Therapeutic Peptides

Abstract

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

Description

[Technical field]

[0001] The present invention relates to peptidomimetics of short peptides derived from leucyl-tRNA synthetase, compositions comprising one or more of said peptidomimetics and their use for the treatment of syndromes caused by mutations in the mt-tRNA (mitochondrial transfer RNA) gene, and to the medical treatment of said syndromes comprising the administration of said one or more peptidomimetics or a composition comprising said one or more peptidomimetics. [Background technology]

[0002] Mitochondrial (mt) diseases due to mutations in transfer RNA (tRNA) genes are responsible for a wide range of syndromes, for which no effective treatment is currently available. The mitochondrial tRNA (mt-tRNA) gene is a "hotspot" for pathological mutations, with over 200 mt-tRNA mutations associated with various disease states. In many cases, these mutations prevent tRNA aminoacylation. Disrupting this key function is thought to affect protein synthesis and expression, folding and function of oxidative phosphorylation enzymes. Mitochondrial tRNA mutations appear in a wide variety of diseases related to cellular energetics, including mitochondrial myopathies, MERRF (myoclonus epilepsy with ragged red fibers syndrome), MIDD (maternally inherited diabetes and deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes). Diseases caused by mt-tRNA mutations can also affect very specific histological types, as in the case of neurosensory nonsyndromic deafness and pigmentary retinopathy, diabetes mellitus and hypertrophic cardiomyopathy.

[0003] In particular, mt-tRNA Leu(UUR) m.3243A>G in the MT-TL1 human gene encoding mt-tRNA LysMutations in mitochondrial genes encoding mt-tRNAs, such as m.8344A>G in the MT-TK human gene encoding mt-tRNAs, cause damage to the mt tRNA structure and impair tRNA interactions with aminoacyl-tRNA synthetases and other molecules (proteins, mRNAs, ribosomes, etc.), thereby resulting in impaired tRNA physiological function.

[0004] The above two mutations together account for the most common and severe human mt-tRNA-related diseases (approximately 85%). Leu(UUR) The mitochondrial tRNA mutation m.3243A>G in the MT-TL1 human gene encoding mt-tRNA is known to cause MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) and MIDD (maternally inherited diabetes mellitus and deafness), whereas mt-tRNA Lys The mutation m.8344A>G in the MT-TK human gene encoding the mitochondrial kinesin kinase inhibitor 1 (MT-TK) is known to cause MERRF (myoclonus epilepsy with ragged red fibers syndrome).

[0005] The disease usually develops during adolescence and early adulthood and affects many energy-demanding organs and tissues, such as the central nervous system, heart, and skeletal muscles, resulting in many symptoms. Symptoms exhibited by patients affected by MELAS include seizures, dementia, stroke-like episodes, muscle weakness, hypoacusia, and cardiac conduction disorders, symptoms exhibited by patients affected by MIDD include diabetes and hearing loss, and symptoms exhibited by patients affected by MERRF include ataxia, myoclonus, muscle atrophy, and dementia.

[0006] The clinical course of the disease is chronic, progressive and ultimately fatal.

[0007] Many diverse approaches have been investigated to counteract the symptoms of these diseases, including DNA manipulation, protein delivery and the development of small molecule drugs. Currently, available treatments are poorly effective and include general enhancers of mt function such as B vitamins (cofactors for enzymes that catalyze essential reactions); vitamin E and coenzyme Q10 (antioxidants); amino acids and other dietary supplements that are empirically administered to patients as a "cocktail" based on biochemical rationale and consensus expert opinion. Current treatments fail to effectively manage symptoms, nor do they specifically address the underlying mechanism of these syndromes, namely mutated tRNA instability.

[0008] Among tRNA-targeted therapies, overexpression of human cognate or noncognate aminoacyl mt-tRNA synthetases has been shown to rescue defective phenotypes in human trans-mt hybrids (cybrids), a well-established cellular model of mt-tRNA mutations.

[0009] Perli et al. showed that plasmids encoding the noncatalytic carboxy-terminal domain (Cterm) (67 residues long) of human mt-LeuRS, either linked or not to well-characterized mt-targeting sequences (from either the Neurospora crassa F0-ATPase subunit 9 precursor or human COX8a), were able to target mt-tRNA Leu(UUR) Mutation m.3243A>G in mt-tRNA Lysdemonstrated that β32_33 effectively rescued the defect of human cybrids carrying m.8344A>G in the cytoplasm (Perli et al EMBO Molecular Medicine 2014,Vol 6 No 2 169-182). Later, in Perli et al. 2016 (Perli et al”short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations” Hum mol genet 2016,Vol 25 No 5 903-915), the authors demonstrated that transfection of plasmids encoding short Cterm-derived β32_33 (16 residues long) or β30_31 (15 residues long) sequences linked to COX8a mt targeting sequences had the same rescue ability as Cterm for both mutant cybrids. The authors also showed that β32_33 conferred higher rescue activity than both β30_31 and Cterm. Finally, the authors demonstrated that the β32_33 and β30_31 peptides were expressed in mutant mt-tRNA Leu(UUR) or mt-tRNA Lys In vitro experiments revealed that tRNA can strongly interact with and stabilize the functional conformation of tRNA. The direct in vitro interaction with and stabilization of the target tRNA suggested that the rescue effect is mediated by a "chaperone" activity.

[0010] More recently, in Perli et al., 2020 (Perli et al, FASEB J, Vol 34 No 6 7675-7686), the authors demonstrated that the exogenously administered β32_33 peptide was itself able to penetrate both the plasma and mitochondrial membranes and exert rescue activity against mutant cells.

[0011] Importantly, the rescuing effect of the mt-LeuRS-derived sequence was similar to that of the cognate mt-tRNA Leu(UUR) as well as non-cognate mt-tRNA LysBased on the observation that the activity of mt-tRNAs against β32_33 and β30_31 was also observed, the authors proposed that these sequences may be active against a wide range of human mt-tRNA mutants. Indeed, transfection of plasmids encoding either the β32_33 or β30_31 sequences has been demonstrated to rescue the phenotypes of multiple mt-tRNA mutants in a yeast model (Di Micco, P. et al 2014 "The yeast N-model suggests the use of short peptides derived from mt LeuRS for the therapy of diseases due to mutations in several mt tRNAs". Biochim. Biophys. Acta, 1843, 3065-3074).

[0012] Conjugation with a mitochondrial permeability sequence (disclosed herein as SEQ ID NO: 10) did not increase rescue activity or mitochondrial localization, indicating that the β32_33 peptide itself is endowed with mitochondrial targeting properties (Perli et al 2020, “Exogenous peptides are able to stabilize mitochondrial tRNAs, penetrate human cell and mitochondrial membranes and rescue severe mitochondrial defects” FASEB J Vol 34 No 6 7675-7686). In this paper, the authors demonstrated that constructs in which the β32_33 peptide sequence was scrambled or positively charged residues were mutated to alanine had no rescue activity, indicating that both the order of amino acids along the sequence and the presence of positive charges are essential determinants of peptide efficacy. Thus, the β32_33 peptide is a promising molecule for the development of therapeutic agents against diseases caused by mt-tRNA point mutations. The authors also show that the β32_33 peptide is a promising lead molecule for the development of non-peptide derivatives endowed with rescue activity against mutations in mt-tRNA.

[0013] Despite the encouraging data on potential therapeutic peptides, there remains a need to provide molecules that not only exhibit rescue activity against mutations in mt-tRNA but are also suitable for use in therapy, and therefore there is a strong clinical need for the identification of molecules suitable for the treatment of mt-tRNA-related diseases such as MELAS, MIDD and MERRF as well as other mt diseases caused by mt-tRNA point mutations. Summary of the Invention

[0014] The results reported in Perli et al. 2020 indicate that the β32_33 peptide is a promising lead molecule for the development of non-peptide derivatives endowed with rescue activity against mutations in mt-tRNA. However, the strong degradation commonly observed in peptide molecules makes the peptide itself unsuitable for therapeutic use unless its stability in blood can be increased.

[0015] Perli et al. (2020) provide clear information on the essential features responsible for the β32_33 peptide rescue activity that can be exploited to develop therapeutic agents against human mt-tRNA mutation-associated diseases. These features are the spatial arrangement of residues and the number of positive charges.

