METHODS FOR THE TREATMENT OF MITOCHONDRIAL GENETIC DISEASES

Abstract

The invention relates to a method for treating mitochondrial genetic diseases. The inventors have worked with primary fibroblasts from patients and control individuals and collected protein lysates for western blotting. Importantly, they observed that the genetic mitochondrial disorders, show a significant increase in phosphorylation of ribosomal protein S6 (pS6) compared to control fibroblasts, indicative of hyperactivated mTOR signaling. Patients with mitochondrial disorders and controls cells were treated for 48 hours with DMSO or BYL719. All lines from patients with mitochondrial diseases show reduced membrane potential, determined by TMRE staining intensity, and abnormal morphology, fragmentation and the presence of depolarized (low TMRE staining) mitochondria. Treatment with BYL719 attenuated these phenotypes in all MELAS fibroblasts while having no overt impact on the control cells. Similar experiments using flow cytometry confirmed membrane potential (TMRE) rescue by BYL719 treatment in MELAS fibroblasts.

Claims

1. A method for treating mitochondrial genetic diseases in a subject in need thereof comprising administrating to the subject a therapeutically effective amount of a PI3K inhibitor.

2. The method according to claim 1, wherein the PI3K inhibitor is a small molecule.

3. The method according to claim 1, wherein the PI3K inhibitor is BYL719 (Alpelisib).

4. The method according to claim 1, wherein the PI3K inhibitor is GDC-0032 (Taselisib).

5. The method according to claim 1, wherein the mitochondrial genetic disease is Leigh Syndrome.

6. The method according to claim 1, wherein the mitochondrial genetic disease is ataxia.

7. The method according to claim 1, wherein the mitochondrial genetic disease is cerebellar hypoplasia.

8. The method according to claim 1, wherein the mitochondrial genetic disease is kearns-sayre syndrome.

9. A method of screening a drug suitable for the treatment of mitochondrial genetic diseases comprising i) providing a test compound ii) determining the ability of said test compound to inhibit the activity of PI3K, and, based on results from the determining step, iii) identifying the test compound as a suitable drug.

Description

FIGURES

[0031] FIG. 1: mTOR Activation and Mitochondrial Defects in Primary Fibroblast Lines from patients with mitochondrial disorders. A) Western blotting of lysates from primary MELAS patient and control fibroblast lines showing mTORC1 overactivation in MELAS patients. Cells were treated for 48 hours with DMSO, BYL719, or cyclosporin A (CsA) and probed for phosphorylated ribosomal protein S6, total rpS6, and GAPDH and quantification. 48 hours of treatment with BYL719 inhibited S6 phosphorylation, while treatment with cyclosporin A had no significant impact. B) Western blotting of lysates from other mitochondrial disorders and control fibroblast lines showing mTORC1 overactivation in MELAS patients. Cells were treated for 48 hours with DMSO or BYL719 and probed for phosphorylated ribosomal protein S6, total rpS6, and GAPDH and quantification. 48 hours of treatment with BYL719 inhibited S6 phosphorylation. C) Representative confocal microscopy images of primary fibroblast lines treated for 48 hours with DMSO or BYL719 and stained for mitochondrial membrane potential (tetramethylrhodamine ethyl ester, TMRE, red), mitochondrial mass (10-N-nonyl acridine orange, 10-NAO, green), and DNA (Hoechst 33342, blue).

[0032] FIG. 2: daily BYL719 administration dramatically improved animal survival. At the age of 21 days, Ndufs4−/− mice were treated with the PI3KCA inhibitor, BYL719 (MedChem Express; 50 mg.Math.kg-1 in 0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.) (n=9) or vehicle (0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.) (n=8).

EXAMPLES

Example 1: Rescue of Mitochondrial Morphology and Membrane Potential by Short-Term BYL719 Treatment

[0033] Material & Methods

[0034] Patients

[0035] Nine patients with genetic mitochondrial disorders (MELAS n=4, Leigh Syndrome n=3, Ataxia and Cerebellar Hypoplasia n=1 and Kearns-Sayre Syndrome n=1) and 4 healthy control individuals had a skin biopsy with isolation of dermal fibroblasts. Punch skin biopsies were collected using standard methods.

[0036] Generation of Primary Dermal Fibroblast Cultures

[0037] To generate dermal fibroblast cultures, biopsies were minced and incubated at room temperature in 0.05% trypsin-EDTA (ThermoFisher 25300054) solution for 30 min with gentle shaking. Cells were collected by centrifuging at 700 g for 10 min, re-suspended in cell culture media containing 25% FBS, and plated onto 24 well plates to establish lines. Fibroblast cultures were grown and maintained in 1×MEM (Corning 10-010-CV) supplemented with 25% FBS and penicillin/streptomycin (Corning 30-001-CI) to a final concentration of 100 IU penicillin and 500 μg/mL streptomycin.

