of an acute condition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] FIG. 1A. Schematic representation of TAT-LAD and LAD fusion proteins, their expression and purification. Schematic representation of TAT-LAD fusion protein and the control proteins-TAT-8-LAD (lacking the MTS moiety) and LAD (lacking the TAT moiety).

[0058] FIG. 1B. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using anti-LAD antibody analysis of purified TAT-LAD, TAT-8-LAD, and LAD fusion proteins. Proteins were purified using affinity chromatography.

[0059] FIG. 1C. Enzymatic activity of purified TAT-LAD, TAT-8-LAD, and LAD fusion proteins. LAD activity values (nmol/min/mg) presented are mean values±SD of three separate enzymatic assays, each carried out in triplicate. LAD: lipoamide dehydrogenase; MTS: mitochondrial targeting sequence; TAT: transactivator of transcription peptide.

[0060] FIG. 2A. Delivery of TAT-LAD into G229CN35X and E375K patients' cells. Western blot analysis of whole-cell protein extracts from G229CN35X-treated cells using antibodies against LAD (1:1000). TAT-LAD fusion protein (arrow) and endogenous mutated LAD correspond to M.W. of 58kDa and 50kDa, respectively, as expected.

[0061] FIG. 2B. Western blot analysis of whole-cell protein extracts from E375K-treated cells, using antibodies against LAD (1:1000) and α-tubulin (1:10,000). Anti-Tubulin served as an internal control for protein loading.

[0062] FIG. 2C. Fluorescence microscopy analysis of G229CN35X cells treated with FITC-labeled TAT-LAD (panels 1-3) and LAD (panel 4) (0.1 μg/μl, final concentration) for 30 min (panel 1), 2hrs (panel 2) and 4hrs (panel 3).

[0063] FIG. 2D. LAD activity in treated G229CN35X cells. Cells were treated with TAT-LAD, TAT-PAH or LAD protein (0.075-0.1 μg/μl, final concentration) for different time periods. LAD activity was analyzed in whole-cell protein extracts by enzymatic activity assay. Activity assays were conducted at least three times. Values are presented as the mean±s.e.m. (left panel) or depict typical results (right panel). The activity values are presented as nmol/min/mg protein.

[0064] FIG. 2E. LAD activity in treated E375K cells. Cells were treated with TAT-LAD, TAT-PAH or LAD protein (0.075-0.1 μg/μl, final concentration) for different time periods. LAD activity was analyzed in whole-cell protein extracts by enzymatic activity assay. Activity assays were conducted at least three times. Values are presented as the mean±s.e.m. (left panel) or depict typical results (right panel). The activity values are presented as nmol/min/mg protein.

[0065] FIG. 3A. Fate of TAT-LAD and TAT-8-LAD within isolated mitochondria. Radioactive-labeled TAT-LAD and TAT-8-LAD were expressed in vitro and analyzed using SDS-PAGE autoradiography, matching their expected molecular sizes, 58 and 54 kd, respectively.

[0066] FIG. 3B. Fate of TAT-LAD and TAT-8-LAD within isolated mitochondria. Mitochondria isolated from cells were incubated for 30 min. with the radio-labeled proteins. Mitochondria were then washed, treated with proteinase K, and analyzed using SDS-PAGE autoradiography. Asterisk marks 50 kd band of processed TAT-LAD fusion protein.

[0067] FIG. 4A. Delivery of TAT-LAD into mitochondria of G229CN35X patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. The LAD activities in the cytosolic and mitochondrial fractions of the treated cells were analyzed. Activity values are presented as nmol/min/mg protein.

[0068] FIG. 4B. Delivery of TAT-LAD into mitochondria of G229CN35X patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. The CS enzymatic activities in the cytosolic and mitochondrial fractions of the treated cells were analyzed. Activity values are presented as nmol/min/mg protein.

[0069] FIG. 4C. Delivery of TAT-LAD into mitochondria of G229CN35X patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. The LAD/CS ratio in their mitochondrial fraction was calculated. The LAD/CS ratio was almost two-fold higher for TAT-LAD than for TAT-LAD.

[0070] FIG. 4D. Delivery of TAT-LAD into mitochondria of G229CN35X patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. Western blot analysis of the sub-cellular fractions showing the intra-cellular distribution of TAT-LAD and their purity, using antibodies against LAD (1:1000) and the specific markers VDAC (porin) (1:5000) for the mitochondria and α-tubulin (1:10000) for the cytoplasm. The marker E1α was also used to confirm purity of the mitochondrial fraction.

[0071] FIG. 4E. Delivery of TAT-LAD into mitochondria of D479V patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. The LAD activities in the cytosolic and mitochondrial fractions of the treated cells were analyzed. Activity values are presented as nmol/min/mg protein.

[0072] FIG. 4F. Delivery of TAT-LAD into mitochondria of D479V patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. The CS enzymatic activities in the cytosolic and mitochondrial fractions of the treated cells were analyzed. Activity values are presented as nmol/min/mg protein.

[0073] FIG. 4G. Delivery of TAT-LAD into mitochondria of D479V patients' cells. Cells were treated with the fusion protein (0.1 μg/μl final concentration) for 4-6 hrs. Sub-cellular fractions (cytosolic and mitochondrial) were obtained by differential centrifugation. The LAD/CS ratio in their mitochondrial fraction was calculated. The LAD/CS ratio was almost two-fold higher for TAT-LAD than for TAT-LAD. Activity values are presented as nmol/min/mg protein.

[0074] FIG. 4H. Western blot analysis of the sub-cellular fractions showing the intra-cellular distribution of TAT-LAD and their purity, using antibodies against LAD (1:1000) and the specific markers VDAC (porin) (1:5000) for the mitochondria and α-tubulin (1:10000) for the cytoplasm. The marker E1α was also used to confirm purity of the mitochondrial fraction.