[0016] In other words, the spatial arrangement of residues and the number of positive charges are presented as essential features of β32_33 peptide rescue activity that should be taken into account by the skilled artisan when designing potential peptidomimetics of said peptides.

[0017] What this statement means is that, according to the prior art, in order to exert a high rescue activity, the β32_33 peptide (βp) needs to have both characteristics.

[0018] This statement is supported by results presented in the same paper, where, as reported in Table 1 of the paper, two peptides derived from β32_33 and differing only in one of two features (spatial arrangement of the residues and number of positive charges, respectively), one obtained by scrambling (i.e. the same amino acid residues but in a different order within the peptide) and the other by substitution of the three positively charged amino acids K1, K2 and R8 (the numbers indicate the position of the amino acids along the peptide side chains), show significantly lower rescue activity relative to the β32_33 peptide.

[0019] The inventors have confirmed that the β32_33 peptide (SEQ ID NO: 4) disclosed in Perli et al. 2016 and Perli et al. 2020 has a short stability in blood, as shown in Figure 4. Panel A of Figure 4 shows that in a first experiment, after 3 hours of incubation in the plasma of two healthy volunteers at 37°C, i.e. human body temperature, the β32_33 prior art peptide (SEQ ID NO: 4) is significantly degraded (at least 30% degraded peptide is observed). Panel B of Figure 4 shows that in a second experiment, the β32_33 peptide is degraded to an even greater extent, since after 1.5 hours of incubation in the plasma of four healthy volunteers, unlike the two in the previous experiment, more than 85% of the starting amount is present. Despite the different degradation rates exhibited by the β32_33 peptide and PMT (SEQ ID NO: 1) in two experiments in which plasma samples from different volunteers were used, the PMT consistently shows a higher stability than the β32_33 peptide, since after 3 hours of incubation in this medium, in the first experiment, 70% of the β32_33 peptide is available, whereas the PMT is 100% available (Figure 4A), and in the second experiment, only 17% of the β32_33 peptide is available, whereas the PMT is more than 63% available (Figure 4B). Interestingly, as shown in panel B of Figure 4, smaller fragments of PMT, PMT-8a and PMT-8b (SEQ ID NO: 2 and 3, respectively), both 8 residues long, are even more stable than the full-length PMT, which is 16 residues long. This result suggests that the plasma stability of PMT may be further increased by ad hoc chemical modifications affecting the 4th (i.e., d-Phe), 5th (i.e., d-Leu), 8th (i.e., d-Arg) and / or 9th (i.e., d-Thr) residues of PMT and / or the peptide bond between the 4th and 5th residues (i.e., d-Phe and d-Leu) and / or between the 8th and 9th residues (i.e., d-Arg and d-Thr). Furthermore, the plasma stability of PMT may be further increased by modifications affecting additional PMT residues in the peptide bond between which it can be expected to be cleaved in human plasma.

[0020] The poor stability of the prior art β32_33 peptide disclosed in Perli 2016 was expected, since it was produced intracellularly by transfection and therefore consists of l-amino acids (the same applies to Perli 2020, where the only d-amino acid disclosed in Perli 2020 is the d-Arg amino acid in the short peptide of 4 amino acids for mt targeting disclosed in the same paper) and does not have further modifications that could increase its stability.

[0021] The poor in vivo stability against proteolysis of unmodified peptides (such as β32_33 of SEQ ID NO: 4) is in fact a major challenge that must be overcome, since it may result in an unrealistically short in vivo biological half-life and subsequently poor bioavailability when used in imaging and therapeutic applications. Many biologically and pharmacologically interesting peptide-based drugs may not be utilized due to poor stability. Those skilled in the art know that a possible way to overcome this limitation is the use of peptide analogs designed to mimic the pharmacophore of natural peptides, while also containing non-natural modifications that act to maintain or improve pharmacological properties. Various strategies have been developed to increase the metabolic stability of peptide-based pharmaceuticals. These include C-terminal and / or N-terminal modifications, introduction of d or other non-natural amino acids, backbone modifications, PEGylation and alkyl chain incorporation, cyclization and peptide bond replacement, all of which have been or can be applied to peptide-based pharmaceuticals.

[0022] While the use of d-amino acids is known in the art, it is also known to those skilled in the art that simply replacing all l-amino acids in a peptide with d-amino acids is generally an ineffective strategy, as the resulting changes in peptide conformation and side chain orientation can prevent correct binding geometry and thus disrupt target binding (e.g., Evans et al Molecules.2020 May;25(10):2314 Methods to Enhance the Metabolic Stability of Peptide-Based PET Radiopharmaceuticals). Furthermore, peptide bond cleavage by plasma or liver hydrolases is only one possible reason underlying the short in vivo lifetime of peptide compounds, which can also be excreted via the kidney and / or sequestrated by plasma or tissue proteins. A prime example of such a protein is serum albumin, which can bind molecules containing hydrophobic regions and transport them to the liver for degradation, thus lowering the amount of such molecules that are released into the bloodstream and freely diffuse into cells and tissues.

[0023] For this reason, the benefits conferred by d-amino acids to peptides are usually pursued without replacing all amino acids with their d-amino acid equivalents. For example, replacing the l-amino acids at the N-terminus of most proteins with the corresponding d-amino acids can significantly increase in vivo stability by preventing recognition of the protein's N-terminus by proteases.

[0024] In addition, Perli et al. 2020 clearly teaches that the spatial arrangement of residues and the number of positive charges are essential features for the rescue activity of the β32_33 peptide of SEQ ID NO: 4, and therefore, the inventors have found, quite surprisingly, that a peptide having the same sequence of the β32_33 peptide in which all l-amino acids are replaced with d-amino acids and thus does not maintain the above-mentioned essential features, The native β32_33 peptide maintained the same ability to penetrate cell and mt membranes upon exogenous administration (Figure 1). mt-tRNA is involved in more than 50% of human mt-tRNA mutation-related diseases (MELAS, MIDD) Leu(UUR) ( Fig. 2 , panels A and C) of a cell model carrying the m.3243A>G mutation in the gene encoding the MERRF-associated mt-tRNA Lys The same rescue of the native β32_33 peptide was maintained in the defective phenotype (Figure 2, panels B and D) of a cell model carrying the m.8344A>G mutation in the gene encoding It is safe in both mutant and wild-type cells when administered exogenously up to 20 μM (Figure 3), and In the first experiment, it was extremely stable in human plasma since it did not undergo detectable degradation after 3 hours (Figure 4, panel A), in contrast to the 30% degraded β32_33 peptide, and in the second experiment, more than 50% PMT was present after 6 hours (Figure 4, panel A), in contrast to the 90% degraded β32_33 peptide. Since not all in vivo sources of degradation can be predicted a priori and therefore superior in vivo PMT stability for the β32_33 peptide could only be demonstrated by plasma stability experiments, these results also demonstrate that, as mentioned above, peptide bonds between d-amino acids are often more stable in vivo relative to peptide bonds between l-amino acids, but this is not an absolute rule.

[0025] Moreover, surprisingly, in relation to the results observed upon conjugation of the peptide of sequence number 4 reported in the prior art, the peptide of sequence number 1 of the present invention (also referred to herein as PMT) with the mt targeting sequence of sequence number 8 (also consisting of only d-amino acids) at the N-terminus, the inventors found that the viability of m.3243A>G mutant cybrids was improved with a 10-fold lower concentration of the peptide of sequence number 5 (also referred to herein as M-PMT) relative to the PMT peptide.

[0026] As shown in the figure, the same properties were exhibited by a fragment of the peptide having SEQ ID NO:1.

[0027] Thus, peptides having sequence numbers 1 or 5 and their fragments, characterized by being composed exclusively of d-amino acids, have been shown to be excellent peptidomimetics of the β32_33 peptide (sequence number 4) disclosed in Perli et al. 2016 and Perli et al. 2020, retaining the rescue activity of the β32_33 peptide and exhibiting enhanced associated properties such as stability.

[0028] Thus, the present invention relates to a peptide having SEQ ID NO: 1 and fragments thereof, in which all l-amino acids of the β32_33 peptide (SEQ ID NO: 4) disclosed in Perli et al. 2016 and Perli et al. 2020 are replaced by d-amino acids, which surprisingly maintain rescue activity against mutations in mt-tRNA and enhanced blood stability compared to the blood stability of the peptide having SEQ ID NO: 4, said peptide having SEQ ID NO: 1 being optionally conjugated to an mt targeting sequence consisting of d-amino acids.

