RU2834339C1 - Treatment of mitochondrial diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to genetics and can be used in treating mitochondrial diseases. Use according to the invention relates to a composition containing deoxynucleosides in an amount of more than one, selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine, for the treatment of mitochondrial DNA depletion and/or deletion syndrome caused by a DNA polymerase gamma-1 (POLG1) subunit defect.

EFFECT: use of the invention enables to achieve an increase in the rate of polymerization of mitochondrial DNA regardless of the mutation in the POLG gene.

16 cl, 3 tbl, 3 dwg, 1 ex

Description

Field of technology

The present invention relates to the field of medicine, in particular to the treatment of mitochondrial diseases, and more particularly to such mitochondrial diseases caused by deletion/depletion of mitochondrial DNA (mtDNA).

Prior art

The mitochondrial genome (mtDNA) is a DNA molecule (deoxyribonucleic acid) containing 16.5 thousand nucleotide pairs (kb, kilobase (pairs)), which is usually present in several copies in individual mitochondria.

A large group of Mendelian mitochondrial diseases are caused by mutations in nuclear genes whose products are involved in the replication or maintenance of mtDNA. These rare disorders are also known as intergenomic communication defects, mtDNA depletion diseases, multiple mtDNA deletion diseases, or mitochondrial depletion and deletion diseases (MDDS). These disorders have specific orphan codes: ORPHA35698 - mitochondrial DNA depletion syndrome and ORPHA254807 - mitochondrial DNA multiple deletion syndrome, and are also recognized in the OMIM (Online Mendelian Inheritance in Man) database: http://www.om im.org/phenotypicSeries/PS603041 for mitochondrial DNA depletion syndrome and http://www.omim.org/phenotypicSeries/PS157640 for multiple mitochondrial DNA deletions. These diseases are a complex group of genetically and clinically heterogeneous diseases characterized by the presence of mtDNA aberrations in one or a combination of affected tissues (e.g. skeletal muscle, liver, brain).

The severity and progression of such diseases is highly variable and can range from mild manifestations (eg, progressive external ophthalmoplegia) to severe phenotypes that can lead to death in infancy or early childhood, as seen in the classic mtDNA depletion syndromes.

Most genes so far associated with defects in intergenomic communication are either directly involved in mtDNA replication or involved in the metabolism of deoxyribonucleoside triphosphates (dNTPs), the building blocks of DNA synthesis. However, an increasing number of mutations leading to MDDS are identified in genes causing mtDNA instability through an as yet unknown pathomechanism (OPA1, MPV17, FBXL4, etc.). Classically, defects in certain genes are thought to specifically result in mtDNA depletion or multiple deletions. For example, defects in DGUOK genes typically result in mtDNA depletion, whereas defects in OPA1 genes typically result in multiple mtDNA deletions. However, there is increasing evidence that mtDNA depletion and multiple deletions may be considered as manifestations of the same pathological processes that affect mtDNA replication and repair. Mutations in genes that until recently had been associated only with mtDNA depletion with onset in infancy (TK2, DGUOK) were found to also cause multiple mtDNA deletions, in some cases acquired.

Because MDDS are multiorgan disorders, a multidisciplinary team comprising of various specialists is required to provide support and symptomatic treatment for associated complications.

To date, effective treatment methods for these complex diseases have not been developed. This is mainly due to the lack of information on the exact parameters of the mechanisms causing such diseases, the variability between one syndrome and another, etc.

Despite the above, attempts have been made to find suitable treatments for MDDS caused by dNTP metabolism defects, in which dNTP availability is thought to be impaired. For example, recent experimental studies have shown that bypassing the defective step of deoxyribonucleotide biosynthesis can overcome mtDNA depletion. In particular, it was reported that supplementation of purine deoxynucleotide (dN) monophosphates (dNMPs) can restore mtDNA depletion in TK2-KO mice [1]. In addition, Camara Y. et al. [2] reported that the use of deoxyribonucleosides and/or specific inhibitors of their catabolism can be an effective pharmacological approach to treat various MDDS caused by dNTP homeostasis defects.