[0075] FIG. 5A. PDHC co-localization and enzymatic activity in TAT-LAD-treated cells from patients. D479V cells were treated with fluorescein isothiocyanate (FITC)-labeled TAT-LAD or LAD (green fluorescence, middle column), washed, fixed, permeabilized, and incubated with anti-E1α antibody. The cells were then washed and incubated with anti-mouse Cy5 antibody (red fluorescence, left column). The cells were analyzed for co-localization using confocal microscopy (yellow merge, right column). Original magnifications: ′60 (LAD) and ′100 (TATLAD).

[0076] FIG. 5B. PDHC co-localization and enzymatic activity in TAT-LAD-treated cells from patients. Cells were incubated with TAT-LAD (0.1 μg/μl, final concentration) for 3, 6, or 24 hours. PDHC activity assays were performed as described in Materials and Methods. PDHC activity in treated E375K cells of patients. Activity values are presented as nmol/min/mg protein. Values presented are mean values±SD. PDHC, pyruvate dehydrogenase complex. Co-localization was also observed in treated G229CN35X patients' cells.

[0077] FIG. 5C. PDHC co-localization and enzymatic activity in TAT-LAD-treated cells from patients. Cells were incubated with TAT-LAD (0.1 μg/μl, final concentration) for 3, 6, or 24 hours. PDHC activity assays were performed as described in Materials and Methods. PDHC activity in treated E375K and D479V cells of patients. Activity values are presented as the percentage of normal PDHC activity measured in healthy fibroblasts in the same experiments. Activity assays were repeated three times. Values are mean values±SD. PDHC, pyruvate dehydrogenase complex. Co-localization was also observed in treated G229CN35X patients' cells.

[0078] FIG. 6. Enzymatic Activity of LAD in plasma of E3 mice injected with TAT-LAD. Behavior and stability of the injected TAT-LAD were followed in the plasma of injected mice by measuring LAD enzymatic activity. Blood samples from E3 injected mice were withdrawn at different time points, and plasma was prepared.

[0079] FIG. 7A.TAT-LAD activity in various organs of E3 mice treated with TAT-LAD: Time dependency. LAD activity is presented as percent increase from the basal activity measured in the non-treated (PBS-injected) E3 mice.

[0080] FIG. 7B. Effect of TAT-LAD vs. LAD control protein in liver.

[0081] FIG. 7C. Effect of TAT-LAD vs. LAD control protein in brain.

[0082] FIG. 7D. Effect of TAT-LAD vs. LAD control protein in heart.

[0083] FIG. 8A. PDHC Activity in organs of E3 mice treated with TAT-LAD. Results are presented as the percent increase over basal PDHC activity in the same organ of the non-treated (PBS-injected) E3 mice.

[0084] FIG. 8B. Effect of TAT-LAD vs. LAD control protein in liver.

[0085] FIG. 8C. Effect of TAT-LAD vs. LAD control protein in brain.

[0086] FIG. 8D. Effect of TAT-LAD vs. LAD control protein in heart.

[0087] FIG. 9A. PDHC Activity vs. LAD activity in livers of E3 mice treated with TAT-LAD.

[0088] FIG. 9B. PDHC Activity vs. LAD activity in brains of E3 mice treated with TAT-LAD.

[0089] FIG. 9C. PDHC Activity vs. LAD activity in hearts of E3 mice treated with TAT-LAD.

[0090] FIG. 10: Complex I activity is restored in cells from patients with complex I deficiency that are treated with TAT-ORF66. “PBS” refers to untreated cells.

DETAILED DESCRIPTION OF THE INVENTION

[0091] In one embodiment, the present invention provides a composition for treating or alleviating a mitochondrial disorder, comprising a fusion protein, wherein the fusion protein comprises a protein transduction domain (PTD) fused to a functional component of an enzyme of a mitochondrial multi-component enzyme complex. In certain preferred embodiments, the fusion protein is produced by recombinant techniques As provided herein, provision of PTO-fusion proteins containing a catalytic domain of a mitochondrial enzyme to a subject in need thereof is capable of treating and alleviating mitochondrial metabolic disorders.

[0092] In certain preferred embodiments, the fusion protein of methods and compositions of the present invention further comprises a mitochondria targeting sequence (MTS). The MTS is preferably selected from the group consisting of (a) the naturally occurring MTS of the mitochondrial enzyme or (b) an MTS of another mitochondrial enzyme that is encoded by the nuclear DNA, translated/produced in the cytoplasm, and transported into the mitochondria. In other preferred embodiments, such as those exemplified herein, the MTS is that of the mitochondrial enzyme whose catalytic domain is present in the fusion protein. Thus, the entire wild-type sequence of the enzyme, or a fragment thereof containing both the MTS and the catalytic domain, may be used in fusion proteins of the present invention. It will be understood to those skilled in the art that the MTS's of various mitochondrial enzymes synthesized in from nuclear genes are largely if not completely interchangeable, and thus may be used in an interchangeable fashion in methods and compositions of the present invention.

[0093] In certain preferred embodiments, the MTS is situated between the PTO and the enzyme or functional component thereof, as the case may be. In certain more preferred embodiments, the portion of the fusion protein C-terminal to the MTS consists of the functional component of an enzyme. In another embodiment, no residues heterologous to the enzyme are present C-terminal to the MTS. In this embodiment, cleavage of the MTS generates an enzyme with the native sequence, thus able to readily integrate into a conformationally-sensitive multi-component enzyme complex.

[0094] In certain preferred embodiments, the PTD is a TAT peptide. In other embodiments, the PTD is another PTD known in the art that is capable of traversing the cellular and mitochondrial membranes of a eukaryotic cell. Non-limiting representative examples of suitable PTD sequences are listed herein.

[0095] Each type of fusion protein represents a separate embodiment of the present invention.

[0096] In another embodiment, the present invention provides a pharmaceutical composition for treating or alleviating a mitochondrial disorder, comprising a pharmaceutically acceptable carrier and as an active ingredient a fusion protein of the present invention.