[0029] The object of the present invention is to A peptide having SEQ ID NO:1 and / or a fragment thereof at least 8 amino acids in length, said peptide consisting entirely of d-amino acids, said peptide or fragment thereof being optionally conjugated at its N-terminus to an mt targeting sequence; A peptide of SEQ ID NO:1 and / or a fragment thereof as defined in the present specification and claims for use as a medicament, said peptide or fragment thereof being optionally conjugated at its N-terminus to an mt targeting sequence; A peptide of SEQ ID NO:1 and / or a fragment thereof as defined herein and in the claims, said peptide or fragment thereof being optionally conjugated at its N-terminus to an mt targeting sequence, for use in the treatment of a human mt-tRNA associated disease and / or a variant thereof as defined herein; A pharmaceutical composition comprising a peptide of SEQ ID NO:1 and / or a fragment thereof as defined herein and in the claims, said peptide or fragment thereof being optionally conjugated at its N-terminus to an mt targeting sequence, and at least one pharma- ceutically acceptable carrier; said pharmaceutical composition for use as a medicament; said pharmaceutical composition for use in treating a human mt-tRNA-associated disease; A process for preparing a pharmaceutical composition as described herein and claimed herein, comprising the step of mixing one or more peptides of SEQ ID NO: 1 and / or fragments thereof as defined above, said peptides or fragments thereof being optionally conjugated at the N-terminus to an mt targeting sequence, with at least one pharma- ceutical acceptable carrier; A method for the treatment of an mt-tRNA associated disease, comprising administering to a subject in need of treatment of an mt-tRNA associated disease a therapeutically effective amount of a peptide of SEQ ID NO: 1 and / or a fragment thereof, said peptide or fragment thereof being optionally conjugated at its N-terminus to an mt targeting sequence, or a pharmaceutical composition as defined herein and in the claims; As well as the use of a peptide of SEQ ID NO:1 and / or a fragment thereof as defined herein and / or in the claims, said peptide or fragment thereof being optionally conjugated at its N-terminus to an mt targeting sequence, or a pharmaceutical composition as defined herein and in the claims, for the preparation of a medicament for the treatment of an mt-tRNA associated disease, wherein one or more of said peptides are mixed with at least a pharma- ceutical acceptable carrier, thereby obtaining a pharmaceutical composition as defined herein and in the claims. It is.

[0030] Glossary As used herein, β32_33 peptide or βp refers to a peptide previously reported to have rescue activity against mutant cells (Perli et al., FASEB J, 2020 and Perli et al. "Hum mol genet 2016, Vol 25 No 5 903-915), and is also reported herein as the peptide having sequence number 4.

[0031] The peptide having SEQ ID NO:1, which is a peptidomimetic therapeutic of the β32_33 peptide, is also referred to herein and in the figures as PMT.

[0032] The peptide having sequence number 5, which is a peptidomimetic therapeutic of the β32_33 peptide conjugated at its N-terminus with a designed mt targeting sequence having sequence number 8, is also referred to as M-PMT in the present specification and figures.

[0033] The PMT fragment of the invention comprising PMT residues 1-8 having SEQ ID NO:2 is also referred to in the present specification and drawings as PMT-8a.

[0034] The PMT fragment of the present invention comprising PMT residues 5-12 having SEQ ID NO:3 is also designated in the present specification and drawings as PMT-8b.

[0035] The M-PMT fragment of the present invention having SEQ ID NO:6, comprising PMT residues 1-8 having SEQ ID NO:2 conjugated at the N-terminus to an mt targeting sequence having SEQ ID NO:8, is also referred to in the present specification and drawings as M-PMT-8a.

[0036] The M-PMT fragment of the invention having SEQ ID NO:7, comprising PMT residues 5-12 having SEQ ID NO:3 conjugated at the N-terminus to an mt targeting sequence having SEQ ID NO:8, is also referred to in the present specification and drawings as M-PMT-8b.

[0037] According to the present specification and scientific literature, a mt targeting or mt penetrating sequence is an N-terminal sequence that specifically directs and localizes the protein / peptide to which it is attached to the mitochondria, i.e. a mitochondrial transporter cell penetrating peptide (including designed and naturally occurring) that can enter mitochondria, or in other words, a peptide that shows efficient cellular uptake and specific mitochondrial localization. In all parts of the specification and claims, SEQ ID NOs: 1, 2, 3, 5, 6 and 7 refer to sequences that consist exclusively of d-amino acids.

[0038] According to the present specification, the peptide of SEQ ID NO: 1 or a fragment thereof having SEQ ID NO: 2 or 3, whether conjugated to an mt targeting sequence at the N-terminus or not conjugated to said mt targeting sequence, is also indicated as a "peptide", whereas the peptides having SEQ ID NO: 5, 6, and 7 may also be referred to as "conjugated peptides" due to the fact that they result from the conjugation of the peptide of SEQ ID NO: 1 or one of its fragments according to the present specification to an mt targeting sequence at the N-terminus.

[0039] As used herein, an mt-tRNA-associated disease or syndrome has the meaning generally intended in the art and refers to a disease or syndrome associated with (caused by) a mutation in mitochondrial tRNA (mt-tRNA), preferably a point mutation in mitochondrial tRNA.

[0040] As used herein, a mutation in the mitochondrial gene (mt) encoding mt-tRNA includes mt-tRNA Leu(UUR) m.3243A>G in the MT-TL1 human gene encoding mt-tRNA Lys m.8344A>G=>MERRF in the MT-TK human gene encoding the mt-tRNA IleThe m.4277T>C mutation in the (MTTI) gene and mitochondrial tRNA deficiency syndrome (MTTS) causes mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes. Val This includes the m.1630A>G mutation in (MTTV).

[0041] The abbreviation "mt" in this specification and claims, as well as in the relevant prior art, stands for "mitochondrial."

[0042] As used herein, an "effective amount" is defined as the amount required to confer a therapeutic effect on the treated subject, and is typically determined based on the age, surface area, weight, and condition of the subject. [Brief description of the drawings]

