THERAPEUTICAL PEPTIDOMIMETIC

Abstract

The present invention relates to peptidomimetics of a short peptide from leucyl-tRNA synthetase, compositions comprising one or more of said peptidomimetics and their use for the treatment of syndromes caused by mutations of mt-tRNA (mitochondrial transfer RNA) genes, and medical treatments of said syndromes comprising the administration of said one or more peptidomimetics or compositions comprising the same.

Claims

1. A peptide having the amino acid sequence SEQ ID NO 1 and/or a fragment thereof of at least 8 amino acids of length, or variants of said peptide or fragment, wherein said peptide entirely consists of d-amino acids; optionally further conjugated at the N-terminus with a mitochondrial (mt)-targeting sequence.

2. The peptide fragment according to claim 1 wherein said peptide fragments has the amino acid sequence SEQ ID NO 2 or SEQ ID NO 3.

3. The peptide and/or fragment thereof according to claim 1 further conjugated at the N-terminus with said mt-targeting sequence.

4. The peptide and/or fragment thereof according to claim 3 wherein said mt-targeting sequence is 3 to 11 amino acids.

5. The peptide and/or fragment thereof according to claim 4 wherein said mt-targeting sequence comprises at least one arginine and/or lysine and/or phenylalanine residue.

6. The peptide and/or fragment thereof according to claim 4 wherein said mt-targeting sequence is selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18, SEQ ID NO 19, SEQ ID NO 20, SEQ ID NO 21, SEQ ID NO 22, SEQ ID NO 23, SEQ ID NO 24, SEQ ID NO 25, SEQ ID NO 26, SEQ ID NO 27, SEQ ID NO 18 and SEQ ID NO 29.

7. The peptide and/or fragment thereof according to claim 3 wherein said mt-targeting sequence entirely consists of d-amino acids.

8. The peptide and/or fragment thereof according to claim 7 wherein said mt-targeting sequence is selected from the group consisting of SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18 or and SEQ ID NO 19.

9. The peptide according to claim 3 wherein said peptide has the amino acid sequence SEQ ID NO 5.

10. The peptide fragment according to claim 3 wherein said peptide fragments has the amino acid sequence SEQ ID NO 6 or SEQ ID NO 7.

11. (canceled)

12. A method for treating a mt-tRNA-related disease comprising administering the peptide and/or fragment thereof for use according to claim 1.

13. The method according to claim 12 wherein said mt-tRNA-related diseases is selected from the group consisting of mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), MIDD (Maternally Inherited Diabetes and Deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

14. The method according to claim 12 wherein said mt-tRNA-related disease is caused by a point mutation in a gene encoding one of the following mt-tRNAs: mt-tRNA.sup.Leu(UUR), mt-tRNA.sup.Lys, mt-tRNA.sup.IIe or mt-tRNA.sup.Val

15. The method according to claim 14 wherein said mutation is m.Math.3243A>G in the MT-TL1 human gene encoding mt-tRNA.sup.Leu(UUR) or m.Math.8344A>G in the MT-TK human gene encoding mt-tRNA.sup.Lys or m.Math.4277T>C mutation in the mt-tRNA.sup.IIe in the human gene MT-TI or m.Math.1630A>G mutation in mt-tRNA.sup.Val in the human gene MT-TV.

16. (canceled)

17. A pharmaceutical composition comprising one or more peptides and/or fragments thereof as defined in claim 1 and at least one pharmaceutically acceptable carrier.

18. (canceled)

19. A method for treating a mt-tRNA-related disease comprising administering the pharmaceutical composition according to claim 17.

20. The method according to claim 19 wherein said mt-tRNA-related diseases is selected from the group consisting of mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), MIDD (Maternally Inherited Diabetes and Deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

21. The method according to claim 19 wherein said mt-tRNA-related disease is caused by a point mutation in a gene encoding one of the following mt-tRNAs: mt-tRNA.sup.Leu(UUR), mt-tRNA.sup.Lys, mt-tRNA.sup.IIe or mt-tRNA.sup.Val.

22. The method according to claim 21 wherein said mutation is m.Math.3243A>G in the MT-TL1 human gene encoding mt-tRNA.sup.Leu(UUR) or m.Math.8344A>G in the MT-TK human gene encoding mt-tRNA.sup.Lys or m.Math.4277T>C mutation in the mt-tRNA.sup.IIe in the human gene MT-TI or m.Math.1630A>G mutation in mt-tRNA.sup.Val in the human gene MT-TV.

23. (canceled)

24. A process for preparation of a pharmaceutical composition comprising admixing one or more peptides and/or fragments thereof as defined in claim 1 with at least one pharmaceutical acceptable carrier.

25. (canceled)

Description

DETAILED DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1. Upon exogenous administration to mutant cells, all the constructs reported in the image are able to penetrate cell membranes and colocalize with mitochondria.

[0051] Top row (constructs): exogenously administered constructs covalently linked to a fluorescent dye (Cy5) are localized within cells. This result indicates that all constructs are able to penetrate cell membranes.

[0052] Middle row (Mitotracker red): mitochondria within the same cells shown in the top row are highlighted by Mitotracker red, a dye able to link specifically and exclusively to mitochondria.

[0053] Bottom row (merge): co-localization of constructs and mitochondria is revealed by a filter setting for both dyes.

[0054] The cells used for the experiments are trans-mitochondrial hybrids (hereafter named cybrids) bearing the m.Math.3243A>G mutation in mt-tRNA.sup.Leu(UUR), which is associated with the MELAS syndrome.

[0055] The constructs used for the experiments are: the 32_33 peptide (p), previously reported to have rescuing activity on mutant cells (Perli et al, FASEB J, 2020 and Perli et al Hum mol genet 2016, Vol 25 No 5 903-915); the peptide-mimetic-therapeutic of SEQ ID NO 1 (PMT); the PMT fragments comprising PMT residues 1-8 of SEQ ID NO 2 (PMT-8a) and 5-12 of SEQ ID NO 3 (PMT-8b); and the 32_33 peptide linked to elamipretide (E), a different peptide previously reported by Sabbah H N et al 2016 to possess mitochondrial targeting properties and putative mitochondrial protective activity [Sabbah H N, Gupta R C, Kohli S, Wang M, Hachem S, Zhang K. Chronic Therapy With Elamipretide (MTP-131), a Novel Mitochondria-Targeting Peptide, Improves Left Ventricular and Mitochondrial Function in Dogs With Advanced Heart Failure. Circ Heart Fail. 2016 February;9 (2):e002206. doi: 10.1161/CIRCHEARTFAILURE.115.002206. PMID: 26839394; PMCID: PMC4743543.]. Names of all constructs are followed by C to indicate that they are linked to Cy5.