[0097] In another embodiment, the present invention provides use of a fusion protein of the present invention for the preparation of a medicament for the treatment of a mitochondrial disorder.

[0098] In another embodiment, the present invention provides a method for treating a mitochondrial disorder, the method comprising the step of administering to a subject in need of such treatment a therapeutically effective amount of a fusion protein of the present invention, thereby treating a mitochondria disorder. Upon entry into a mitochondrion of the subject, the fusion protein restores the missing enzymatic activity.

[0099] In another embodiment, the present invention provides a method for introducing a mitochondrial enzyme activity into a mitochondria of a subject, the method comprising the step of administering to a subject in need of such treatment a. therapeutically effective amount of a fusion protein of the present invention, thereby introducing a mitochondrial enzyme activity into a mitochondria of a subject in need thereof

[0100] As provided herein m Examples 1-4, TAT-LAD is able to enter cells and their mitochondria rapidly and efficiently. Moreover, it is able to raise LAD activity within LAD-deficient cells and their mitochondria back to normal activity values and higher. Most importantly, it is able to replace the mutated enzyme and be naturally incorporated into α-ketoacid dehydrogenase complexes such as the PDHC. We sow here that PDHC activity of LAD deficient cells treated with TAT-LAD changed from ˜10% to 70-75% of normal activity after only 3 Hr′ of incubation. These high enzymatic activity values decreased following 24 Hr′ of incubation but stably remained well above basal activity. Thus, in a clinical context, a single application may be sufficient for a patient presenting with a life-threatening decompensation episode.

[0101] One advantage of using TAT-fusion proteins for treatment of mitochondrial disorders is their ability to be delivered into virtually all cells with no specificity. When trying to replace a mutated mitochondrial enzyme there is no need for specific targeting but rather to deliver the enzyme into each cell/tissue, reaching primarily high-energy demanding tissues such as muscles, liver, and central nervous system (CNS), which are usually the most affected in these types of disorders.

[0102] Moreover, LAD-TAT exhibited a very rapid mode of action, raising whole-cell LAD activity in LAD-deficient cells back to normal values after only 30 min incubation and even higher values upon prolongation of treatment (FIG. 2D-E). Normal LAD activity in fibroblasts ranges between 60-140 nmol/min/mg and in asymptomatic carriers of LAD deficiency between 25-50 nmo/min/mg (Berger, 1996).

[0103] The PDHC is a macromolecular multi-component enzymatic machine. Its assembly process involves numerous different subunits. Optimal positioning of individual components within this multi-subunit complex directly affects the efficiency of the overall enzymatic reaction and the stability of its intermediates (Vettakkorumakankav, 1996; Berger, 1996; Del Gaizo 2003b). Given the structure of the complex, restoration of activity of a whole complex reduced due to a single mutated nonfunctioning component would not have been expected to be treatable by exogenous administration of the mutated component. Interestingly, as demonstrated herein, TAT-mediated replacement of the E3 component was sufficient to increase the enzymatic activity of the whole complex of the POHC (FIG. 5).

[0104] As provided herein, PTD fusion proteins of the present invention raised POHC activity four- to fivefold in a sustained fashion, through the last timepoint at 24 hours (FIG. 5 5B). When treating a metabolic disease such as LAD deficiency, there is no need to augment enzyme activity back to 100%; rather, it need by raised above the energetic threshold required for a normal metabolism. Even slight augmentation in LAD activity can raise ATP synthesis rate and can favorably affect the neurological involvement in LAD deficiency. Therefore the changes demonstrated herein in LAD activity, LAD/CS ratio, and POHC activity are likely to significantly affect clinical presentation in patients at least to the level of asymptomatic LAD deficiency carriers.

[0105] Today, one major impediment of ERT is the inability of the administered enzyme to cross the blood-brain barrier (BBB). This fundamental obstacle has severely limited development of ERT for metabolic disorders in which the CNS is affected (Brady, 2004). TAT-fusion proteins are able to cross BBB, thus making them a favorable choice for development of ERT for metabolic disorders involving the CNS.

[0106] As provided herein in Examples 5-7, the LAD deficiency of E3 mice is treatable by PTO-LAD proteins of the present invention. It is noteworthy that experiments with the E3 mice have established substantial evidence that alternations in α-ketoacid dehydrogenases (the complexes containing LAD) may play a role in the pathogenesis of neurodegenerative diseases. Decreases in activity of the LAD-associated complexes α-ketoglutarate dehydrogenase and pyruvate dehydrogenase, in brain, represent a common element in several age-associated neurodegenerative diseases, including Alzheimer's and Parkinson's diseases (Gibson et al., 2000 and Sullivan and Brown, 2005). Studies of adult LAD-deficient mice have suggested that a partial decrease of LAD, which is sufficient to diminish activity of its associated enzyme complexes (Johnson et al., 1997), results in an elevated level of susceptibility to chemical neurotoxicity (Klivenyi et al., 2004). Moreover, variations in the DLD gene (the mouse analogue of LAD) have been linked to Alzheimer's disease (Brown et al, 2004 and Brown et al 2007). Furthermore, PTD-LAD fusion proteins of the present invention are shown herein to restore LAD and PDHC activity to brain, thus showing that they can cross the BBB and functionally integrate into PDHC there. These results clearly show that PTD-LAD fusion proteins of the present invention are capable of treating neurodegenerative diseases.

EXPERIMENTAL DETAILS SECTION

Materials and Experimental Methods (Examples 1-4)

[0107] Cell Culture

[0108] Fibroblast primary culture cells of patients bearing the mutated genotypes G229CN35X, E375K/E357K and D479V/D479V were established from forearm skin biopsies. Cells were maintained in DMEM (Biological Industries, Beit-Haemek, Israel) supplemented with 15% Fetal Bovine Serum (HyClone, Logan UT, USA), penicillin/streptomycin and L-glutamine (Biological Industries, Beit-Haemek, Israel) in a humified atmosphere with 5% CO2 at 37° C. All cell cultures tested negative for mycoplasma contamination. All experiments involving patients' cells were approved by the Hadassah University Hospital ethical review committee.