[0043] [Figure 1]Upon exogenous administration to mutant cells, all constructs reported in the images are able to penetrate the cell membrane and localize with mitochondria. Top row (constructs): Exogenously administered constructs covalently linked to a fluorescent dye (Cy5) localize inside the cells. This result shows that all constructs are able to penetrate the cell membrane. Middle row (Mitotracker red): Mitochondria in the same cells shown in the top row are highlighted by Mitotracker red, a dye that can bind specifically and exclusively to mitochondria. Bottom row (overlay): Filter settings for both dyes reveal colocalization of constructs with mitochondria. The cells used in the experiments are transmitochondrial hybrids (hereafter referred to as cybrids) carrying the m.3243A>G mutation in mt-tRNALeu(UUR), associated with MELAS syndrome.The constructs used in the experiments were: β32_33 peptide (βp), previously reported to have rescue activity against mutant cells (Perli et al, FASEB J, 2020 and Perli et al Hum mol genet 2016, Vol 25 No 5 903-915); peptidomimetic therapeutic agent (PMT) of SEQ ID NO:1; a PMT fragment comprising PMT residues 1-8 (PMT-8a) of SEQ ID NO:2 and PMT residues 5-12 (PMT-8b) of SEQ ID NO:3; and β32_33 peptide linked to elamipretide (E), a different peptide previously reported by Sabbah HN et al 2016 to have mitochondrial targeting properties and putative mitochondrial protective activity [Sabbah HN, Gupta RC, 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 Feb;9(2):e002206.doi:10.1161 / CIRCHEARTFAILURE.115.002206.PMID:26839394;PMCID:PMC4743543.]. The names of all constructs are followed by "-C" to indicate that they are linked to Cy5. Cells were incubated with 0.25 μM of constructs for 24 hours. Half an hour before imaging, cells were stained with Mitotracker Red. Finally, the fluorescent signals were detected by laser scanning confocal microscopy. PCC: Pearson's correlation coefficient (mean ± SEM of six images). [Diagram 2]After exogenous administration, PMT significantly improves cell viability and mitochondrial respiration of mutant cells. Top: Viability of cells treated with compounds. X-axis and Y-axis indicate the compound used and the percentage of live cells after treatment, respectively. Bottom: Oxygen consumption of cells treated with compounds. X-axis and Y-axis indicate the compound used and the amount of oxygen consumed in fmol per minute per cell, respectively. The first bar of each graph represents cells without pathological phenotype treated with vehicle only. WT: wild type; l-8344: cells with extremely low levels of the mutation 8344A>G in mt-tRNALys. The second bar of each graph represents cells with pathological phenotype with either m.3243A>G in mt-tRNALeu(UUR) causing MELAS or m.8344A>G in mt-tRNALys causing MERRF, treated with vehicle only. All other bars in each graph represent cells with a pathological phenotype treated with different compounds. 3243: m.3243A>G mutant cells; H-8344: high m.8344A>G mutation load. The cells used in the experiment are cybrids, as in FIG. 1. The compounds used in the experiment are the same as those listed in FIG. 1 (i.e., βp; PMT; PMT-8a; PMT-8b; and E-βp) + elamipretide (E). Since elamipretide is described as a mitochondrial targeting sequence, the E-βp peptide was used to verify whether the combination of the prior art peptide (SEQ ID NO: 4) or the peptide of SEQ ID NO: 1 with elamipretide has a synergistic effect. The results of the experiment show that elamipretide does not confer any additional beneficial effect on the tested peptides (SEQ ID NO: 1 and SEQ ID NO: 4). In this case, the compound is not linked to Cy5, which is used only for the fluorescence experiments. V indicates cells treated with empty vehicle. For viability assessment, cells were plated in either glucose or galactose medium because a survival phenotype can be discerned in cells growing on galactose, which forces cells to rely on mitochondrial respiration, but not in cells growing on glucose.After 24 h of incubation, the number of viable cells in galactose medium was normalized to the number of viable cells in glucose at the same time point, which corresponds to normal growth conditions. Data are compared to the values ​​of mutant cells incubated with vehicle only. Mean ± SEM of at least three independent experiments is shown. Oxygen consumption rate was measured for cells grown on glucose, since the variation of this parameter can be evaluated in cells growing in this medium after 36 h of treatment. Data are shown compared to the values ​​of vehicle mutant cells. Mean ± SEM of three independent experiments is shown. p<0.05, §§§§p<0.0001 for §m.3243A>G vs. WT cells; °°p<0.01, °°°p<0.001 for H-8344 vs. l-8344 cells; *p<0.05, **p<0.01, ***p<0.001 for cells incubated with compound vs. cells incubated with vehicle only. [Diagram 3]Exogenously administered PMT is neither cytotoxic nor mitochondrial toxic up to 20 μM in mutant and wild-type cells. Images show the effect of increasing PMT concentrations on healthy and mutant cells compared to the effect of cytotoxic (C1) and mitochondrial toxic (C2) agents, assessed using the Mitochondrial ToxGlo™ Assay. The X- and Y-axes show the compounds incubated with the cells and the ratio of fluorescent to luminescent signals, respectively. In this assay, an increase in fluorescence indicates a decrease in cell membrane integrity, and a decrease in luminescence indicates a decrease in cellular ATP levels. In each graph, the first bar represents the effect of cell treatment with digitonin (C1), a cytotoxic agent. This causes both an increase in fluorescence and a decrease in luminescence, indicating cytotoxicity. The second bar represents the effect of cell treatment with sodium azide (C2), a mitochondrial toxic agent. This causes a decrease in luminescence with no effect on fluorescence, indicating mitochondrial toxicity. The other bars represent the effect of cell treatment with 5 μM, 10 μM or 20 μM PMT. This has no effect on either fluorescence or luminescence, indicating the absence of cytotoxicity or mitochondrial toxicity. The horizontal black line represents the fluorescence / luminescence signal ratio of untreated cells. WT: wild type; 3243: cells with the mutation m.3243A>G in mt-tRNALeu(UUR); L-8344 and H-8344: cells with low and high loadings of the mutation m.8344A>G in mt-tRNALys. The cells used in the experiments are cybrids, as in Figures 1 and 2. The compounds used are different concentrations of PMT not linked to Cy5; a cytotoxic agent (digitonin); and a mitochondrial toxic agent (sodium azide). Cybrids were seeded in 96-well plates in normal growth conditions (i.e., glucose medium) and treated with different PMT concentrations. In parallel, control cells (both wild type and mutant) were incubated with either 400ug / ml digitonin (C1) or 100ul sodium azide (C2) for 3 hours. Fluorescence and luminescence were measured using a GloMax Multi+Luminometer.The signals observed after each treatment (i.e., 5-10-20 µM PMT; C1; and C2) were normalized using the values ​​of non-treated cells and expressed as fluorescence / emission ratios. Data are the mean ± SEM of two independent experiments. [Figure 4]PMT, PMT-8a and PMT-8b undergo slower degradation in human plasma than the β32_33 peptide. Panel A. Representative chromatographic profiles of β32_33 and PMT obtained after 3 hours of compound incubation with human plasma from two healthy subjects. The X-axis indicates the time (min) that each compound is eluted by the chromatographic column. The Y-axis indicates the signal intensity of the two peptides. Panel B. Decay over time of β32_33, PMT, PMT-8a and PMT-8b incubated with human plasma from four healthy subjects for up to 72 hours. The X-axis indicates the time (hr) that the samples are analyzed. The Y-axis indicates the signal intensity of the four peptides. Samples of plasma were obtained from healthy volunteers and used immediately for analysis. Either β32_33 peptide or PMT for the experiments in panel A, and β32_33 peptide, PMT, PMT-8a or PMT-8b for the experiments in panel B, were incubated in plasma at a final concentration of 0.2 mM at 37°C for 3 h (A) or up to 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), 200 μL aliquots 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 μL of 0.1% formic acid containing 5% acetonitrile and analyzed on a Water Acquity H-Class UPLC system equipped with a single quadrupole mass detector with an electrospray ionization source. Samples were separated on a reversed-phase C18 column by running 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 ion recording (SIR): m / z = 917.88, corresponding to the [M+2H]2+ ion obtained from either β32_33 or PMT; m / z = 961.42, corresponding to the [M+H]+ ion obtained from PMT-8a; and m / z = 869.48, corresponding to the [M+H]+ ion obtained from PMT-8b. [Diagram 5]Comparison of the structures of the β32_33 peptide and the PMT. Chemical structures of the β32_33 peptide (A) and the PMT (B,C). N, O, and H atoms are explicitly shown, but carbon atoms are not. Single and double chemical bonds present on the plane are shown by single and double lines, respectively. Filled wedges indicate bonds that protrude toward the viewer. Dashed wedges indicate groups that point away from the viewer. For each chiral center, the S or R configuration is shown. For each pair of corresponding amino acids (e.g., Lys1, Lys2, Ser3, etc.), the side chains that are oriented toward the viewer in the β32_33 peptide (A) are oriented away from the viewer in the PMT (B,C), and the side chains that are oriented away from the viewer in the β32_33 peptide (A) are oriented toward the viewer in the PMT (A). In panel C, chemical groups in the PMT that have different orientations relative to the β32_33 peptide (A) are highlighted with grey ellipses. Because the relative positions of all amino acid side chains to the main chain are opposite in the β32_33 peptide and the PMT, interactions between the β32_33 peptide and the target mt-tRNA, including both main chain and side chain atoms, may not be conserved in the PMT. [Figure 6]After exogenous administration, M-PMT significantly improves the viability of mutant cells at concentrations down to 0.5 μM. Viability of cells treated with compounds. The concentrations of the different compounds used in the experiment and the percentage of live cells after treatment are shown on the X-axis and Y-axis, respectively. The first bar corresponds to wild-type cells treated with vehicle only. The second bar represents cells with m.3243A>G in mt-tRNALeu(UUR), which causes MELAS, treated with vehicle only. Further bars show the effect of decreasing concentrations of PMT and M-PMT on mutant cell viability. WT: wild-type cells. 3243: m.3243A>G mutant cells. The cells used in the experiment are cybrids. The compounds used in the experiment are PMT at 5, 2 and 0.5 μM concentrations, and M-PMT at 5, 2 and 0.5 μM concentrations. V indicates cells treated with empty vehicle. For viability assessment, cells were plated in either glucose or galactose medium. The reason for this is that a survival phenotype can be recognized in cells growing on galactose, which forces cells to rely on mitochondrial respiration, but not in cells growing on glucose. After 24 h of incubation, the number of live cells in galactose medium was normalized to the number of live cells in glucose (corresponding to normal growth conditions) at the same time point. Data are compared to values ​​for mutant cells incubated with vehicle only. Means ± SEM of at least two independent experiments are shown. p<0.0001 for m.3243A>G vs. WT cells; *p<0.05 for cells incubated with compound vs. cells incubated with vehicle only. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Array Description SEQ ID NO:1 PMT All amino acids are d-amino acids KKSFLSPRTALINFLV SEQ ID NO:2 PMT-8a All amino acids are d-amino acids KKSFLSPR SEQ ID NO:3 PMT-8b All amino acids are d-amino acids LSPRTALI SEQ ID NO: 4 β32_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 l-amino acids) SEQ ID NO:5 corresponds to SEQ ID NO:1 conjugated with the mitochondrial targeting sequence FRFK, where all amino acids are d-amino acids: FRFKKKSFLSPRTALINFLV SEQ ID NO:6 corresponds to SEQ ID NO:2 conjugated with the mitochondrial targeting sequence FRFK, in which all amino acids are d-amino acids: FRFKKKSFLSPR SEQ ID NO:7 corresponds to SEQ ID NO:2 conjugated with the mitochondrial targeting sequence FRFK, in which all amino acids are d-amino acids. SEQ ID NO:8 Artificial mitochondrial targeting / transmitting sequence 1 FRFK, all amino acids are d-amino acids SEQ ID NO: 9 Artificial mitochondrial targeting / transmitting sequence 2 FRA x K, all amino acids are d-amino acids SEQ ID NO: 10 Mitochondrial targeting / transmitting sequence 3 Fd(R)FK, where R is the only D amino acid, Horton KL et al., 2008 SEQ ID NO: 11 Artificial mitochondrial targeting / transmitting sequence 4 A x RA x K, all amino acids are d-amino acids SEQ ID NO: 12 Artificial mitochondrial targeting / transmitting sequence 5 FRFKFRFK, all amino acids are d-amino acids SEQ ID NO: 13 Artificial mitochondrial targeting / transmitting sequence 6 FRA x KFRA x K, all amino acids are d-amino acids SEQ ID NO: 14 Artificial mitochondrial targeting / transmitting sequence 7 A x RA x KA xRA x K, all amino acids are d-amino acids SEQ ID NO: 15 Artificial mitochondrial targeting / transmitting sequence 8 RKKRRQRRR, all amino acids are d-amino acids SEQ ID NO: 16 Artificial mitochondrial targeting / transmitting sequence 9 FRF2K, all amino acids are d-amino acids SEQ ID NO: 17 Artificial mitochondrial targeting / transmitting sequence 10 FRY Me K, all amino acids are d-amino acids SEQ ID NO: 18 Artificial mitochondrial targeting / transmitting sequence 11 FRYK, all amino acids are d-amino acids SEQ ID NO: 19 Artificial mitochondrial targeting / transmitting sequence 12 YRYK, all amino acids are d-amino acids SEQ ID NO: 20 Mitochondrial targeting / transmitting sequence 13 Fd(R)A x K and R are the only d-amino acids, Horton KL et al., 2008 SEQ ID NO: 21 Mitochondrial targeting / transmitting sequence 14 A x d(R)A x K and R are the only d-amino acids, Horton KL et al., 2008 SEQ ID NO: 22 Mitochondrial targeting / transmitting sequence 15 Fd(R)FKFd(R)FK, where R is the only d amino acid, Horton KL et al., 2008 SEQ ID NO: 23 Mitochondrial targeting / transmitting sequence 16 Fd(R)A x KFd(R)A x K and R are the only d-amino acids, Horton KL et al., 2008 SEQ ID NO: 24 Mitochondrial targeting / transmitting sequence 17 A x d(R)A x KA x d(R)A x K and R are the only d-amino acids, Horton KL et al., 2008 SEQ ID NO: 25 Mitochondrial targeting / transmitting sequence 18 RKKRRQRRR, Horton KL et al., 2008 SEQ ID NO: 26 Mitochondrial targeting / transmitting sequence 19 Fd(R)F2K, where R is the only d amino acid, Horton KL et al., 2008 SEQ ID NO: 27 Mitochondrial targeting / transmitting sequence 20 Fd(R)Y Me K and R are the only d-amino acids, Horton KL et al., 2008 SEQ ID NO: 28 Mitochondrial targeting / transmitting sequence 21 Fd(R)YK, where R is the only d amino acid, Horton KL et al., 2008 SEQ ID NO: 29 Mitochondrial targeting / transmitting sequence 22 Yd(R)YK, where R is the only d amino acid, Horton KL et al., 2008