[0056] Cells were incubated for 24 hours with 0.25 M of constructs. Half an hour before imaging, cells were stained with Mitotracker Red. Finally, fluorescence signals were detected with a laser scanning confocal microscope. PCC: Pearson's Correlation Coefficient (meanSEM of six images).

[0057] FIG. 2. Following exogenous administration, the PMT significantly improves cell viability and mitochondrial respiration of mutant cells.

[0058] Top row: Viability of compound-treated cells. The X and Y axes show the compounds used and the percentage of viable cells following treatment, respectively.

[0059] Bottom row: Oxygen consumption of compound-treated cells. The X and Y axes show the compounds used and the amount of consumed oxygen expressed in fMoles per minute per cell, respectively.

[0060] The first bar of each graph represents cells without a pathological phenotype, treated with vehicle only. WT: wild-type; I-8344: cells with extremely low levels of mutation m.Math.8344A>G in mt-tRNA.sup.Lys. The second bar of each graph represents cells with a pathological phenotype, bearing either the MELAS-causing m.Math.3243A>G in mt-tRNA.sup.Leu(UUR) or the MERRF-causing m.Math.8344A>G in mt-tRNA.sup.Lys, treated with vehicle only. All the other bars of each graph represent cells with a pathological phenotype, treated with different compounds. 3243: m.Math.3243A>G mutant cells; H-8344: high m.Math.8344A>G mutation load.

[0061] The cells used for the experiments are cybrids, as in FIG. 1.

[0062] The compounds used for the experiments are the same as those listed in FIG. 1 (i.e., p; PMT; PMT-8a; PMT-8b; and E-p) plus elamipretide (E). The E-p peptide was used in order to verify whether the combination of the prior art peptide (SEQ ID NO 4) or of the peptide of SEQ ID NO 1 with elamipretide had a synergic effect as elamipretide has been described as a mitochondrial targeting sequence. The results of the experiments show that elamipretide does not provide additional advantageous effects to the tested peptides (SEQ ID NO 1 and SEQ ID NO 4) In this case compounds are not linked to Cy5, which is only used for fluorescence experiments. V indicates cells treated with an empty vehicle.

[0063] For viability assessment, cells were plated in either glucose or galactose medium. The reason for this is that a viability phenotype can be appreciated in cells growing on galactose, which forces cells to rely on mitochondrial respiration, but not in cells growing on glucose. After 24 hours incubation, the number of viable cells in the galactose medium was normalized to the number of viable cells in glucose at the same time point, which represents the normal growth condition. Data are compared with the value of mutant cells incubated with vehicle only. MeanSEM of at least three independent experiments is shown.

[0064] Oxygen consumption rate was measured on cells grown on glucose, since variations in this parameter can be appreciated in cells growing in this medium, after 36 hours of treatment. Data are shown compared with the value of the vehicle mutant cells. MeanSEM of three independent experiments is shown.

[0065] .sup.p<0.05, .sup.p<0.0001 for m.Math.3243A>G vs WT cells; .sup.00p<0.01, .sup.ooop<0.001 for H-8344 vs I-8344 cells; *p<0.05, **p<0.01, ***p<0.001 for cells incubated with compounds vs vehicle only.

[0066] FIG. 3. Exogenously administered PMT is neither cyto-or mito-toxic up to 20 M in mutant and wild-type cells.

[0067] Images show the effect of increasing PMT concentrations on healthy and mutant cells, compared with the effect of a cytotoxic (C1) and a mitotoxic (C2) agent, evaluated using the Mitochondrial ToxGlo Assay.

[0068] The X and Y axis show the compounds incubated with cells and the ratio between fluorescent and luminescent signal, respectively. In this assay, fluorescence increase indicates a decrease in cell membrane integrity, and luminescence decrease indicates a decrease in cellular ATP levels. In each graph, the first bar represents the effect of cell treatment with the cytotoxic reagent digitonin (C1); this causes both an increase in fluorescence and a decrease in luminescence, which indicates cellular toxicity. The second bar represents the effect of cell treatment with the mitotoxic agent sodium azide (C2); this causes a decrease in luminescence and has no effect on fluorescence, which indicates mitochondrial toxicity. The other bars represent the effect of cell treatment with 5, 10 or 20 M PMT; this has no effect on either fluorescence or luminescence, indicating absence of cyto-or mito-toxicicity. The horizontal black line represents fluorescence/luminescence signal ratio of untreated cells. WT: wild type; 3243: cells bearing the mutation m.Math.3243A>G in mt-tRNA.sup.Leu(UUR); L-8344 and H-8344: cells bearing low and high load of mutation m.Math.8344A>G in mt-tRNA.sup.Lys.

[0069] Cells used for the experiments are cybrids, as in FIGS. 1 and 2. Compounds used are: PMT not linked to Cy5, at different concentrations; a cytotoxic agent (digitonin); and a mitotoxic agent (sodium azide).

[0070] Cybrids were plated on a 96-well plate in normal growth conditions (i.e., glucose medium) and treated with different PMT concentrations. In parallel, control cells (both wild type and mutated) were incubated with either 400 g/ml digitonin (C1) or 100 l sodium azide (C2) for three hours. Fluorescence and luminescence were measured with a GloMax Multi+Luminometer. Signals observed following each treatment (i.e., 5-10-20 M PMT; C1; and C2) were normalized using values of untreated cells and expressed as fluorescence/luminescence ratio. Data are the meanSEM of two independent experiments.

[0071] FIG. 4. The PMT, PMT-8a and PMT-8b undergo slower degradation than the 32_33 peptide in human plasma.

[0072] Panel A. Representative chromatographic profiles of 32_33 and PMT obtained after compound incubation for 3 h with human plasma from two healthy subjects. The X axis shows the time (in minutes) at which each compound is eluted by the chromatographic column. The Y axis indicates the signal intensity of the two peptides.

[0073] Panel B. Time course decay of 32_33, PMT, PMT-8a and PMT-8b incubated up to 72 h with human plasma from four healthy subjects. The X axis shows the time (in hours) at which the sample is analysed. The Y axis indicates the signal intensity of the four peptides. Samples of plasma were obtained from healthy volunteers and immediately used for the analysis. Either 32_33 peptide or PMT for the experiment in panel A, and 32_33 peptide, PMT, PMT-8a or PMT-8b for the experiment in panel B, was incubated in plasma at a final concentration of 0.2 mM, at 37 C., up to 3 h (A) or 72 h (B). At the indicated time points, i.e., T0 and 3 h (A) or T0, 1.5, 3, 6 and 72 h (B), aliquots of 200 L were treated with 3 volumes of acetonitrile containing 1% formic acid and extracted by a solid phase extraction system to remove proteins and phospholipids. Samples were dried under vacuum, resuspended in 100 L of 0.1% formic acid containing 5% acetonitrile, and analyzed with a Water Acquity H-Class UPLC system equipped with a single-quadruple mass detector with electrospray ionization source. Samples were separated onto a reverse phase C18 column by performing a gradient with two mobile phases consisting of 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.5 mL/min. Quantification was performed by Selected lon Recording (SIR): at m/z=917.88, corresponding to the [M+2 H].sup.2+ion obtained from either 32_33 or PMT; at m/z=961.42, corresponding to the [M+H].sup.+ion obtained from PMT-8a; and at m/z=869.48, corresponding to the [M+H].sup.+ion obtained from PMT-8b.