[0109] Construction of Plasmids Expressing TAT-LAD and LAD Proteins.

[0110] TAT fusion proteins were generated using the pTAT plasmid, provided by Dr. S. F. Dowdy. The plasmid contains a gene encoding a 6-histidine His-tag, followed by the TAT peptide (AA 47-57). To construct a pTAT plasmid with LAD fused to the His-tagged TAT peptide, the gene for human LAD precursor was amplified by PCR from a placental cDNA library using the oligonucleotides set forth in SEQ ID NO: 1 (forward) and SEQ ID NO: 2 (reverse). The PCR product was cloned downstream of the TAT sequence into a BamHI/XhoI-digested pTAT vector.

[0111] The TAT-A-LAD expression plasmid was constructed by PCR amplification of the mature LAD sequence from the TAT-LAD plasmid using the oligonucleotides set forth in SEQ ID NO: 5 (forward) and SEQ ID NO: 6 (reverse). The PCR product was cloned downstream of the TAT sequence into a BamHI/XhoIcut pTAT plasmid.

[0112] A control LAD protein lacking the TAT peptide was also cloned. The LAD expression vector was generated by subcloning the LAD fragment into a modified pTAT vector lacking the TAT sequence; nucleotide and amino acid sequences of the control LAD protein are set forth in (SEQ ID NO: 45-46, respectively). All clones were confirmed by sequencing analysis. Examples of the sequences used are given below:

[0113] The TAT-LAD DNA sequence—(includes His tag, TAT peptide, and the gene for human LAD precursor) is set forth in SEQ ID NO: 3. The amino acid sequence is set forth in SEQ ID NO: 4.

[0114] The naturally-occurring LAD MTS has the sequence set forth in SEQ ID NO: 39. The sequence used in TAT-LAD is identical except that it lacks the N-terminal Met and is set forth in SEQ ID NO: 41.

[0115] Expression and Purification of Proteins

[0116] E. coli BL21-CodonPlus (ADE3) competent cells transformed with plasmids encoding the fusion proteins were grown at 37° C. in SLB medium containing kanamycin (50 μg/ml), tetracycline (12.5 μg/ml) and chloramphenicol (34 μg/ml). At an OD.sub.600 of 0.8, protein expression was induced by adding IPTG (1 mM, final concentration). After a 24-hr incubation at 22° C., cells were harvested by centrifugation (2000×g for 15 min at 4° C.) followed by sonication in binding buffer (PBS pH7.4, PMSF 1 mM and10 mM imidazole (Sigma-Aldrich, St. Louis, USA)). The suspensions were clarified by centrifugation (35,000×g for 30 min at 4° C.), and the supematants containing the fusion proteins were purified under native conditions using HiTrapTM Chelating HP columns (Amersham-Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with binding buffer. Columns were washed by stepwise addition of increasing imidazole concentrations. Finally, target proteins were eluted with elution buffer (PBS pH7.4 and 500 mM Imidazole). All purification procedures were carried out using the AKTA™ FPLC system (Amersham-Pharmacia Biotech, Uppsala, Sweden). Removal of imidazole was performed by dialysis against PBS (pH 7.4). Proteins were kept frozen in aliquots at −20° C. until use.

[0117] Western Blot Analysis

[0118] Proteins (5-20 μg protein/lane) were resolved on 12% SOS-PAGE gels and transferred onto an Immobilon-PTM Transfer membrane (Millipore, Bradford, USA). Western blots were performed using anti-LAD (Elpeleg 1997), anti-His (Amersham-Pharmacia Biotech, Uppsala, Sweden), anti-α-Tubulin (Serotec, Oxford, UK) and anti-VDAC (porin) (Calbiochem, Darmstadt, Germany) antibodies at 1:1000, 1:10,000, 1:10,000, or 1:5000 dilutions, respectively.

[0119] Delivery of Fusion Proteins Into Cells

[0120] Cells were plated on 6-well plates or in 250 ml flasks (NUNC Brand Products, Roskilde, Denmark). When cells reached 90% confluency, medium was replaced with fresh medium containing 0.05-0.1 mg/ml (final concentration) TAT-fusion proteins for various time periods. After incubation, cells were washed with PBS, trypsinized, pelleted and kept at −80° C. till further use. Pellets were then resuspended in PBS containing 0.5% Triton X-100 and 1 mM PMSF (Sigma-Aldrich, St. Louis, USA), kept on ice for 10 minutes and centrifuged at 15,000×g for 10 minutes. The supernatants were analyzed by western blotting analysis or for enzyme activity.

[0121] Isolation of Sub-Cellular Fractions

[0122] Mitochondrial fractions were isolated from cultured cells using a differential centrifugation technique (Bourgeron 1992). Cells were washed with PBS, tripsinized and pelleted. The cells' pellets were kept frozen at −80° C. till use. Pellets were resuspended in ice-cold Tris-HCl buffer (10 mM, pH7.6, 1 mM PMSF) and homogenized with a Dounce homogenizer (Teflon-glass). The homogenates were combined with sucrose (0.25 M, final concentration) and centrifuged for 10 min at 600×g at 4° C. The supernatants were collected and centrifuged for 10 min at 14,000×g at 4° C. The resulting pellets containing the mitochondria were resuspended in PBS containing 0.5% Triton X-100 and 1 mM PMSF and incubated on ice for 15 min 20 before being analyzed for enzymatic activities and Western blots. Purity of sub-cellular fractions was confirmed by Western blotting using the following specific marker antibodies: α-tubulin for cytoplasm and VDAC (porin) for mitochondria.