[0045] Abbreviations in the above sequence: F2: diphenylalanine; A X YMe: cyclohexylalanine; YMe: methylated tyrosine. If the only d-amino acid is arginine, the amino acid is indicated in the sequence as d(R).

[0046] As discussed in the Summary of the Invention, the peptides of the invention are peptidomimetic compounds, hereinafter referred to as "PMT". PMT contains only d-amino acids (designated by the one-letter code preceded by a lowercase "d" letter) and its sequence is set forth in SEQ ID NO:1: 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) It is.

[0047] As shown in the figure and discussed in the experimental section below, PMT and its fragments, when administered exogenously, can permeabilize plasma and mitochondrial membranes (Figure 1) and rescue defective phenotypes in cell models harboring mt-tRNA mutations (Figure 2).

[0048] Furthermore, exogenously administered PMT is safe up to 20 μM in both mutant and wild-type cells, and finally, PMT is extremely stable in human plasma, since after 3 h of incubation in human plasma medium, in the first experiment PMT is 100% available whereas 70% of the β32_33 peptide is available (Figure 4A), and in the second experiment PMT is over 63% available whereas only 17% of the β32_33 peptide is available (Figure 4B).

[0049] The present invention relates to peptides having sequence number 1, characterized in that they consist exclusively of d-amino acids, and fragments thereof of the indicated sizes, which have been shown to be excellent peptidomimetics of the β32_33 peptide in terms of biological activity, i.e. rescuing the defective phenotype of a cell model with an mt-tRNA mutation (Perli et al., FASEB J, 2020 and Perli et al., "Hum mol genet 2016, Vol 25 No 5 903-915), and which exhibit the advantageous feature of being more stable in plasma than the natural peptide, and therefore the present invention relates to peptides having sequence number 1, characterized in that they consist exclusively of d-amino acids, and fragments thereof, in particular fragments of at least 8 amino acids.

[0050] 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, may be conjugated at the N-terminus to an mt targeting sequence. Figure 6 shows that conjugation to an mt targeting sequence such as SEQ ID NO: 8 surprisingly improves the efficacy (i.e. rescue activity) of the peptidomimetic of the invention by about 10-fold.

[0051] Therefore, an object of the present invention is also a peptide consisting of a dmt targeting sequence conjugated to the N-terminus of a peptide having SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0052] Indeed, in contrast to the data disclosed in Perli et al., 2020, where the use of an mt targeting sequence did not increase the rescue activity and mitochondrial localization of a peptide having SEQ ID NO:4, conjugation of the peptide of the present invention with an mt targeting sequence dramatically increased the rescue activity of the d-peptide of the present invention.

[0053] Preferably, the mt targeting sequence of the present invention is a sequence of 3 to 11 amino acids, preferably 3 to 6 amino acids, containing at least one arginine and / or at least one lysine and / or at least one phenylalanine residue.

[0054] Preferably, said at least one arginine and / or at least one phenylalanine residue is d-arginine and / or d-lysine and / or d-phenylalanine.

[0055] According to the invention, the mt targeting sequence may be a sequence selected from SEQ ID NO: 8 to SEQ ID NO: 29. In one embodiment of the invention, the fragment of the peptide of SEQ ID NO: 1 conjugated at its N-terminus to one of said mt targeting sequences is the peptide of SEQ ID NO: 2 or SEQ ID NO: 3.

[0056] In a preferred embodiment, the mt targeting sequence consists only of d-amino acids, and in a further preferred embodiment, the 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.

[0057] In a further preferred embodiment, said mt targeting sequence consisting only of d-amino acids is SEQ ID NO:8.

[0058] In a preferred embodiment, the peptides conjugated at their N-terminus to the mt targeting sequence having SEQ ID NO:8 are peptides having SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.