[0074] FIG. 5. Comparison between the structures of 32_33 peptide and PMT.

[0075] Chemical structure of the 32_33 peptide (A) and PMT (B, C). N, O and H atoms are explicitly indicated, whereas carbon atoms are implicit. Single and double chemical bonds lying on the plane are shown by single and double lines, respectively. Solid wedges indicate bonds projecting out towards the viewer. Broken wedges indicate groups receding away from the viewer. For each chiral center the S or R configuration is indicated. For each pair of corresponding amino acids (e.g., Lys1, Lys2, Ser3, ecc.) side-chains that are directed towards the viewer in the 32_33 peptide (A), are directed away from the viewer in the PMT (B, C), and side-chains that are directed away from the viewer in the 32_33 peptide (A), are directed towards the viewer in the PMT (A). In panel C, chemical groups of the PMT that have a different orientation with respect to the 32_33 peptide (A) are highlighted by grey ovals.

[0076] Since the relative position of all amino acid side-chains with respect to the main chain is opposite in the 32_33 peptide and in the PMT, interactions between the 32_33 peptide and target mt-tRNA involving both main chain and side chain atoms cannot be conserved in the PMT.

[0077] FIG. 6. Following exogenous administration, the M-PMT significantly improves viability of mutant cells at concentrations down to 0.5 M.

[0078] Viability of compound-treated cells. The concentrations of the different compounds used for the experiments and the percentage of viable cells following treatment are shown in the X and Y axes, respectively. The first bar corresponds to wild type cells treated with vehicle only. The second bar represents cells bearing the MELAS-causing m.Math.3243A>G in mt-tRNA.sup.Leu(UUR), treated with vehicle only. The additional bars show the effect of the PMT and M-PMT at decreasing concentrations on mutant cell viability. WT: wild-type cells. 3243: m.Math.3243A>G mutant cells.

[0079] The cells used for the experiments are cybrids.

[0080] The compounds used for the experiment are: PMT at a 5, 2 and 0.5 M concentration, and M-PMT at 5, 2 and 0.5 M concentration. V indicates cells treated with an empty vehicle.

[0081] For viability assessment, cells were plated in either glucose or galactose medium. The reason for this is that a viability phenotype can be appreciated in cells growing on galactose, which forces cells to rely on mitochondrial respiration, but not in cells growing on glucose. After 24 hours incubation the number of viable cells in galactose medium was normalized to the number of viable cells in glucose (that represents the normal growth condition) at the same time point. Data are compared with the value of mutant cells incubated with vehicle only. MeansSEM of at least two independent experiments are shown.

[0082] .sup.ooop<0.0001 for m.Math.3243A>G vs WT cells; *p<0.05 for cells incubated with compounds vs vehicle only.

SEQUENCES DESCRIPTION

[0083] SEQ ID NO 1 PMT all amino acids are d-amino acids KKSFLSPRTALINFLV

[0084] SEQ ID NO 2 PMT-8a all amino acids are d-amino acids KKSFLSPR

[0085] SEQ ID NO 3 PMT-8b all amino acids are d-amino acids LSPRTALI

[0086] SEQ ID NO 4 32_33 KKSFLSPRTALINFLV (Perli et al, FASEB J, 2020 and Perli et al Hum mol genet 2016, Vol 25 No 5 903-915) (all amino acids are I-amino acids)

[0087] SEQ ID NO 5 corresponds to SEQ ID NO 1 conjugated with mitochondrial targeting sequence FRFK, all amino acids are d-amino acids FRFKKKSFLSPRTALINFLV

[0088] SEQ ID NO 6 corresponds to SEQ ID NO 2 conjugated with mitochondrial targeting sequence FRFK, all amino acids are d-amino acids FRFKKKSFLSPR

[0089] SEQ ID NO 7 corresponds to SEQ ID NO 2 conjugated with mitochondrial targeting sequence FRFK, all amino acids are d-amino acids FRFKLSPRTALI

[0090] SEQ ID NO 8 artificial mitochondrial targeting/penetrating sequence 1 FRFK, all amino acids are d-amino acids

[0091] SEQ ID NO 9 artificial mitochondrial targeting/penetrating sequence 2 FRA.sub.xK, all amino acids are d-amino acids

[0092] SEQ ID NO 10 mitochondrial targeting/penetrating sequence 3 Fd (R) FK, only R is a D amino acid, Horton K L et al, 2008

[0093] SEQ ID NO 11 artificial mitochondrial targeting/penetrating sequence 4 A.sub.xRA.sub.xK, all amino acids are d-amino acids

[0094] SEQ ID NO 12 artificial mitochondrial targeting/penetrating sequence 5 FRFKFRFK, all amino acids are d-amino acids

[0095] SEQ ID NO 13 artificial mitochondrial targeting/penetrating sequence 6 FRA.sub.xKFRA.sub.xK, all amino acids are d-amino acids

[0096] SEQ ID NO 14 artificial mitochondrial targeting/penetrating sequence 7 A.sub.xRA.sub.xKA.sub.xRA.sub.xK, all amino acids are d-amino acids

[0097] SEQ ID NO 15 artificial mitochondrial targeting/penetrating sequence 8 RKKRRQRRR, all amino acids are d-amino acids

[0098] SEQ ID NO 16 artificial mitochondrial targeting/penetrating sequence 9 FRF.sub.2K, all amino acids are d-amino acids

[0099] SEQ ID NO 17 artificial mitochondrial targeting/penetrating sequence 10 FRY.sub.MeK, all amino acids are d-amino acids

[0100] SEQ ID NO 18 artificial mitochondrial targeting/penetrating sequence 11 FRYK, all amino acids are d-amino acids

[0101] SEQ ID NO 19 artificial mitochondrial targeting/penetrating sequence 12 YRYK, all amino acids are d-amino acids

[0102] SEQ ID NO 20 mitochondrial targeting/penetrating sequence 13 Fd(R)A.sub.xK, only R is a d amino acid, Horton K L et al, 2008