[0123] LAD and Citrate Synthase (CS) Activity Assays

[0124] LAD and CS activities were determined for whole-cell protein extracts, sub-cellularfractions or purified TAT-fusion proteins.

[0125] LAD activity was determined as described m Berger, 2005. The reaction was performed in potassium phosphate buffer (50 mmol/l, pH 6.5) containing EDTA (1 mmol/l) and NADH (1.5 mmol/l) (Sigma-Aldrich, St. Louis, USA). Following addition of Lipoamide (2 mmol/l) (Sigma-Aldrich, St. Louis, USA), the decrease in absorbance from a steady state was measured spectrophotometrically at 340 nm (Uvikon XL, Bio-Tek Instruments, Milan, Italy).

[0126] CS activity was determined by following spectrophotometrically (412 nm) the appearance of free SH-group of the released CoA-SH upon the addition of 10 mM oxaloacetate to sub-cellular fractions to which 100 uM acetyl-CoA and 2 mM DTNB (Dithionitrobenzoic acid; Sigma-Aldrich, St. Louis, USA) was added.

[0127] Analysis of Cells Treated with TAT-LAD by Fluorescence and Confocal Microscopy

[0128] TAT-LAD and LAD proteins were fluorescently labeled with Fluorescin (FITC) using a protein labeling kit (EZ-Label, PIERCE Biotechnology, Rockford Ill., USA) according to the manufacturer' s protocol. Unbound fluorescent dye was removed by dialysis against PBS. Cells grown on coverslips to 50-70% confluency were treated with FITC-labeled TAT-LAD or LAD (0.1 mg/mL final concentration) for various time periods. When indicated, cells were further incubated with the mitochondrial selective fluorescent dye MitoTracker-Red CMXRosTM (Molecular Probes, Eugene, USA, 200 nM). Cells were then washed with PBS, fixed in 3.7% formaldehyde in PBS for 10 min at room temperature, and washed again. In fluorescence experiments, cells were analyzed directly without fixation. Cells were analyzed with a fluorescence microscope. (NIKON 90 Nikon Corporation, Tokyo, Japan) or a confocal laser scanning microscope (NIKON Cl, Nikon Corporation, Tokyo, Japan).

[0129] PDHC Activity Assay

[0130] PDHC activity was determined using radioactive pyruvate as follows: Frozen cell pellets were suspended and sonicated in 0.25 ml potassium-phosphate buffer (10 mM, pH 7.4). The reaction was performed in 0.4 ml reaction buffer containing 200-300 μg protein whole-cell extracts and was terminated by adding 1M perchloric acid. The .sup.14CO.sub.2 was collected in Hyamine Hydroxide™ (Packard, USA) and counted in a liquid scintillation (UltimaGold™, Packard, USA) counter (Kontron Instruments, Zurich, Switzerland). Controls with no coenzymes were conducted simultaneously to account for background .sup.14CO.sub.2 release.

[0131] Delivery and Processing of the Fusion Proteins

[0132] Mitochondria isolated from healthy fibroblasts and radioactive-labeled TAT-LAD protein and control TAT-LAD protein were used. In vitro translation of the proteins was performed using the TnT Quick Coupled Transcription/Translation System™ (Promega, Madison, Wis.) in the presence of [.sup.35S]-methionine (Amersham Biosciences, Piscataway, N.J.). Isolated mitochondria were incubated with the radio-labeled proteins (1 mg/ml mitochondria, 1:10 volume-to-volume ratio) for 30 minutes at 30° C., then pelleted, washed with buffer A, and treated with 2.5 μg/ml proteinase K (Roche Diagnostics, Mannheim, Germany) for 10 minutes on ice. Phenylmethylsulphonylfluoride was added (1 mmol, final concentration) to stop the reaction. Mitochondria were then re-pelleted, washed, and analyzed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels that were fixed, dried and visualized using a Phosphorimager™ (BAS-2500; Fujifilm, Valhalla, N.Y.).

Example 1

Construction, Expression, Purification and In-Vitro Activity of TAT-LAD and LAD Proteins

[0133] Over-expression and purification of the fusion protein TAT-LAD was accomplished by inserting the precursor human LAD sequence into the pTAT vector. Expression vectors encoding TAT-.1-LAD, lacking the MTS sequence, and a control LAD protein lacking the TAT peptide were also constructed (FIG. 1A). These proteins were all expressed and highly purified under the same conditions. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis and Western blotting confirmed the identity of these highly purified proteins (FIG. 1B). These purified LAD-based fusion proteins were found to be highly active in an in vitro LAD enzymatic activity assay (FIG. 1C).

Example 2

Delivery of TAT-LAD into LAD Deficient Cells

[0134] The next experiment examined the ability of protein transduction domains (PTD's) such as TAT to deliver the human LAD enzyme into cultured cells from patients with LAD deficiency. Purified TAT-LAD was incubated for different time periods with cells from patients heterozygous for the G229C/Y35X and E375K LAD mutations. Whole-cell protein extracts were prepared and analyzed by Western blotting using anti-LAD antibodies. TAT-LAD fusion protein (58 kDa) rapidly entered G229C/Y35X cells and was detectable after 30 minutes of incubation (FIG. 2A). In cells homozygous for the E375K mutation (FIG. 2B), its delivery was somewhat slower; it was detected within the cells after a 2-hour incubation. Endogenous mutated LAD (50 kDa) was detected only in G229C/Y35X cells and not in E375K cells (FIG. 2A). In both cell lines, steady state was reached after 2-3 hours; thus, the amount of the fusion protein remained similar through the 6-hour (FIG. 2A) and 24-hour (FIG. 2B) timepoints.