[0059] In view of the significant rescue activity exhibited by the peptidomimetics disclosed herein, the present invention also relates to a peptide having SEQ ID NO: 1 according to any of the disclosed embodiments and / or a fragment thereof, preferably conjugated at its N-terminus to an mt targeting sequence according to any one of the embodiments disclosed above, for use as a medicament.

[0060] In one embodiment, the present invention relates to the following chemical modifications: - modification of residues d-Phe 4, d-Leu 5, d-Arg 8 and / or d-Thr 9 of SEQ ID NO: 1; - modification of residues d-Phe 8, d-Leu 9, d-Arg 12 and / or d-Thr 13 of SEQ ID NO:5; - modification 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 absent in the PMT-8b fragment that does not undergo any degradation in human plasma), - modification of the peptide bond between d-Phe 8 and d-Leu 9 of SEQ ID NO: 5 or 6 (see above); - modification of the peptide bond between d-Arg 8 and d-Thr 9 of SEQ ID NO: 1 and between d-Arg 4 and d-Thr 5 of SEQ ID NO: 3 (in that this peptide bond is absent in the PMT-8a fragment, which does not undergo any degradation in human plasma); - modification of the peptide bond between d-Arg 12 and d-Thr 13 of SEQ ID NO:5 and between d-Arg 8 and d-Thr 9 of SEQ ID NO:7 (see above) The present invention also relates to variants of SEQ ID NOs: 1, 2, 3, 5, 6 and 7 which include one or more of:

[0061] All the above modifications are aimed at improving the PMT or fragments thereof (optionally conjugated with mt targeting sequences) as listed in Table 1, plasma stability while retaining rescue activity, or variants of PMT including chemical modification of additional residues in the peptide bond that would be shown not to undergo degradation therebetween by analysis of PMT fragments resulting from incubation in human plasma, aiming at improving PMT plasma stability while retaining rescue activity. Said variants may also preferably be conjugated at the N-terminus with mt targeting sequences according to any of the embodiments disclosed above. Preferably, said variants are conjugated with mt targeting sequences of SEQ ID NO:8.

[0062] In particular, the present invention relates to a peptide having SEQ ID NO:1 and / or a fragment thereof, optionally conjugated at the N-terminus to an mt targeting sequence according to any of the disclosed embodiments, for use in the treatment of mt-tRNA associated diseases.

[0063] As explained in the prior art and in the Summary of the Invention and Glossary, human mt-tRNA-associated diseases are diseases caused by mutations, in particular point mutations in various mt-tRNA-encoding genes that result in mutations in the mt-tRNA itself.

[0064] The diseases present a variety of different symptoms that usually affect highly oxygen consuming tissues such as the brain, heart, and muscles, i.e. tissues where the role of mitochondria is crucial. Non-limiting examples of mt-tRNA-associated diseases according to the present invention include mitochondrial myopathies, MERRF (myoclonus epilepsy with ragged red fibers syndrome), MIDD (maternally inherited diabetes and deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

[0065] In one embodiment of the present invention, the mt-tRNA-related disease is characterized by the following mitochondrial tRNA mt-tRNA Leu(UUR) , mt-tRNA Lysmt-tRNA Ile and mt-tRNA Val It is caused by a point mutation in the gene that codes for one of the following:

[0066] In particular, mt-tRNA (Leu)(UUR) and mt-tRNA (Lys) is responsible for approximately 85% of mt-tRNA-related diseases.

[0067] In one embodiment of the present invention, a peptide having SEQ ID NO: 1 as defined herein and / or a fragment thereof, optionally conjugated at its N-terminus to a mt targeting sequence according to any of the embodiments disclosed above, is used for the treatment of an mt-tRNA associated disease, said mt-tRNA associated disease being characterized by the inability to tolerate mt-tRNA targeting. Leu(UUR) m.3243A>G in the MT-TL1 human gene encoding mt-tRNA Lys m.8344A>G in the MT-TK human gene or mt-tRNA in the human gene MT-TI Ile or the m.4277T>C mutation in the human gene MT-TV Val It is caused by a point mutation selected from the m.1630A>G mutation in

[0068] When the disease is caused by one of the above mutations, the disease is MIDD, MELAS or MERRF.

[0069] A further object of the present invention is a pharmaceutical composition comprising one or more peptides and / or fragments thereof according to any one of claims 1 to 5 and at least one pharma- ceutically acceptable carrier.

[0070] Non-limiting examples of suitable pharmaceutical compositions are for systemic, oral, injectable, aerosol, oropharyngeal, and nasal administration.

[0071] The compositions of the present invention may be in the form of a solid, semi-solid, liquid, emulsion, gel, sprayable product, and the like.

[0072] The compositions of the present invention may also comprise one or more of peptides having SEQ ID NOs: 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 its variants.

[0073] Accordingly, the present invention also relates to the pharmaceutical compositions disclosed and claimed herein for use as medicines, in particular for use in the treatment of mt-tRNA associated diseases.

[0074] Non-limiting examples of mt-tRNA related diseases according to the present invention include mitochondrial myopathies, MERRF (myoclonus epilepsy with ragged red fibers syndrome), MIDD (maternally inherited diabetes and deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes).

[0075] In one embodiment of the present invention, the mt-tRNA-associated disease is characterized by the following mt-tRNA: Leu(UUR) , mt-tRNA Lys mt-tRNA Ile and mt-tRNA Val In one embodiment of the present invention, the pharmaceutical composition as defined herein is for the treatment of an mt-tRNA-associated disease, wherein the mt-tRNA-associated disease is caused by a point mutation in a gene encoding one of the mt-tRNA Leu(UUR) m.3243A>G in the MT-TL1 human gene encoding mt-tRNA Lys m.8344A>G in the MT-TK human gene or mt-tRNA in the human gene MT-TI Ile or the m.4277T>C mutation in the human gene MT-TV Val It is caused by a point mutation in the genotype, the m.1630A>G mutation in

[0076] When the disease is caused by one of the above mutations, the disease is MIDD, MELAS or MERRF.

[0077] The present invention also relates to a process for the preparation of a pharmaceutical composition as defined above and in the claims, comprising mixing one or more peptides having SEQ ID NO: 1 as defined herein and in the claims, optionally conjugated at the N-terminus with a mt targeting sequence according to any one of the embodiments disclosed above, and / or fragments thereof, with at least one pharma- ceutically acceptable carrier. The peptides of the present invention can be synthesized by any technique commonly used in the art for the preparation of d-peptides, and can be purified to pharmaceutical grade using conventional techniques. Once prepared and purified, the d-peptides of the present invention are formulated into the corresponding pharmaceutical composition according to techniques well known in the art with conventional carriers, excipients, etc. See, for example, Volume "Remington's Pharmaceutical Sciences 15a Ed."

[0078] The composition of the present invention may further contain other compatible auxiliary components that are not listed above and are usually found in pharmaceutical compositions at the use level established in the art.Thus, for example, the composition may contain additional compatible pharmacoactive materials for combination therapy, or may contain materials that are useful for physically formulating various dosage forms of the present invention, such as excipients, preservatives, antioxidants, thickeners, stabilizers, etc.

[0079] The present invention also relates to the use of a peptide having SEQ ID NO: 1 as defined and claimed herein and / or a fragment thereof, optionally conjugated at its N-terminus with an mt targeting sequence according to any one of the embodiments disclosed above, in in vitro methods of pharmacological toxicological studies, for example for the detection of PMT off-targets, for the evaluation of tissue-specific PMT effects, for the investigation of PMT activity towards further diseases.

[0080] As an example, for evaluation of tissue-specific PMT effects, peptides having SEQ ID NO: 1 and / or one or more fragments thereof, optionally conjugated at their N-terminus to an mt targeting sequence according to any one of the embodiments disclosed above, are contacted with specific tissue cells or tissues or organoids optionally carrying one or more mutations in the mt-tRNA gene that result in a mutation in the corresponding mt-tRNA affecting the phenotype of the cells, tissues or organoids described below, and their ability to rescue the abnormal phenotype of the cells, tissues, organoids caused by said mutations is evaluated.

[0081] Alternatively, PMT or a fragment thereof can be tested on healthy cells, tissues or organoids to identify its undesirable off-target effects against untreated controls, or PMT or a fragment thereof can be tested in combination with other compounds to identify potentially therapeutically effective combinations of active ingredients.