[0103] SEQ ID NO 21 mitochondrial targeting/penetrating sequence 14 A.sub.xd(R)A.sub.xK, only R is a d amino acid, Horton K L et al, 2008

[0104] SEQ ID NO 22 mitochondrial targeting/penetrating sequence 15 Fd(R)FKFd(R)FK, only R is a d amino acid, Horton K L et al, 2008

[0105] SEQ ID NO 23 mitochondrial targeting/penetrating sequence 16 Fd(R)A.sub.xKFd(R)A.sub.xK, only R is a d amino acid, Horton K L et al, 2008

[0106] SEQ ID NO 24 mitochondrial targeting/penetrating sequence 17 A.sub.xd(R)A.sub.xKA.sub.xd(R)A.sub.xK, only R is a d amino acid, Horton K L et al, 2008

[0107] SEQ ID NO 25 mitochondrial targeting/penetrating sequence 18 RKKRRQRRR, Horton K L et al, 2008

[0108] SEQ ID NO 26 mitochondrial targeting/penetrating sequence 19 Fd(R)F.sub.2K, only R is a d amino acid, Horton K L et al, 2008

[0109] SEQ ID NO 27 mitochondrial targeting/penetrating sequence 20 Fd(R)Y.sub.MeK, only R is a d amino acid, Horton K L et al, 2008

[0110] SEQ ID NO 28 mitochondrial targeting/penetrating sequence 21 Fd(R)YK, only R is a d amino acid, Horton K L et al, 2008

[0111] SEQ ID NO 29 mitochondrial targeting/penetrating sequence 22 Yd(R)YK, only R is a d amino acid, Horton K L et al, 2008

[0112] Abbreviations in the sequences above: F2: diphenylalanine; A.sub.x: Cyclohexylalanine; YMe: methylated tyrosine. When the sole d-aminoacid is arginine, the aminoacid is indicated in the sequence as d(R).

DETAILED DESCRIPTION OF THE INVENTION

[0113] As discussed in the summary of the invention, the peptide of the invention is a peptide-mimetic compound, hereafter indicated as PMT. The PMT comprises only d-amino acids (indicated by one-letter code preceded by lower-case d letter), the sequence of which is SEQ ID NO 1:

[0114] d(K)d(K)d(S)d(F)d(L)d(S)d(P)d(R)d(T)d(A)d(L)d(I)d(N)d(F)d(L)d(V).

[0115] As shown in the figures and discussed in the experimental part below, the PMT, as well as fragments of the same, is able to penetrate cell and mitochondrial membranes upon exogenous administration (FIG. 1), and to rescue the defective phenotype of cell models carrying mt-tRNA mutations (FIG. 2).

[0116] Additionally, exogenously administered PMT is safe up to 20 M in both mutant and wild-type cells and finally, the PMT is extremely stable in human plasma, since after 3 hours incubation in this medium, in a first experiment the PMT is 100% available vs. 70% of the 32_33 peptide (FIG. 4A), and in a second experiment the PMT is>63% available vs. only 17% of the 32_33 peptide (FIG. 4B).

[0117] The invention relates to a peptide having SEQ ID NO 1, said peptide being characterised by consisting exclusively of d-amino acids and to fragments thereof, in particular fragments of at least 8 amino acids, as said peptide and fragments thereof of the indicated size have shown to be excellent peptide-mimetics of the 32_33 peptide (Perli et al, FASEB J, 2020 and Perli et al Hum mol genet 2016, Vol 25 No 5 903-915) in terms of biological activity i.e. rescuing defective phenotype of cell models carrying mt-tRNA mutations, the mimetics showing the advantageous feature of being more stable in plasma than the natural peptide.

[0118] In an advantageous embodiment of the invention, said peptide having SEQ ID NO 1 and fragments thereof, in particular fragments of at least 8 amino acids, can be conjugated at the N-terminus with an mt-targeting sequence. FIG. 6 shows that conjugation with an mt-targeting sequence, such as SEQ ID NO 8, surprisingly improves the effectiveness (i.e., the rescuing activity) of the peptidomimetics of the invention of about 10 folds.

[0119] Hence, object of the invention are also peptides consisting of a d mt-targeting sequence conjugated at the N-terminus of the peptides having SEQ ID NO 1, SEQ ID NO 2 and SEQ ID NO 3.

[0120] In fact, contrary to the data disclosed in Perli et al 2020, wherein the use of a mt-targeting sequence did not increase the rescuing activity and mitochondrial localisation of the peptide having SEQ ID NO 4, the conjugation of the peptides of the invention with a mt-targeting sequence did dramatically increase the rescuing activity of the d-peptides of the invention.

[0121] Preferably, the mt-targeting sequence of the invention, is a sequence of 3-11 amino acids, preferably of 3 to 6 amino acids, and comprises at least one arginine and/or at least one lysine and/or at least one phenylalanine residue.

[0122] Preferably at least one of said arginine and/or at least one phenylalanine residues are d-arginine and/or d-lysine and/or d-phenylalanine.

[0123] According to the invention the mt-targeting sequence can be a sequence selected from SEQ ID NO 8 to SEQ ID NO 29. In an embodiment of the invention the fragments of the peptide of SEQ ID NO 1, conjugated at N-terminus with one of said mt-targeting sequence are the peptides of SEQ ID NO 2 or SEQ ID NO 3.

[0124] In a preferred embodiment, the mt-targeting sequence consists only of d-aminoacids, in a further preferred embodiment said mt-targeting sequence is selected from SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, SEQ ID NO 15, SEQ ID NO 16, SEQ ID NO 17, SEQ ID NO 18 or SEQ ID NO 19.

[0125] In a further preferred embodiment, said mt-targeting sequence consisting only of d-aminoacids is SEQ ID NO 8.

[0126] In a preferred embodiment the peptides conjugated at N-terminus with the mt-targeting sequence having SEQ ID NO 8, are the peptides having SEQ ID NO 5, SEQ ID NO 6 and SEQ ID NO 7.

[0127] Given the important rescuing activity shown by the peptide-mimetics herein disclosed, the invention also relates to the peptide having SEQ ID NO 1 and/or fragments thereof according to any of the embodiments disclosed, preferably conjugated at the N-terminus with an mt-targeting sequence according to any one of the embodiments disclosed above, for use as a medicament.