Despite these efforts, there remains a need for therapeutic approaches to these and other cases of MDDS.

The essence of the invention

The authors of the present invention have established that the introduction of deoxyribonucleosides allows the restoration of mtDNA levels in MDDS diseases not caused by a defect in dNTP metabolism.

Until now, there has been a general belief that nucleoside administration can only be effective in the treatment of mitochondrial diseases caused by a defect in dNTP metabolism. In fact, the prior art assumed that a disease caused by a defect in dNTP metabolism could be overcome by administering a "defective" nucleoside [2]. Thus, until now, it was believed that only diseases caused by a deficiency of a specific nucleotide could be treated by administering a sufficient amount of this "defective" nucleotide/nucleoside.

In contrast to the prior art methods, the present inventors introduced deoxyribonucleosides into fibroblast samples from patients previously diagnosed with MDDS, carrying certain mutations affecting the catalytic subunit of the polymerase gamma 1 protein (one of the enzymes involved in the mtDNA replication complex). Surprisingly, it was found that introducing deoxyribonucleosides into these samples restored mtDNA levels to "normal" (healthy) levels (see Table 3, below) regardless of the mutation carried by the patient. It could not have been predicted that the abnormal function of the POLG1 gene encoding the enzyme (which ultimately negatively affects the proper functioning of the replication complex), caused by the different mutations, could be equally overcome by introducing deoxyribonucleosides in the case of cells obtained from all three patients. It is significant that restoration of mtDNA levels by the introduction of deoxyribonucleosides does not depend on the mutation responsible for the defect in the mtDNA replication complex.

Thus, according to a first aspect, the present invention provides a composition comprising one or more canonical deoxyribonucleosides for use in the treatment of a mitochondrial DNA wasting and/or deletion syndrome, provided that the syndrome is not due to a defect in deoxyribonucleoside triphosphate (dNTP) metabolism. This aspect can also be formulated as a use of a composition comprising one or more canonical deoxyribonucleosides for the preparation of a medicament for the treatment of a mitochondrial DNA wasting and/or deletion syndrome, provided that the syndrome is not due to a defect in deoxyribonucleoside triphosphate (dNTP) metabolism. This aspect can also be formulated as a method for the treatment of a mitochondrial DNA wasting and/or deletion syndrome not due to a defect in deoxyribonucleoside triphosphate (dNTP) metabolism, the method comprising administering a therapeutically effective amount of one or more canonical deoxyribonucleosides to a subject in need thereof.

Experimental data were obtained in cells from patients suffering from various clinical manifestations caused by POLG deficiency.

POLG is the gene encoding the catalytic subunit of the mitochondrial DNA polymerase called DNA polymerase gamma. In eukaryotic cells, mitochondrial DNA is replicated by DNA polymerase gamma, a trimeric protein complex consisting of a 140-kDa catalytic subunit encoded by the POLG gene and a dimeric 55-kDa accessory subunit encoded by the POLG2 gene. The catalytic subunit has three enzymatic activities: DNA polymerase activity, 3'-5' exonuclease activity that corrects misincorporated nucleotides, and 5'-dRP lyase activity required for DNA base excision repair. In the examples below, patients suffered from POLG deficiency due to mutations in the exonuclease or polymerase domain (R309C (in the exonuclease domain), and G848S and V1177L (in the polymerase domain)) and in the linker region (W748S).

The present inventors have surprisingly found that administration of deoxyribonucleosides "works" in the case of all the studied mutations of POLG and that, regardless of whether the mutation affects the function or structure of POLG, there is a marked improvement in the enzymatic activity of the mutated form of POLG to such an extent that the mtDNA levels are restored and are in the same state as in a healthy subject. In other words, administration of deoxyribonucleosides hyperstimulates the enzymatic form of POLG, which before said administration was partially deprived of polymerase activity.