[0082] By "phenotype-affecting" in the above sentence it is intended that said mutation may be a mutation that causes an abnormal phenotype and thus causes an mtRNA-associated disease.

[0083] "Rescue" of the abnormal phenotype can be partial rescue (from a more severe phenotype to a less severe phenotype, i.e., relative to a control untreated sample) and complete rescue (from an abnormal phenotype to a normal phenotype, i.e., relative to a control sample not carrying the mutation).

[0084] The peptides of the invention can also be used in vitro in combination with one or more additional compounds, as described above, to identify compounds that may have a pharmacological effect on mtRNA-related diseases.

[0085] Furthermore, the present invention relates to a method for the treatment of an mt-tRNA associated disease, comprising administering to a subject in need of treatment of an mt-tRNA associated disease a therapeutically effective amount of a peptide having SEQ ID NO: 1 as defined herein and / or a fragment thereof (which is a peptidomimetic of the art known peptide having SEQ ID NO: 4) or a pharmaceutical composition as defined herein, optionally conjugated at its N-terminus with an mt targeting sequence according to any one of the embodiments disclosed above.

[0086] Non-limiting examples of mt-tRNA related diseases treatable by the methods of the present invention include mitochondrial myopathies, MERRF (myoclonus epilepsy with ragged red fibers syndrome), MIDD (maternally inherited diabetes and deafness) and MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes).

[0087] In one embodiment of the present invention, the mt-tRNA-associated disease is characterized by the following mt-tRNA: Leu(UUR) , mt-tRNA Lys mt-tRNA Ile and mt-tRNA Val These are caused by point mutations in the genes encoding one of the following:

[0088] In one embodiment of the present invention, the present invention relates to the treatment of mt-tRNA associated diseases, said mt-tRNA associated diseases being caused by point mutations, said point mutations being in the mt-tRNA Leu(UUR) m.3243A>G in the MT-TL1 human gene encoding mt-tRNA Lys m.8344A>G in the MT-TK human gene or mt-tRNA in the human gene MT-TI Ile or the m.4277T>C mutation in the human gene MT-TV Val The m.1630A>G mutation in

[0089] When the disease is caused by one of the above two mutations, the disease is MIDD, MELAS or MERF.

[0090] A further object of the present invention is the use of a peptide having SEQ ID NO: 1 as defined herein and / or a fragment thereof, optionally conjugated at its N-terminus with an mt targeting sequence according to any one of the embodiments disclosed above, for the preparation of a medicament for the treatment of an mt-tRNA associated disease, wherein one or more of said peptides having SEQ ID NO: 1 as defined herein and / or a fragment thereof are mixed with at least a pharma- ceutically acceptable carrier, thereby obtaining a pharmaceutical composition as defined herein and claimed.

[0091] As noted above, mt targeting sequences consisting entirely of d-amino acids are preferred.

[0092] All cells used in the experiments reported below were obtained from patients who, in accordance with current legislation, gave their free and informed consent for said uses.

[0093] example material and method Peptide synthesis All constructs were synthesized by Pepscan (Pepscan Presto, Lelystad, The Netherlands) with a purity of >85%.

[0094] The compounds used in this study are listed in Table 1. [Table 1-1] [Table 1-2] [Table 1-3]

[0095] cell line mt-tRNA Leu(UUR) m.3243A>G mutation in mt-tRNA Lys Previously established osteosarcoma-derived (143B.TK-) cybrid cell lines from patients carrying either the m.8344A>G mutation in the pathological mt-tRNA gene and controls were used (kindly provided by Drs. Valeria Tiranti and Valerio Carelli). Leu(UUR) The mutants had a mutation load of over 98%. Lys The mutants had a mutational burden of either ≈80% (H-8344) or ≈30% (l-8344). High mutation burden mutants were pathogenic, whereas low mutation levels showed no detectable phenotype [Perli et al, Hum Mol Genet, 2016].

[0096] cell culture Cybrid cells were cultured at 37 °C in a humidified atmosphere of 95% air and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g / l d-glucose, 10% fetal bovine serum (FBS), 2 mM l-glutamine, 50 μg / ML uridine, 100 U / mL penicillin and 100 mg / mL streptomycin (referred to as glucose medium). 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 recognized in cells grown on galactose, which forces cells to rely on mitochondrial respiration, but not in cells grown on glucose.

[0097] Fluorescence microscopy Constructs made from the compounds listed in Table 1 linked to Cy5 fluorophores via maleimide crosslinkers were administered to subconfluent cybrid cell cultures at 0.25 μM in glucose medium. Approximately 24 hours after treatment with the different constructs, cells were incubated with 200 nM Mitotracker Red FM (LifeTechnologies Italia, Monza, Italy) for 30 min at 37° C. Cells were then visualized by confocal microscopy. Images of 800×800px (at 88 nm / px) were acquired on an Olympus iX83 FluoView1200 laser scanning confocal microscope using a 60×NA1, 2 water objective (Olympus Italia SRL Milano, Italy), zoom 3×, 559 nm and 635 nm lasers, and filter settings for MitoTracker Red and Cy5. Fluorescent images were analyzed with ImageJ software (14, https: / / imagej.nih.gov / ij / , 1997-2018) to determine the Pearson correlation coefficient.

[0098] Cell viability To test the proliferation potential, cells were harvested and cultured at 30 × 10 cells in glucose medium in 60 mm dishes with one of the compounds (each at 5 μM concentration). 4 Cells were seeded in 0.25% trypsin and 0.2% EDTA for 24 h. Cells were switched into glucose or galactose and after 24 h, cell viability was measured by trypan blue dye exclusion assay. Cells were harvested with 0.25% trypsin and 0.2% EDTA, washed, suspended in PBS in a 1:1 ratio in the presence of trypan blue solution (Sigma-Aldrich) 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.

[0099] Respirometric assay The oxygen consumption rate (OCR) of cybrids incubated with compounds was assessed with a Clark-type oxygen electrode (Hansatech Instruments, Norfolk, UK). After incubation with compounds, both control and mutant cybrids were maintained in glucose medium for 36 h and then cultured as intact cells (3 × 10 6 ) and OCR was measured.

[0100] Mitochondrial toxicity Mitochondrial ToxGlo™ Assay (Promega Italia Srl. Milan, Italy) was used to measure mitochondrial toxicity caused by PMT according to the manufacturer's protocol. Cybrids were seeded in 96-well plates and treated with different concentrations of PMT (5, 10 and 20 μM). After 24 hours of treatment, control cells (both wild type and mutant) were incubated for 3 hours with either 400 ug / ml digitonin (cytotoxic agent) or 100 μl sodium azide (mitochondrial toxic agent) as a positive control for cytotoxicity or mitochondrial toxicity, respectively. Subsequently, cells were incubated with the specific reagents and fluorescence or luminescence was measured with a GloMax Multi+Luminometer (Promega Italia Srl., Milan, Italy).

[0101] statistical analysis All data are expressed as mean ± SEM. Data were analyzed by standard ANOVA procedures followed by multiple pairwise comparisons adjusted with Bonferroni correction. Significance was considered at <0.05. Numerical estimates were obtained using Graphpad Prism version 7 (Graphpad Inc San Diego, CA, USA).

[0102] plasma stability To assess whether the compounds (βp and d-βp) are stable in blood or are hydrolyzed by plasma peptidases, we set up a chromatographic assay to evaluate compound concentrations after incubation in human plasma.

[0103] Blood was collected by venipuncture in vacutainers containing EDTA as an anticoagulant. Two different samples from healthy volunteers were used. Plasma was separated by centrifugation and used immediately for the experiments. Each compound was dissolved in 500uL of plasma at a final concentration of 0.2mM and divided into two aliquots, one of which was analyzed immediately to assess basal compound levels and the other was incubated at 37°C for 3 hours under gentle shaking. To perform chromatographic analysis, samples were treated with 3 volumes of acetonitrile containing 1% formic acid and then extracted to remove proteins and phospholipids by using an Ostro™ pass-through sample preparation system. Samples were dried under vacuum and then resuspended in 100uL of 0,1% formic acid containing 5% acetonitrile and then directly injected into the chromatographic column.