[0128] In an embodiment the invention also relates to variants of SEQ ID NOs 1, 2, 3, 5, 6 and 7 comprising one or more of the following chemical modifications: [0129] modification/s of residues d-Phe 4, d-Leu 5, d-Arg 8 and/or d-Thr 9 of SEQ ID NO 1; [0130] modification/s of residues d-Phe 8, d-Leu 9, d-Arg 12 and/or d-Thr 13 of SEQ ID NO 5; [0131] modifications of the peptide bond between d-Phe 4 and d-Leu 5 of SEQ ID NO 1 or 2(in that this peptide bond is not present in the PMT-8b fragment, which does not undergo degradation at all in human plasma), [0132] modifications of the peptide bond between d-Phe 8 and d-Leu 9 of SEQ ID NO 5 or 6 (see above); [0133] modifications of the peptide bond between d-Arg 8 and d-Thr 9 of SEQ ID NO 1, d-Arg 4 and d-Thr 5 of SEQ ID NO 3, (in that this peptide bond is not present in the PMT-8a fragment, which does not undergo degradation at all in human plasma); [0134] modifications of the peptide bond between d-Arg 12 and d-Thr 13 of SEQ ID NO 5, d-Arg 8 and d-Thr 9 of SEQ ID NO 7 (see above).

[0135] All the above modifications are aimed at improving PMT or fragments thereof (optionally conjugated with the mt-targeting sequence) as listed in table 1, plasma stability while retaining rescuing activity; or variants of the PMT comprising chemical modifications of additional residues the peptide bonds between which will be shown not to undergo degradation by the analysis of the PMT fragments resulting from incubation in human plasma aimed at improving PMT plasma stability while retaining rescuing activity. Also said variants can be preferably conjugated at the N-terminus with an mt-targeting sequence according to any of the embodiments disclosed above. Preferably said variants are conjugated with mt-targeting sequence of SEQ ID NO 8.

[0136] In particular, the invention relates to the peptide having SEQ ID NO 1 and/or fragments thereof, optionally conjugated at the N-terminus with an mt-targeting sequence according to any of the embodiments disclosed, for use in the treatment of mt-tRNA-related diseases.

[0137] As explained in the state of the art as well as in the summary of the invention and in the glossary, human mt-tRNA-related diseases are diseases caused by mutations, in particular point mutations of various mt-tRNA coding genes which result in mutations in the mt-tRNA itself.

[0138] Said diseases show a panel of different symptoms normally affecting highly oxygen consuming tissues such as brain, heart, muscles etc., i.e. tissues in which the role of mitochondria is extremely relevant. A non-limiting example of mt-tRNA-related diseases according to the invention includes mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), MIDD (Maternally Inherited Diabetes and Deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

[0139] In an embodiment of the invention, the mt-tRNA-related disease is caused by a point mutation in a gene encoding one of the following mitochondrial tRNAs mt-tRNA.sup.Leu(UUR) mt-tRNA.sup.Lys mt-tRNA.sup.IIe and mt-tRNA.sup.Val

[0140] In particular, mt-tRNA.sup.(Leu)(UUR), and mt-tRNA.sup.(Lys), which are responsible of about 85% of the mt-tRNA-related disease.

[0141] In an embodiment of the invention, the peptide having SEQ ID NO 1 and/or fragments thereof as defined herein, optionally conjugated at the N-terminus with an mt-targeting sequence according to any of the embodiments disclosed above, is in the treatment of mt-tRNA-related diseases, wherein said mt-tRNA-related disease is caused by a point mutation selected from m.Math.3243A>G in the MT-TL1 human gene encoding mt-tRNA.sup.Leu(UUR) or m.Math.8344A>G in the MT-TK human gene encoding mt-tRNA.sup.Lys or m.Math.4277T>C mutation in the mt-tRNA.sup.IIe in the human gene MT-TI or m.Math.1630A>G mutation in mt-tRNA.sup.Val in the human gene MT-TV.

[0142] When the disease is caused by one of the mutations indicated above, said disease is MIDD, MELAS or MERRF.

[0143] A further object of the present invention is a pharmaceutical composition comprising one or more peptide and/or fragments thereof as defined in any one of claims 1 to 5 and at least one pharmaceutically acceptable carrier.

[0144] Non limited examples of suitable pharmaceutical composition are for systemic, oral, injectable, aerosol, oropharyngeal, nasal administration.

[0145] The composition of the invention can be in the form of a solid, semi-solid, liquid, emulsion, gel, nebulizable product and the like.

[0146] The composition of the invention can also comprise one or more of the peptides having SEQ ID NO 1, 2 3, 5, 6 and/or 7, complexed in the form of nanovesicles, liposomes and nanoparticles, based on either inorganic compounds or proteins, including human ferritin and variants thereof.

[0147] The invention hence relates also to the pharmaceutical composition herein disclosed and claimed for use as a medicament, in particular for use in the treatment of mt-tRNA-related diseases.

[0148] A non-limiting example of mt-tRNA-related diseases according to the invention includes mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), MIDD (Maternally Inherited Diabetes and Deafness)) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

[0149] In an embodiment of the invention, the mt-tRNA-related disease is caused by a point mutation in a gene encoding one of the following mt-tRNAs: mt-tRNA.sup.Leu(UUR), mt-tRNA.sup.Lys mt-tRNA.sup.IIe and mt-tRNA.sup.Val. In an embodiment of the invention, the pharmaceutical composition as defined herein, is in the treatment of mt-tRNA-related diseases, wherein said mt-tRNA-related disease is caused by a point mutation is m.Math.3243A>G in the MT-TL1 human gene encoding mt-tRNA.sup.Leu(UUR) or m.Math.8344A>G in the MT-TK human gene encoding mt-tRNA.sup.Lys or m.Math.4277T>C mutation in the mt-tRNA.sup.IIe in the human gene MT-TI or m.Math.1630A>G mutation in mt-tRNA.sup.Val in the human gene MT-TV.

[0150] When the disease is caused by one of the mutations indicated above, said disease is MIDD, MELAS or MERRF.

[0151] The invention also relates to a process for the preparation of the pharmaceutical composition as defined above and in the claims comprising admixing one or more peptide having SEQ ID NO 1 and/or fragments thereof, optionally conjugated at the N-terminus with an mt-targeting sequence according to any one of the embodiments disclosed above, as defined in the description and in the claims with at least one pharmaceutical acceptable carrier. The peptide/s of the invention can be synthesized by any technique commonly used in the art for the preparation of d-peptides and it can be purified, with conventional techniques, to pharmaceutical grade. Once prepared and purified, the d-peptide/s of the invention are formulated in the corresponding pharmaceutical compositions according to well-known techniques in the field together with the conventional carrier/s, excipient/s and the like; see for example the volume Remington's Pharmaceutical Sciences 15a Ed.

[0152] The compositions of the present invention may additionally contain other compatible adjunct components conventionally found in pharmaceutical compositions, not recited above, at their art-established usage levels. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials for combination therapy or may contain materials useful in physically formulating various dosage forms of the present invention, such as excipients, preservatives, anti-oxidants, thickening agents, stabilizers and the like.