The experimental data presented below (summarized in Table 3 below) suggest that the introduction of deoxyribonucleosides may be sufficient to increase the rate of mtDNA polymerization regardless of the mutation in the POLG gene. However, these data also suggest that any other MDDS disease known to be characterized by mtDNA loss (either by reduced mtDNA copy number or by multiple mtDNA deletions) and which is caused by mutations in proteins of the replication complex itself (POLG1, POLG2, which encodes the polymerase gamma accessory unit; PEO1, MGME1, and DNA2, among others) or indirectly involved in mtDNA replication (such as MPV17) can also be effectively treated by hyperstimulation of POLG enzymatic activity via deoxyribonucleoside administration: increased POLG activity results in a substantial increase in mtDNA levels, which can "neutralize" the mtDNA loss regardless of the cause of such loss (a particular mutation in a particular protein) and restore "normal" levels.

As a result, a dNs-based treatment strategy could partially or completely counteract mtDNA depletion in any defect that impairs mtDNA replication due to partially or completely functional polymerase activity.

Based on the experimental data presented below, it can be concluded that the restoration of mitochondrial DNA levels can be independent of the severity of the patient's disease, which gives the invention great therapeutic value.

Other advantages associated with the administration of deoxyribonucleosides in the treatment of an object with MDDS according to the present invention are the cost (low) and the absence of special requirements for their preservation. In addition, canonical deoxyribonucleosides are natural compounds, usually present in all living organisms.

Brief description of graphic materials

Fig. 1 shows the amino acid sequence of the POLG1 protein according to the NCBI (National Center for Biotechnology Information) database. The following mutations are underlined: at position 309 - arginine to cysteine, at position 748 - tryptophan to serine, at position 848 - glycine to serine, at position 1143 - glutamic acid to glycine, at position 1177 - valine to leucine.

Figure 2 shows the metabolic pathways involved in mtDNA wasting and deletion syndromes (MDDS). Proteins whose dysfunction is associated with MDDS are labeled with number 1 (involved in dNTP metabolism), number 2 (belonging to the replication complex), or number 3 (associated with MDDS via an unknown pathomechanism). Although the genes SUCLA2 and SUCLG1 have been linked to the enzyme nucleotide diphosphate kinase, their association with dNTP metabolism has not been clearly confirmed. Also shown are other proteins involved in dNTP metabolism that have not yet been associated with MDDS, and specific inhibitors of deoxyribonucleoside catabolic enzymes: tetrahydrouridine (THU); 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride (TPI), immucillin H (IH), and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Abbreviations: ABAT: 4-aminobutyrate aminotransferase; ADA: adenosine deaminase; ANT1: adenine nucleotide translocase 1; CDA: cytidine deaminase; cdN: cytosolic deoxyribonucleotidase; dAdo: deoxyadenosine; dCK: deoxycytidine kinase; dCTD: dCMP deaminase; dCtd: deoxycytidine; dGK: deoxyguanosine kinase; dGuo: deoxyguanosine; dlno: deoxyinosine; DNA2: DNA helicase 2; dThd: thymidine; dUrd: deoxyuridine; ENT1: equilibrated nucleoside transporter 1; FBXL4: F-box leucine-rich repeat-containing protein 4; mdN: mitochondrial deoxyribonucleotidase; MFN2: mitofusin-2; MGME1: mitochondrial genome maintenance exonuclease 1; MPV17: mitochondrial inner membrane MPV17; NDPK: nucleotide diphosphate kinase; NMPK: nucleotide monophosphate kinase; OPA1: optic atrophy 1; PNP: purine nucleoside phosphorylase; POLG1: polymerase gamma subunit 1; POLG2: polymerase gamma subunit 2; RNR: ribonucleotide reductase; SAMHD1: SAM domain and HD domain-containing protein 1; SUCLA2: beta-subunit, succinate-CoA ligase; SUCLG1: a-subunit, succinate-CoA ligase; TK1: thymidine kinase 1; TK2: thymidine kinase 2; TP: thymidine phosphorylase; TS: thymidylate synthase; Twinkle: mitochondrial Twinkle helicase.