[0135] Delivery of TAT-LAD into cells was also followed using direct fluorescence analysis. TAT-LAD was fluorescently labeled with Fluorescin (FITC), incubated with G229CN35X cells for different time periods, and analyzed by fluorescence microscopy. FITC-labeled LAD protein lacking the PTD moiety was used as a control protein. TAT-LAD was efficiently delivered into the cells (FIG. 2C, panels 1-3) whereas fluorescence signals were not detected in cells treated with the control LAD protein (FIG. 2C, panel 4). These results correlated with the Western blot analysis (FIG. 2A-B). TAT-LAD was detected rapidly within cells (after only 30. min of incubation; FIG. 2C, panel 1) and there were no differences in fluorescence signal intensity after longer incubation periods (FIG. 2C, panels 2-3).

[0136] To test the ability of a PTD to deliver an active human LAD enzyme into LAD-deficient cells, purified TAT-LAD was incubated with G229CN35X and E375K cells for different time periods. These experiments utilized the control LAD protein and TAT-PAH protein, which is a control TAT-fusion enzyme that lacks LAD activity.

[0137] Protein extracts of treated cells were analyzed for their LAD activity. Activity of LAD within the cells increased dramatically in concordance with incubation time, reaching steady state after 2-3 Hr′ (FIG. 2D-E). These results resembled those observed by the Western analysis. This augmentation in LAD activity within patients' cells was dose-dependent, and was not observed following addition of control LAD protein.

[0138] In G229CN35X cells, LAD activity increased by 2.5-fold (from 31 nmol/min/mg to 78 nmol/min/mg) after only 30 min of incubation and reached equilibrium of 230-250 nmol/l min/mg, an 8-fold increase, after 2-3 Hr′ (FIG. 2D). G229CN35X cells incubated with control proteins TAT-PAH or LAD showed no change in basal LAD activity, >20 nmol/min/mg, which is lower than normal values (Saada 2000). In E375K cells (FIG. 2E), the same trends were observed. LAD activity increased by −9 fold (increasing from 5 nmol/min/mg to 423 nmol/min/mg) after 2 Hr′ of incubation and reached equilibrium of 630-690 nmol/min/mg after 4 Hr′ of incubation, which lasted through the last time 24 Hr′ incubation. E375K cells that were incubated with the control protein LAD showed no change in their basal LAD activity.

[0139] Though treated with identical protein concentrations, E375K and G229CN35X cells responded differently as maximum activity values were much higher in E375K than m G229CN35X cells, indicating possible differences in treatment efficiency in patients bearing different genotypes.

Example 3

Delivery of TAT-LAD into Mitochondria

[0140] The next step was to examine the ability of TAT-LAD to be delivered across the mitochondrial membrane and naturally processed in mitochondria. In vitro-translated [.sup.35S]-methionine-labeled TAT-LAD was incubated with isolated mitochondria from healthy fibroblasts. The mitochondria were treated with proteinase K to digest proteins nonspecifically adsorbed to the outer membrane, thereby ensuring that the mitochondrial extract contained only proteins within the mitochondria. As a control, .sup.35S-methionine-labeled TAT-LAD protein lacking the MTS (and consequently lacking the natural processing site within it) was used. As seen in FIG. 3A, TAT-LAD and TAT-Δ-LAD were both expressed at their expected molecular sizes of 58 and 54 kd, respectively. After treatment, both TAT-LAD and TAT-Δ-LAD were detected within the mitochondria after 30 minutes of incubation (FIG. 3B), because of the PTD sequence that these proteins carry. However, only the TAT-LAD fusion protein was processed to its mature size, as indicated by the appearance of an additional 50-kd band on the SOS-PAGE autoradiograph (FIG. 3B, asterisk). As expected, the TAT-Δ-LAD protein (lacking the MTS) was not processed, and appeared as a single band at its full unprocessed size. Thus, TAT-LAD is able to be delivered into mitochondria and processed therein.

[0141] It was next examined whether TAT-LAD was able to reach mitochondria after being delivered into intact cells. Purified TAT-LAD was incubated with G229CN35X and D479V cells for different time periods. After incubation, mitochondrial and cytoplasmic sub-cellular fractions were prepared and analyzed for presence of TAT-LAD and for LAD enzymatic activity. CS activity was utilized as a mitochondrial marker. Western blot of sub-cellular fractions indicated the presence of TAT-LAD (58kDa) in both cytosolic and mitochondrial fractions of treated G229CN35X and D479V cells following 4 and 6 Hr′ of incubation (FIGS. 4D and H, respectively). Purity of sub-cellular fractions was confirmed using antibodies against the sub-cellular markers α-tubulin (50 kDa) for the cytoplasm and VDAC (porin) (31 kDa) for the mitochondria.

[0142] In support of these findings, there was a significant increase in LAD activity in both cytosolic and mitochondrial fractions of cells treated with TAT-LAD.

[0143] In G229CN35X cells, LAD activity in mitochondrial fractions increased by 7-fold (from 28 nmolmin/mg to 205 nmolmin/mg) after a 4 Hr′ incubation (FIG. 4A).

[0144] Enzymatic activity remained about the same after 6 Hr′ (193 nmolmin/mg) demonstrating that equilibrium had been reached. This dramatic increase in LAD activity was also measured in cytosolic fractions, changing from 10 nmomin/mg to 222 and 339 nmolmin/mg after 4 Hr′- and 6 Hr′ incubations, respectively (FIG. 4A). Similar results were observed with D479V cells. LAD activity in mitochondrial fractions changed from 28 nmolmin/mg to 165 and 117 nmolmin/mg after 4 Hr′- and 6 Hr′ incubations, respectively (FIG. 4E). In cytosolic fractions, activity changed from 20 nmolmin/mg to 125 and 193 nmolmin/mg after 4 Hr′ - and 6 Hr′ incubations, respectively.