[0153] The invention also relates to the use of the peptide having SEQ ID NO 1 and/or fragments thereof, optionally conjugated at the N-terminus with an mt-targeting sequence according to any one of the embodiments disclosed above, as herein defined and claimed in in vitro methods of pharmaco-toxicological studies, e.g., for the detection of PMT off targets, for the assessment of tissue specific PMT effect, for the investigation of PMT activity on additional diseases.

[0154] By way of example, for the assessment of tissue specific PMT effect the peptide having SEQ ID NO 1 and/or one or more fragments thereof optionally conjugated at the N-terminus with an mt-targeting sequence according to any one of the embodiments disclosed above, is put in contact with specific tissue cells or tissues or organoids optionally bearing one or more mutation in mt-tRNA genes resulting in mutations in the corresponding mt-tRNAs that affect the phenotype of said cells, tissues or organoids, and their capability of rescuing the cellular, tissue, organoid abnormal phenotype caused by said mutation/s is assessed.

[0155] Alternatively, the PMT or fragments thereof can be tested on healthy cells, tissues or organoids in order to identify undesired off-target effects thereof vs. untreated controls or the PMT or fragments thereof can be tested in combination with other compounds in order to identify potentially therapeutically effective active principle combinations.

[0156] By affect the phenotype in the sentences above it is intended that said mutation/s cause an abnormal phenotype and can therefore be mutation/s causing mtRNA-related diseases.

[0157] Rescuing the abnormal phenotype can be a partial rescue (from a more severe to a less severe phenotype, i.e., with respect to control untreated samples) as well as a full rescue (from abnormal to normal phenotype i.e., with respect to control samples not bearing the mutation/s).

[0158] The peptide/s of the invention can also be used in vitro, as described above, in combination with one or more additional compound in order to identify compounds that can have a pharmacological effect on mtRNA-related diseases

[0159] Additionally, the invention relates to a method for the treatment of mt-tRNA-related diseases comprising administering to a subject in need thereof, a therapeutically effective amount of peptide having SEQ ID NO 1 and/or fragments thereof optionally conjugated at the N-terminus with an mt-targeting sequence according to any one of the embodiments disclosed above, as defined in the description and in the claims (which are peptidomimetics of the peptide known in the art having SEQ ID NO 4) or of the pharmaceutical composition as defined in the description and in the claims.

[0160] A non-limiting example of mt-tRNA-related diseases treatable with the method of the invention comprises mitochondrial myopathy, MERRF (Myoclonic Epilepsy with Ragged Red Fibers), MIDD (Maternally Inherited Diabetes and Deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

[0161] In an embodiment of the invention, the mt-tRNA-related disease is caused by a point mutation in a gene encoding one of the following mt-tRNAs: mt-tRNA.sup.Leu(UUR), mt-tRNA.sup.Lys mt-tRNA.sup.IIe and mt-tRNA.sup.Val, which are responsible of more than the 85% of human mt-tRNA-related diseases.

[0162] In an embodiment of the invention, the invention relates to the treatment of mt-tRNA-related diseases, wherein said mt-tRNA-related disease is caused by a point mutation, wherein said mutation is m.Math.3243A>G in the MT-TL1 human gene encoding mt-tRNA.sup.Leu(UUR) or m.Math.8344A>G in the MT-TK human gene encoding mt-tRNA.sup.Lys or m.Math.4277T>C mutation in the mt-tRNA.sup.IIe in the human gene MT-TI or m.Math.1630A>G mutation in mt-tRNA.sup.Val in the human gene MT-TV.

[0163] When the disease is caused by one of the two mutations indicated above, said disease is MIDD, MELAS or MERF.

[0164] A further object of the invention is the use of a peptide having SEQ ID NO 1 and/or fragments thereof optionally conjugated at the N-terminus with an mt-targeting sequence according to any one of the embodiments disclosed above, as defined in the description and in the claims for the preparation of a medicament for the treatment of mt-tRNA-related diseases wherein one or more of said peptide having SEQ ID NO 1 and/or fragments thereof as defined in the description and in the claims is admixed at least with a pharmaceutically acceptable carried thereby obtaining a pharmaceutical composition as defined in the description and in the claims.

[0165] As stated above, mt-targeting sequences consisting of d-aminoacids only are preferred. All cells used in the experiments reported below were obtained from patients that have given their free and informed consent to said use according to current legislation.

EXAMPLES

Materials and Methods

Peptide synthesis

[0166] All constructs were synthesized with purity>85% by Pepscan (Pepscan Presto, Lelystad, The Netherlands).

[0167] The compounds used for the study are listed in Table 1.

TABLE-US-00001 SEQ ID Compounds Peptide Reference NO p,here KKSFLSPRTALINFLV Perlietal,FASEB 4 namedalso- J,2020(I-amino 32_33 acids) PMT(d-p) d(K)d(K)d(S)d(F)d(L)d(S)d(P)d(R) Unpublished 1 d(T)d(A)d(L)d(I) d(N)d(F)d(L)d(V) PMT-8a(d-p- d(K)d(K)d(S)d(F)d(L)d(S)d(P)d(R) Unpublished 2 8a) PMT-8b(d-p- d(L)d(S)d(P)d(R)d(T)d(A)d(L)d(I) Unpublished 3 8b) M-PMT(d-M- d(F)d(R)d(F)d(K)d(K)d(K)d(S)d(F) Unpublished p) d(L)d(S)d(P)d(R)d(T)d(A)d(L)d(I) d(N)d(F)d(L)d(V) M-PMT-8a(d- d(F)d(R)d(F)d(K)d(K)d(K)d(S)d(F) Unpublished 6 M-p-8a) d(L)d(S)d(P)d(R) M-PMT-8b(d- d(F)d(R)d(F)d(K)d(L)d(S)d(P)d(R) Unpublished p-8b) d(T)d(A)d(L)d(I) mt-targeting d(F)d(R)d(F)d(K) Unpublished 8 sequence1 mt-targeting d(F)d(R)d(A.sub.x)d(K) Unpublished sequence2 mt-targeting Fd(R)FK HortonKLetal, 10 sequence3 2008; mt-targeting d(A.sub.x)d(R)d(A.sub.x)d(K) Unpublished 11 sequence4 mt-targeting d(F)d(R)d(F)d(K)d(F)d(R) Unpublished 12 sequence5 d(F)d(K) mt-targeting d(F)d(R)d(A.sub.x)d(K)d(F)d(R) Unpublished 13 sequence6 d(A.sub.x)d(K) mt-targeting d(A.sub.x)d(R)d(A.sub.x)d(K)d(A.sub.x)d(R)d(A.sub.x) Unpublished 14 sequence7 d(K) mt-targeting d(R)d(K)d(K)d(R)d(R)d(Q)d(R)d Unpublished 15 sequence8 (R)d(R) mt-targeting d(F)d(R)d(F.sub.2)d(K) Unpublished 16 sequence9 mt-targeting d(F)d(R)d(Y.sub.Me)d(K) Unpublished 17 sequence10 mt-targeting d(F)d(R)d(Y)d(K) Unpublished 18 sequence11 mt-targeting d(Y)d(R)d(Y)d(K) Unpublished 19 sequence12 mt-targeting Fd(R)A.sub.xK HortonKLetal, 20 sequence13 2008 mt-targeting A.sub.xd(R)A.sub.xK HortonKLetal, 21 sequence14 2008; mt-targeting Fd(R)FKFd(R)FK HortonKLetal, 22 sequence15 2008 mt-targeting Fd(R)A.sub.xKFd(R)A.sub.xK HortonKLetal, 23 sequence16 2008; mt-targeting A.sub.xd(R)A.sub.xKA.sub.xd(R)A.sub.xK HortonKLetal, 24 sequence17 2008 mt-targeting RKKRRQRRR HortonKLetal, 25 sequence18 2008 mt-targeting Fd(R)F.sub.2K HortonKLetal, 26 sequence19 2008 mt-targeting Fd(R)Y.sub.MeK HortonKLetal, 27 sequence20 2008 mt-targeting Fd(R)YK HortonKLetal, 28 sequence21 2008 mt-targeting Yd(R)YK HortonKLetal, 29 sequence22 2008 Elamipretide SabbahHNetal // 2016 Elamipretide-32_33 Unpublished // Abbreviations: F2: diphenylalanine; A.sub.x: Cyclohexylalanine; YMe: methylated tyrosine.
Cell lines