[0104] Chromatographic analysis was 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 quadrupole mass detector (ACQUITY QDa) with an electrospray ionization source. The analysis was performed on a reversed-phase C18 column (75 mm × 3.2 mm i.d., 2.5 μm particle size). The mobile phases were 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 elution was performed by linearly increasing the concentration of solvent B to 70% in 7 min. Mass spectrometric detection was performed in positive electrospray ionization mode with nitrogen as the nebulizer gas. The analysis was performed in total ion current (TIC) mode with a mass range of 100–1200 m / z. The capillary voltage was 0.8 kV, the cone voltage was 8 V, the ion source temperature was 120° C., and the probe temperature was 600° C. Quantification of each compound was performed by selected ion recording (SIR) at m / z 917.88, which corresponds to

[0105] result PMT and PMT fragments (PMT-8a and PMT-8b) permeabilize the plasma membrane and colocalize with mitochondria. The uptake and localization of Cy5-conjugated constructs in cybrids was assessed by flow cytometry, confocal microscopy and immunoblot analysis on isolated mitochondria (Figure 1). Confocal microscopy was performed using a specific live cell stain for mitochondria (Mitotracker FM Red). After 12 hours, the fluorescent signals of all constructs were clearly detectable in cybrids. All constructs showed cellular uptake and clear overlap with mitochondrial reticular structures, as demonstrated by Pearson's correlation coefficients reflecting mitochondrial specificity (Figure 1). These results indicate that upon exogenous administration to mutant cells, all constructs reported in the images are able to penetrate the cell membrane and localize with mitochondria.

[0106] m.3243A>G mt-tRNA Leu(UUR) and m.8344A>G mt-tRNA Lys Effect of PMT and PMT fragments (PMT-8a and PMT-8b) on viability of mutant cybrids To assess the effects of compounds on viability, cybrids were grown in glucose-free medium supplemented with galactose (galactose medium), conditions that force cells to rely on the mt respiratory chain for ATP synthesis and cause a severe reduction in growth in the presence of mutations.

[0107] We observed that PMT could induce significant improvements in cell viability and apoptosis rate in both m.3243A>G and m.8344A>G mutant cybrids compared with untreated mutant cells (Figure 2, upper panel). The rescue activity of PMT was comparable to that of the β32_33 peptide. PMT-8b also significantly improved cell viability and apoptosis rate in the m.3243A>G mutant.

[0108] m.3243A>G mt-tRNA Leu(UUR) and m.8344A>G mt-tRNA LysEffects of PMT and PMT fragments (PMT-8a and PMT-8b) on oxygen consumption in mutant cybrids To investigate whether the increased cell viability is related to improved mt bioenergetics, we analyzed the respiratory capacity of mutant and control cells using Clark-type electrodes. We demonstrated that PMT determined a significant increase in the oxygen consumption rate in both pathological mutants (Figure 2, lower panel). This activity is comparable to that of the β32_33 peptide in m.3243A>G mutant cybrids and even higher than that of the β32_33 peptide in m.8344A>G mutant cybrids.

[0109] Lack of cytotoxicity and mitochondrial toxicity of PMT and PMT fragments (PMT-8a and PMT-8b) To assess the toxicity of increasing concentrations of exogenously administered PMT and PMT fragments, we performed the Mitochondrial ToxGlo™ Assay. PMT, PMT-8a and PMT-8b fragments express the m.3243A>G mt-tRNA Leu(UUR) Mutant cybrid, m.8344A>G mt-tRNA Lys The results were neither cytotoxic nor mitochondrially toxic in mutant cybrids and healthy control cells up to 20 μM.

[0110] PMT has greater stability in human plasma than the β32_33 peptide. To evaluate their plasma stability, two experiments were performed. In the first experiment, β32_33 peptide or PMT were incubated in plasma samples from two healthy volunteers and the amount of each compound was measured before (T0) and 3 hours after plasma incubation (3 h). As shown in Figure 4A, after 3 hours of plasma incubation, PMT undergoes no visible degradation, whereas only 70% of the β32_33 peptide is still available. In the second experiment, β32_33 peptide, PMT, PMT-8a or PMT-8b were incubated in plasma samples from four healthy volunteers and the amount of each compound was measured before plasma incubation (T0) and at different time points after plasma incubation (i.e., 1.5, 3, 6 and 72 h). As shown in Figure 4B, PMT has a higher plasma stability than β32_33 peptide at all time points, but unlike the previous experiment, it underwent detectable degradation. Conversely, after 72 h, PMT-8a underwent no detectable degradation and 80% of the PMT-8b fragment was present, suggesting that the eight C-terminal residues of PMT, none of which were present in PMT-8a and only four of which were present in PMT-8b and are predominantly hydrophobic in nature (e.g., d-Ala 10, d-Leu 11, d-Ile 12, d-Phe 14, d-Leu 15, and d-Val 16), may be at least partially involved in peptide sequestration by plasma proteins with hydrophobic pockets, such as serum albumin.

[0111] M-PMT improves the survival rate of m.3243A>G mt-tRNALeu(UUR) at a 10-fold lower concentration than PMT. To assess the effects of PMT and M-PMT on viability, cybrids were grown in glucose-free medium supplemented with galactose (galactose medium), conditions that force cells to rely on the mt respiratory chain for ATP synthesis and cause a severe reduction in growth in the presence of the mutations.

[0112] M-PMT significantly improved the viability of m.3243A>G mutant cybrids at 5, 2, and 0.5 μM concentrations compared with untreated mutant cells (Figure 6), whereas the PMT peptide exerted rescue activity at 5 μM, but not at 2 and 0.5 μM concentrations.

Claims

1. A peptide having SEQ ID NO: 1 and / or a fragment thereof of at least 8 amino acids in length, or a variant of said peptide or fragment, wherein said peptide consists entirely of d-amino acids, or a variant thereof.

2. A fragment of the peptide of claim 1, wherein the fragment of the peptide has SEQ ID NO: 2 or SEQ ID NO:

3.

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

4. The peptide and / or fragment thereof according to claim 3, wherein the mt targeting sequence is a sequence of 3 to 11 amino acids.

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

6. 5. The peptide and / or fragment thereof of claim 4, wherein the mt targeting sequence is selected from 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:28 or SEQ ID NO:

29.

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

8. The peptide and / or fragment thereof of claim 7, wherein the 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:19 or SEQ ID NO:

19.

9. The peptide of claim 3, wherein the peptide has SEQ ID NO:

5.

10. The peptide fragment of claim 3, wherein the fragment of the peptide has SEQ ID NO: 6 or SEQ ID NO:

7.

11. A pharmaceutical composition comprising one or more peptides and / or fragments thereof according to any one of claims 1 to 10.

12. 12. The pharmaceutical composition of claim 11 for use as a medicament.

13. The pharmaceutical composition according to claim 11 for the treatment of an mt-tRNA-associated disease.

14. 14. The pharmaceutical composition of claim 13, wherein the mt-tRNA-associated disease is selected from mitochondrial myopathy, MERRF (myoclonus epilepsy with ragged-red fibers syndrome), MIDD (maternally inherited diabetes and deafness), and MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes).

15. The mt-tRNA-associated disease is characterized by the following mt-tRNA: mt-tRNA Ｌｅｕ（ＵＵＲ） , mt-tRNA Ｌｙｓ mt-tRNA Ｉｌｅ and mt-tRNA Ｖａｌ The pharmaceutical composition of claim 13, wherein the IL-14 gene is caused by a point mutation in a gene encoding one of the following:

16. The mutation is mt-tRNA Ｌｅｕ（ＵＵＲ） m. 3243A>G in the MT-TL1 human gene encoding mt-tRNA Ｌｙｓ m. 8344A>G in the MT-TK human gene encoding mt-tRNA in the human gene MT-TI Ｉｌｅ m. 4277T>C mutation in the human MT-TV gene or mt-tRNA Ｖａｌ 16. The pharmaceutical composition of claim 15, wherein the m. 1630A>G mutation in

17. 15. The pharmaceutical composition of claim 14, wherein the disease is MIDD, MELAS, or MERF.

18. A method for the preparation of a pharmaceutical composition, comprising mixing one or more peptides and / or fragments thereof according to claims 1 to 10 with at least one pharmaceutically acceptable carrier.

19. Use of a peptide and / or fragments thereof according to any one of claims 1 to 10 in an in vitro method of pharmacological toxicological research.

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