[0145] In addition, CS enzymatic activity was determined in G229CN35X cells (FIG. 4B) and D479V cells (FIG. 4F). CS is a mitochondrial matrix enzyme that participates in the Krebs cycle, converting Acetyl-CoA to Citrate. CS enzymatic activity assay was used as a control reference to verify the purity of mitochondrial sub-fractions and also to calculate LAD/CS ratio to standardize LAD enzymatic activity values. In both cell lines, CS activity in cytosolic fractions was barely detectable, while in the mitochondrial fractions it was within the range of normal levels for fibroblasts, thus verifying the purity of sub-cellular fractions. Furthermore, CS activity was constant and almost identical in all mitochondrial fractions, enabling proper standardization of LAD activity values. Mitochondria of G225C/Y35X exhibited LAD/CS ratios of 0.102 before incubation and 0.740 and 0.678 after 4 Hr′- and 6 Hr′ incubations with TAT-LAD, respectively (FIG. 4C). In mitochondria of D479V treated cells, the LAD/CS ratio changed from 0.142 to 0.715 and 0.561 after 4 Hr′- and 6 Hr′incubation, respectively (FIG. 3G).

[0146] Co-localization experiments were used to further confirm delivery of TAT-LAD into the mitochondria of LAD-deficient cells. FITC-labeled TAT-LAD was incubated with G229CN35X cells grown on coverslips for different time periods. Cells were then incubated with the mitochondrial-selective fluorescent dye MitoTracker-Red CMXRosTM and analyzed by confocal microscopy. As shown in FIG. 5A, TAT-LAD (green fluorescence, middle column) co-localized with mitochondria (red fluorescence, left column) within the first 30 minutes of incubation, as indicated by the yellow staining in the merge (right column).

Example 4

A PTD-LAD Fusion Protein Augments PDHC Activity in LAD-Eeficient Cells

[0147] The final and most crucial test for TAT-LAD's ability to successfully treat LAD deficiency by ERT is the enzyme's ability to substitute for the mutated endogenous enzyme, including successful integration into its natural multi-component enzymatic complexes such as pyruvate dehydrogenase complex (PDHC). LAD deficiency affects three mitochondrial multi-component enzymatic complexes, whose activity could be restored by TAT-LAD. The ability of TAT-LAD to successfully replace the endogenous defective enzyme and increase the activity of PDHC was tested in D479V and E375K cells.

[0148] PDHC activity was increased in the two genotypically different cells. In E375K cells, PDHC activity increased significantly by 12-fold after 3 hours of incubation (from 0.029 to 0.367 nmol/min/mg), remaining approximately four- to fivefold higher than the low basal values for at least 24 hours (FIG. 5B). Presented as a percentage of normal PDHC activity of healthy fibroblasts, the PDHC activity in D479V cells increased from 9% to 69% of normal activity after 3 hours of incubation, remaining at 50% of the normal level for at least 24 hours. In E375K cells, PDHC activity increased from 5 to 75% of normal activity after 3 hours incubation, declining to about 30% after 24 hours of incubation (FIG. 5C). Of note, these PDHC activity values are in close correlation with LAD enzymatic activity values measured in mitochondria of treated cells, reaching maximum levels after 3 Hr′ incubation with TAT-LAD.

[0149] PTD-LAD fusion proteins are thus able to treat LAD deficiency by augmenting PDHC activity in LAD-deficient cells.

Example 5

Enzymatic Activity of LAD in Plasma of E3 Mice Injected with TAT-LAD

Materials and Experimental Methods (Examples 5-6)

[0150] The mouse model of LAD deficiency is described in Klivenyi, P. et al (Mice deficient in dihydrolipoamide dehydrogenase show increased vulnerability to MPTP, malonate and 3-nitropropionic acid neurotoxicity. J Neurochem 88: 1352-1360, 2004) and Johnson, M T et al (Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality. Proc Natl Acad Sci USA 94: 14512-14517, 1997). These mice are heterozygotes to a recessive loss-of-function mutation affecting LAD gene (Did, in mice) expression at the mRNA level (instability) (Did+/−mice or E3 mice). Homozygous mice die in-utero at a very early gastrulation stage. These mice are phenotypically normal, though their LAD activity is reduced by ˜50%, affecting all the LAD-dependent enzyme complexes. Similarly, humans heterozygous for LAD deficiency exhibit −50% LAD activity, but usually have no clinical symptoms. These mice are currently used in experiments in the field of neurodegenerative disorders including Alzheimer's, Parkinson's and Huntington's disease.

[0151] A single dose (0.2 mg per mouse) of highly purified TAT-LAD was injected into the tail vein of E3 mice, and several tissues were extracted and analyzed for LAD and PDHC activities at different time points. Several mice were used at each time point.

Results

[0152] To test the ability of TAT-LAD to treat LAD deficiency in vivo, purified TAT-LAD was injected intravenously into E3 mice, and its effect on LAD and PDHC activities was measured in several tissues. This experiment concentrated our on 3 major organs that have the highest energy demands and thus are often affected in mitochondrial disorders—the liver, the heart (muscles) and the brain.

[0153] First, behavior and stability of the injected fusion protein TAT-LAD in the plasma of injected mice were characterized by measuring LAD enzymatic activity. Blood samples from E3 injected mice were withdrawn at different time points, and plasma was prepared.

[0154] No LAD activity is present in the plasma of either normal healthy mice or E3 mice, so the LAD activity at the first time point was set as the reference. Following the first time point, a decrease in LAD activity was observed in the plasma of E3 mice, over time (FIG. 6). To determine whether a component or factor exists in the plasma that reduced LAD activity over time; mouse plasma was incubated with TAT-LAD in vitro under the same concentrations: at 37° C. and for the same time periods. LAD activity remained stable in these plasma samples. Thus, the decrease in LAD enzymatic activity in the plasma was a result of delivery of TAT-LAD into the organs and tissues of mice. Indeed, these results correlate with the LAD activity measured within the organs (FIG. 7 below). The LAD control protein, lacking the TAT delivery moiety, also decreased its activity in plasma overtime, suggesting possible clearance mechanisms in this case.