[0168] Previously established osteosarcoma derived (143B.TK-) cybrid cell lines from patients, which bear either the m.Math.3243A>G mutation in mt-tRNA.sup.Leu(UUR) or the m.Math.8344A>G mutation in mt-tRNA.sup.Lys, and controls were used (generous gift from Dr Valeria Tiranti and Dr Valerio Carelli). The pathological mt-tRNA.sup.Leu(UUR) mutant had a mutation load>98%. The mt-tRNA.sup.Lys mutant had a mutation load of either 80% (H-8344) or 0% (I-8344). The high mutation load mutant was pathological, whereas the low mutation level did not show any detectable phenotype [Perli et al, Hum Mol Genet, 2016].

Cell culture

[0169] Cybrid cells were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 4.5 g/l d-glucose, 10% foetal bovine serum (FBS), 2 mM I-glutamine, 50 g/ML uridine, 100 U/mL penicillin, and 100 mg/mL streptomycin (referred to as glucose medium) in a humidified atmosphere of 95% air and 5% CO2 at 37 C. For cell viability experiments, cells were grown either in glucose medium or in glucose-free DMEM, supplemented with 5 mM galactose, 110 mg/mL sodium pyruvate, and 10% FBS (referred to as galactose medium). The reason for using the latter medium is that a pathological phenotype can be appreciated in cells growing on galactose, which forces cells to rely on mitochondrial respiration, but not in cells growing on glucose.

Fluorescence microscopy

[0170] Constructs made of compounds listed in Table 1 linked to the Cy5 fluorophore via maleimide cross-linker, were administered to sub-confluent cybrid cell cultures at 0.25 M in glucose medium. About 24 hours after treatment with different constructs, cells were incubated with 200 nM Mitotracker Red FM (LifeTechnologies Italia, Monza, Italy) for 30 minutes at 37 C. Subsequently, cells were visualized by confocal microscopy. Images of 800800 px (at 88 nm/px) were acquired at the Olympus iX83 FluoView1200 laser scanning confocal microscope using a 60NA1,2 water objective (Olympus Italia SRL Milano, Italy), zoom 3, 559 nm, and 635 nm lasers and filter setting for MitoTracker Red and Cy5. The fluorescence images were analyzed with the ImageJ software (14, https://imagej.nih.gov/ij/, 1997-2018) to determine the Pearson's correlation coefficient.

Cell viability

[0171] To test the growth capability, cells were harvested and seeded at 3010.sup.4 in 60 mm dishes in glucose medium for 24 hours with the addition of one of the compounds (each at 5 M concentration). Cells were switched in glucose or galactose and after 24 hours cell viability was measured by the Trypan blue dye exclusion assay. Cells were harvested with 0.25% trypsin and 0.2% EDTA, washed, suspended in PBS in the presence of Trypan blue solution (Sigma-Aldrich) at 1:1 ratio and counted using a hemocytometer. The number of viable cells in galactose medium was expressed as a percentage of the number of cells in glucose medium.

Respirometry assay

[0172] Oxygen consumption rate (OCR) of cybrids incubated with compounds was evaluated with Clark type oxygen electrode (Hansatech Instruments, Norfolk, UK). After incubation with compounds, both control and mutant cybrids were maintained in glucose medium for 36 hours, then OCR was measured in intact cells (310.sup.6) in 1 mL DMEM lacking glucose supplemented with 10% sodium pyruvate.

Mitochondrial toxicity

[0173] Mitochondrial toxicity exerted by PMT was measured using the Mitochondrial ToxGlo Assay (Promega Italia Srl., Milano, Italy) according to the manufacturer's protocol. Cybrids were plated on a 96-well plate and treated with different concentrations of PMT (5, 10 and 20 M). Twenty-four hours after treatment, control cells (both wild type and mutated) were incubated with either 400 ug/ml digitonin (a cytotoxic agent) or 100 l sodium azide (mitotoxic agent) for three hours, as positive control for cyto-or mito-toxicity, respectively. Subsequently, cells were incubated with specific reagents and fluorescence or luminescence were measured with a GloMax Multi+Luminometer (Promega Italia Srl., Milano, Italy).

Statistical analysis

[0174] All data are expressed as meanSEM. Data were analyzed by standard ANOVA procedures followed by multiple pair-wise comparison adjusted with Bonferroni corrections. Significance was considered at<0.05. Numerical estimates were obtained with Graphpad Prism 7 version (Graphpad Inc San Diego, CA, USA).

Plasma stability

[0175] In order to evaluate whether compounds (p and d-p) are stable in blood or hydrolyzed by plasma peptidases, we set up a chromatographic assay to evaluate compound concentration after incubation in human plasma.

[0176] Blood was drawn by venipuncture in vacutainer containing EDTA as an anticoagulant.