[0038] Analysis of Mitochondrial Membrane Potential and Morphology

[0039] Cells at similar population doubling (PD-10+/−2) were plated 1:4 from confluent cultures onto coverglass chamberslides and allowed to grow until ˜80% confluent. Media was replaced and supplemented with 5 μmon BYL719 (Chem Express) in DMSO (Fisher BP321-1), or equal volume DMSO, for 48 hours. Cells were stained for 15 min in media with 100 nM tetramethylrhodamine ethyl ester (TMRE, Fisher BDB564696) and 5 μg/mL Hoechst 33342 (Biotium 89139-126) and imaged on a Leica SP5 confocal microscope. Samples were treated and imaged in one session using identical imaging parameters. Flow cytometry analysis was performed by staining cells with only TMRE or 10-N-nonyl acridine orange (10-NAO), dissociating with trypsin-EDTA containing dye for 10 min at room temperature, collecting cells by centrifugation, resuspension in cold PBS, and analysis on a BD Canto II flow cytometer using 488 nm excitation with 585/42 BP and 530/30 BP filters for TMRE and 10-NAO, respectively. A single gate was set to cells using forward and side scatter and all settings unchanged throughout data collection. 10,000 or more events were collected for every sample.

[0040] Analysis of S6 Phosphorylation by Western Blotting

[0041] Cells at similar population doubling (PD-10+/−2) were plated 1:4 from confluent cultures and allowed to grow until ˜80% confluent. Media was replaced with media containing 5 μmol/L BYL719 (Chem Express) in DMSO or equal volume DMSO for 48 hours. Cultures were rinsed with 1×PBS, treated for 10 min with 0.05% Trypsin, collected by centrifugation at 4° C., and pellets were flash frozen on dry ice. Protein lysates were collected by directly adding 1×RIPA buffer (Pierce 89900) containing protease and phosphatase inhibitors (Pierce PI78441) to cell pellets, sonicating in 10 one-second bursts, on ice, with an XL-2000 QSonica at maximum output, and centrifuging to remove cell debris. Protein concentration was determined by BCA assay (Pierce PI23228), equal protein run on 4-12% bis-tris 26 well NuPage midigels (Fisher WG1403), and transferred to nitrocellulose blots (Fisher IB23001). Blots were blocked in LICOR Odyssey blocking buffer (LICOR 427-40100), probed with primary antibodies anti-pS6, anti-S6, and anti-GAPDH (Cell Signaling 4858P, 2217S, and 2118S, respectively) followed by secondary antibody IRDye800 donkey anti-rabbit (LICOR 925-32213) and imaged using LICOR Odyssey Clx scanning imager as previously described.sup.9. Data quantified using NIH ImageJ.sup.11.

[0042] Statistical Analysis

[0043] All data were presented as means+/−SEM. Comparisons between groups were performed using student t-tests, 2-tails. P<0.05 was considered significant. Statistical comparisons of capacity curves were performed using the log-rank test as indicated.

[0044] Results

[0045] Hyperactive mTOR Signaling in Primary Culture of Fibroblasts from Patients with Mitochondrial Disease

[0046] In our previous work we found mTOR to be hyperactivated in whole brain lysates of the Leigh syndrome mouse model.sup.6. To examine if the mTOR pathway was also hyperactive in genetic mitochondrial disorders, we cultured primary fibroblasts from patients and control individuals and collected protein lysates for western blotting. Importantly, we observed that the genetic mitochondrial disorders, show a significant increase in phosphorylation of ribosomal protein S6 (pS6) compared to control fibroblasts, indicative of hyperactivated mTOR signaling (FIG. 1A). We observed also the same findings in all cell lines with mitochondrial disorders (FIG. 1B).

[0047] Patients with mitochondrial disorders and controls cells were then treated for 48 hours with DMSO or BYL719. 48 hours of BYL719 treatment reduced pS6 levels in all cell lines (FIGS. 1A and 1B).

[0048] Rescue of Mitochondrial Morphology and Membrane Potential by Short-Term BYL719 Treatment.

[0049] To examine the impact of mTOR inhibition on mitochondrial morphology and membrane potential, a general measure of mitochondrial function, we treated dermal fibroblasts with BYL719 or DMSO. Cells were stained with TMRE, a marker of mitochondrial membrane potential, and 10-NAO, a marker of the inner mitochondrial membrane which acts as a membrane potential insensitive marker of mitochondrial mass (FIG. 1C). All lines from patients with mitochondrial diseases show reduced membrane potential, determined by TMRE staining intensity, and abnormal morphology, fragmentation and the presence of depolarized (low TMRE staining) mitochondria. Treatment with BYL719 attenuated these phenotypes in all MELAS fibroblasts while having no overt impact on the control cells (FIG. 1C). Similar experiments using flow cytometry confirmed membrane potential (TMRE) rescue by BYL719 treatment in MELAS fibroblasts (data not shown).