Example 6

TAT-LAD Increases LAD Activity in Organs of LAD-Deficient Mice

[0155] Organs were harvested from the mice described in the previous Example, and LAD activity there was measured. FIG. 7A depicts the percentage increase from the basal activity measured in the heterozygous mice, namely E3, non-treated mice, injected only with PBS. A single intravenous injection of TAT-LAD (0.2 mg per mouse) significantly increased LAD enzymatic activity within the liver, heart and most importantly—in the brain after only 30 minutes. The shapes of the curves were similar in the brain and heart and slightly different in the liver (FIG. 7C-D and B, respectively).

[0156] Even more robust increases were observed at steady state. In liver, LAD activity reached a steady state at about 40% of non-treated mice and remained at the same level for up to 6 hours, while in brain and heart, steady-state LAD activity was higher, peaking at 4 hours at levels of 80% and 100%, respectively. The LAD control protein, lacking the TAT delivery moiety, injected in the same amount and under identical conditions, did not significantly increase in LAD activity in the organs. In addition and also of importance was the fact that 24 hours following the injection, LAD activity was still 10% higher than the basal activity.

[0157] Thus, PTO-LAD fusion proteins are able to fully restore deficient LAD activity in a LAD-deficient disease model and thus are able to treat acute decompensation episodes. The long-term magnitude of the increase after only a single treatment, 10%, is also sufficient to affect the clinical status of many cases.

Example 7

TAT-LAD Increases PDHC Activity in Organs of LAD-Deficient Mice

Materials and Experimental Methods

[0158] Principle of PDHC activity measurement in mice's tissues. A kit from MitosciencesTM (Catalog No. MSP18) for measuring PDHC enzymatic activity was used. PDHC was immuno-captured from tissue lysates, and its enzymatic activity is measured. This ensured that any increase in the measured PDHC activity resulted only from the TAT-LAD that had become integrated into the PDHC complex. The enzymatic assay measures reduction in NAD.sup.+ to NADH by an increase in absorbance at 340 nm.

Results

[0159] The next experiment directly tested the ability of PTD-LAD fusion proteins to substitute for the mutated endogenous enzyme, following successful integration into its natural multi-component enzymatic complexes, in the organs of the TAT-LAD-injected mice described in Example 5. FIG. 8A depicts the percentage increase over basal PDHC activity of untreated E3 mice (mock-treated by injection with PBS) in each organ. Brains and hearts (FIG. 8C-D, respectively) of treated E3 mice both responded robustly to TAT-LAD treatment; peaking at 4 hours, with a 145% increase in PDHC enzymatic activity; liver samples (FIG. 8B) peaked at 2 hours with a 135% increase in the activity. A substantial and significant increase in PDHC enzymatic activity (40-65%) was also evident in the three organs at 24 hours post-treatment. Treatment with the control protein LAD did not affect the basal PDHC activity. Interestingly, the percent increase in PDHC activity was much greater than that of LAD activity in the tissues, highlighting the potency of the fusion proteins used (FIG. 9A-C).

[0160] Thus, a single application of a PTD-LAD fusion protein is able to significantly increase PDHC activity in a disease model of LAD deficiency. PTO-LAD fusion proteins are thus able to treat and ameliorate LAD deficiency pathologies.

Example 8

TAT-ORF66 Restores Complex I Activity in the Cells of a Patient with NADH:Ubiquinone Oxidoreductase (Complex n Deficiency

Materials and Experimental Methods

[0161] In order to construct a plasmid expressing a TAT-C60RF66 fusion, the gene for human C60RF66 was amplified by PCR from lymphocytes complementary DNA library, using the oligonucleotides set forth in SEQ IN NO: 47 (forward) and SEQ IN NO: 48 (reverse). The PCR product was cloned downstream of the TAT sequence into a BamHI/XhoI-digested pTAT fragment.

Results

[0162] A missense mutation in a conserved residue of the C60RF66 gene has been identified in a consanguineous family that presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (Complex I) deficiency. In muscle of patients, levels of the C60RF66 protein and of fully assembled Complex I were markedly reduced. Transfoction of the patients' fibroblasts with wild-type C60RF66 cDNA restored complex I activity (Saada A et al, C60RF66 is an assembly factor of mitochondrial complex I. Am J Hum Genet 82(1):32-8, 2008).

[0163] The mRNA sequence of C60RF66 is set forth in SEQ ID NO: 7 (GenBank Accession 10# NM_014165).

[0164] The amino acid sequence of the product of C60RF66 is set forth in SEQ ID NO: 8 (GenBank Accession # NM_014165). The first 34 residues of the protein, (SEQ ID NO: 9), are predicted by the TargetP software to form the mitochondrial-targeting sequence (Saada A et al, ibid).

[0165] To test the ability of a TAT-fusion protein to treat Complex I deficiency, a TAT-C60RF66 fusion protein was constructed and highly purified. Primary fibroblast cells isolated from a patient with the missense mutation in the C60RF66 gene were incubated with TAT-ORF66 for 48 hr, and mitochondria were isolated and analyzed for complex I activity. The TAT-fusion protein was able to restore 80% of wild-type complex I activity in the mitochondria (FIG. 10).

[0166] Thus, Complex I deficiency is treatable using TAT-fusion proteins.

[0167] The findings presented herein demonstrate that a variety of mitochondrial enzymes can be successfully treated by ERT using PTD-based fusion proteins. Deficiencies in LAD, an enzyme that forms part of several multi-component enzymatic complexes, and C60RF66, an assembly factor of Complex I, were successfully treated. Of note, the enzymes were able to translocate into the mitochondria and function in the conformation-sensitive context of these enzymatic complexes with their activity intact, following removal of the heterologous parts of the molecule.

[0168] The findings presented herein demonstrate that a variety of mitochondrial metabolic disorders are treatable by ERT using PTD-based fusion proteins, as evidenced by treatment of both LAD deficiency and Complex I deficiency.

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