[0177] Two different samples from healthy volunteers were used. Plasma was separated by centrifugation and immediately used for the experiments. Each compound was dissolved in 500 uL plasma at a final concentration of 0.2 mM, and split into two aliquots, one of which was immediately analyzed to assess the basal compound level; the other was incubated at 37 C. for 3 hours under gentle shaking. In order to perform chromatographic analysis the samples were treated with 3 volumes of acetonitrile containing 1% formic acid, and then extracted by using the Ostro pass-through sample preparation system to remove proteins and phospholipids. Samples were dried under vacuum and then resuspended in 100 uL of 0.1% formic acid containing 5% acetonitrile, then directly injected onto the chromatographic column.

[0178] Chromatographic analyses were performed on a Water Acquity H-Class UPLC system (Waters, Milford, MA, USA), including a quaternary solvent manager (QSM), a sample manager with a flow through needle system (FTN), a photodiode array detector (PDA) and a single-quadruple mass detector with electrospray ionization source (ACQUITY QDa). Analyses were performed on a reverse phase C18 column (75 mm3.2 mm i.d., 2.5 m particle size). The mobile phase was solvent A, 0.1% formic acid in water, and solvent B, 0.1% formic acid in acetonitrile. The flow rate was 0.5 mL/min, the column temperature was set at 25 C. and the elution was performed by linearly increasing the concentration of solvent B up to 70% in 7 minutes. Mass spectrometric detection was performed in the positive electrospray ionization mode, using nitrogen as the nebulizer gas. Analyses were performed in the Total lon Current (TIC) mode with a mass range of 100-1200 m/z. The capillary voltage was 0.8 kV, cone voltage 8 V, ion source temperature 120 C. and probe temperature 600 C. Quantification of each compound was performed by Selected lon Recording (SIR) at m/z 917.88, corresponding to the

RESULTS

PMT and PMT Fragments (PMT-8a and PMT-8b) Penetrate Cell Membranes and Co-Localize with Mitochondria

[0179] The uptake and localization of Cy5-conjugated constructs in cybrids was assessed by flow cytometry, confocal microscopy, and immunoblot analysis on isolated mitochondria (FIG. 1). Confocal microscopy was performed using a specific live cell staining for mitochondria (Mitotracker FM Red). After 12 hours the fluorescent signal of all constructs was clearly detectable within cybrids. All constructs showed cellular uptake and a clear overlap with mt reticulum, as demonstrated by Pearson's correlation coefficients reflecting the mitochondrial specificity (FIG. 1). These results indicate that, upon exogenous administration to mutant cells, all of the constructs reported in the image are able to penetrate cell membranes and colocalize with mitochondria.

Effect of PMT and PMT Fragments (PMT-8a and PMT-8b) on the Viability of m.Math.3243A>G mt-tRNA.SUP.Leu(UUR) .and m.Math.8344A>G mt-tRNA.SUP.Lys .Mutant Cybrids.

[0180] To evaluate the effect of the compounds on viability, cybrids were grown in glucose-free medium supplemented with galactose (galactose medium), a condition that both forces cells to rely on the mt respiratory chain for ATP synthesis and causes a significant growth reduction in the presence of mutations.

[0181] We observed that the PMT was able to induce a significant improvement of cell viability and apoptotic rate in both m.Math.3243A>G and m.Math.8344A>G mutant cybrids, as compared with non treated mutant cells (FIG. 2, top panels). The rescuing activity of PMT was comparable to that of the 32_33 peptide. The PMT-8b also significantly improved cell viability and apoptotic rate in m.Math.3243A>G mutant.

Effect of PMT and PMT Fragments (PMT-8a and PMT-8b) on Oxygen Consumption of m.Math.3243A>G mt-tRNA.SUP.Leu(UUR) .and m.Math.8344A>G mt-tRNA.SUP.Lys .mutant cybrids

[0182] To investigate whether increased cell viability was related to improved mt bioenergetics, we analyzed the respiratory capability of mutant and control cells by using the Clark type electrode. We demonstrated that the PMT determined a significant increase of oxygen consumption rate in both pathological mutants (FIG. 2, bottom panels). This activity is comparable to that of the 32_33 peptide in m.Math.3243A>G mutant cybrids, and even higher than that of the 32_33 peptide in m.Math.8344A>G mutant cybrids.

Lack of Cyto-and Mitotoxicity of PMT and PMT Fragments (PMT-8a and PMT-8b)

[0183] We performed the Mitochondrial ToxGlo Assay to evaluate toxicity of increasing concentrations of exogenously administered PMT and PMT fragments. The PMT, PMT-8a and PMT-8b fragments resulted to be neither cytotoxic nor mitotoxic up to 20 M in m.Math.3243A>G mt-tRNA.sup.Leu(UUR) mutant cybrids, m.Math.8344A>G mt-tRNA.sup.Lys mutant cybrids and healthy control cells.

The PMT has Higher Stability than the 32_33 Peptide in Human Plasma.

[0184] To assess their plasma stability, two experiments were performed. In a first experiment, the 32_33 peptide or PMT was incubated in plasma samples from two healthy volunteers and the amount of each compound was measured before (T0) and after 3 hours plasma incubation (3 h). As shown in FIG. 4A, after 3 hours plasma incubation the PMT does not undergo visible degradation whereas only 70% of the 32_33 peptide is still available. In a second experiment, the 32_33 peptide, PMT, PMT-8a or PMT-8b was incubated in plasma samples from four healthy volunteers and the amount of each compound was measured before (T0) and at different time points after plasma incubation (i.e., 1.5, 3, 6 and 72 h). As shown in FIG. 4B, the PMT has higher plasma stability than the 32_33 peptide at all time points, although, at variance with the previous experiments, it did undergo detectable degradation. Conversely, after 72 h, the PMT-8a did not undergo detectable degradation, and 80% of the PMT-8b fragment was present, suggesting that the eight C-terminal residues of the PMT, which are not present in the PMT-8a and only four of which are present in the PMT-8b, and the character of which is mostly hydrophobic (e.g., d-Ala 10, d-Leu 11, d-IIe 12, d-Phe 14, d-Leu 15 and d-Val 16) may be at least partially responsible for peptide sequestration by plasma proteins endowed with hydrophobic pockets, such as serum albumin.

M-PMT Ameliorates Viability of m.Math.3243A>G mt-tRNA.SUP.Leu(UUR) .at 10-Fold Lower Concentration with Respect to PMT.

[0185] To evaluate the effect of PMT and M-PMT on viability, cybrids were grown in glucose-free medium supplemented with galactose (galactose medium), a condition that both forces cells to rely on the mt respiratory chain for ATP synthesis and causes a significant growth reduction in the presence of mutations.

[0186] The M-PMT significantly improved viability of m.Math.3243A>G mutant cybrids, as compared with non-treated mutant cells, at 5, 2 and 0.5 M concentration (FIG. 6), whereas the PMT peptide exerted rescuing activity at 5 M but not 2 and 0.5 M concentration.