Example 2: PIK3CA Inhibition in a Mouse Model of Mitochondrial Disorder

[0050] Material & Methods

[0051] Leigh Syndrome is a severe mitochondrial disease that occurs in about 1:40,000 newborns and is associated with retarded growth, muscular deficits including myopathy and dyspnea, lactic acidosis, and a characteristic progressive necrotizing encephalopathy of the vestibular nuclei, cerebellum, and olfactory bulb (Budde et al., 2002). Ndufs4 encodes a subunit of Complex I of the mitochondrial electron transport chain; mutations in the NDUFS4 gene cause LS in humans (Budde et al., 2000), and the Ndufs4 knockout mouse is a murine model of LS (Kruse et al., 2008). Ndufs4−/− mice have decreased Complex I levels and activity in multiple tissues and show severe and progressive symptoms of mitochondrial disease that mirror human LS. LS results in death at an average of 6-7 years in humans, and Ndufs4 KO mice show a similar early-life mortality with an average lifespan of just 60 days. Heterozygous Ndufs4 knockout mice on a C57Bl/6NIA background were bred to produce homozygous KO animals. Animals were fed ad libitum and housed at a constant ambient temperature in a 12-hour light cycle. Animal procedures were approved by the “Services Vétérinaires de la Prefecture de Police de Paris” Departmental Director and by the ethical committee of the Paris Descartes University.

[0052] At the age of 21 days, Ndufs4−/− mice were treated with the PI3KCA inhibitor, BYL719 (MedChem Express; 50 mg.Math.kg-1 in 0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.) (n=9) or vehicle (0.5% carboxymethylcellulose (Sigma Aldrich), daily p.o.) (n=8).

[0053] Results

[0054] As previously reported, Ndufs4.sup.−/− mice first displayed neurological symptoms with difficulty walking, dyspnea, blindness and finally die around P60.

[0055] Interestingly, we observed that daily BYL719 administration dramatically improved animal survival (Figure. 2). In fact, while all Ndufs4.sup.−/− placebo-treated mice died within 80 days, the BYL719 treated Ndufs4.sup.−/− mice were alive 120 days later with an overtly normal appearance.

[0056] These results demonstrate the effectiveness of PIK3CA inhibition in a mouse model of mitochondrial disorder.

REFERENCES

[0057] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure. [0058] 1. DiMauro S, Schon E A. Mitochondrial respiratory-chain diseases. N Engl J Med 2003; 348:2656-68. [0059] 2. Schon E A, DiMauro S, Hirano M. Human mitochondrial DNA: roles of inherited and somatic mutations. Nat Rev Genet 2012; 13:878-90. [0060] 3. Macmillan C, Lach B, Shoubridge E A. Variable distribution of mutant mitochondrial DNAs (tRNA(Leu[3243])) in tissues of symptomatic relatives with MELAS: the role of mitotic segregation. Neurology 1993; 43:1586-90. [0061] 4. Dimauro S, Mancuso M, Naini A. Mitochondrial encephalomyopathies: therapeutic approach. Ann N Y Acad Sci 2004; 1011:232-45. [0062] 5. Schleit J, Johnson S C, Bennett C F, et al. Molecular mechanisms underlying genotype-dependent responses to dietary restriction. Aging cell 2013; 12:1050-61. [0063] 6. Johnson S C, Yanos M E, Kayser E B, et al. mTOR inhibition alleviates mitochondrial disease in a mouse model of Leigh syndrome. Science 2013; 342:1524-8. [0064] 7. Johnson S C, Yanos M E, Bitto A, et al. Dose-dependent effects of mTOR inhibition on weight and mitochondrial disease in mice. Front Genet 2015; 6:247. [0065] 8. Kreis H, Oberbauer R, Campistol J M, et al. Long-term benefits with sirolimus-based therapy after early cyclosporine withdrawal. J Am Soc Nephrol 2004; 15:809-17. [0066] 9. Canaud G, Bienaime F, Tabarin F, et al Inhibition of the mTORC pathway in the antiphospholipid syndrome. N Engl J Med 2014; 371:303-12. [0067] 10. Canaud G, Bienaime F, Viau A, et al. AKT2 is essential to maintain podocyte viability and function during chronic kidney disease. Nat Med 2013; 19:1288-96. [0068] 11. Schneider C A, Rasband W S, Eliceiri K W. NIH Image to ImageJ: 25 years of image analysis. Nature methods 2012; 9:671-5.