Fig. 3 is a schematic diagram of the experimental study to determine mtDNA levels in cells collected at specific time points (t0, t7, t14, t21, and t26). The stability of dNs was monitored in cell media after 2-3 days of culture (t16-t17).

Detailed description of the invention

The present invention is based on the discovery that restoration of mtDNA levels can be achieved by administering deoxyribonucleosides to patients suffering from MDDS caused by a defect other than a defect in dNTP metabolism. Nucleosides are glycosylamines, which can be considered as nucleotides without a phosphate group. A nucleoside consists only of a nucleobase (also called a nitrogenous base) and a 5-carbon sugar (ribose or deoxyribose), whereas a nucleotide consists of a nucleobase, a five-carbon sugar and one or more phosphate groups. In a nucleoside, the base is linked to ribose or deoxyribose via a beta-glycosidic bond. Examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine and inosine.

As explained above, MDDS diseases are a group of disorders caused by defects in mitochondrial DNA (mtDNA) replication and include disorders characterized by either a reduction in the number of mtDNA copies (mtDNA depletion syndromes) or multiple deletions of mtDNA (mtDNA deletion syndromes). They are officially recognized categories in the catalog of orphan diseases and in the Online Mendelian Inheritance in Man, which are reference sites for specialists in the field of such pathologies.

This group of mitochondrial diseases constitutes a well-known group of disorders caused by mutations in a specific group of genes and is therefore well known to specialists in the field of mitochondrial disorders. In some cases, the name by which this group of diseases is known may change: mtDNA depletion and deletion syndromes [3,4]; intergenomic communication (or signaling) defects [5]; mtDNA replication defects [6]; etc. Two or more such names sometimes coexist in the same publication.

Diseases caused by mtDNA replication defects may be due to mutations in:

- A-genes encoding proteins related to the mtDNA replication complex (designated by number "2" in Fig. 2) [7,8,9,10,11];

- B-genes encoding proteins involved in the catabolism or anabolism of nucleosides/nucleotides (designated by the number "1" in Fig. 2) [12,13,14,15];

- C-genes encoding proteins of unknown function, or whose function does not belong to categories A or B and cannot be biochemically linked to the mtDNA replication process (designated as "3" in Fig. 2) [16,17,18,19,20,21,22,23,24].

This classification is clearly recognized by experts in this field of technology [3,4,6,25,26,27].

dNTPs are required at the replication fork as substrates for DNA synthesis. Mammalian cells obtain precursors for DNA synthesis and repair from two different metabolic sources: the cytosolic primary synthesis step and the salvage pathway, the latter relying on two parallel sets of enzymes located in the cytoplasm and the mitochondrial matrix. dNTP synthesis is coupled to nuclear DNA replication, the moment when cellular requirements for DNA precursors are highest. The dNTP pool decreases markedly after completion of the replication phase (S phase) or in non-dividing cells. The cytosolic primary synthesis step relies on cytosolic ribonucleotide reductase (RNR) activity (with the exception of dTMP, for which a mitochondrial thymidylate synthase has recently been identified). RNR catalyzes the allosterically balanced reduction of all four ribonucleoside diphosphates to the corresponding deoxyribonucleosides (dNs), providing the cell with high concentrations of dNTPs during the S phase of the cell cycle. RNR is a heterotetramer containing two copies of a large subunit (R1) and two small subunits (R2 or p53R2). While R2 is degraded by the proteasomal pathway in late mitosis, p53R2 is present throughout the cell cycle and its expression is stable also in non-dividing cells. In contrast to nuclear genome replication, mtDNA synthesis occurs independently of cell division. Although the primary cytosolic step of synthesis mediated by p53R2 is significantly shorter than that mediated by R2 during S phase, it has been shown to be essential for mtDNA maintenance, since mutations in the p53R2 gene result in mtDNA depletion.

The salvage pathway is based on the sequential phosphorylation of precursor nucleosides to form dNMP, dNDP, and ultimately dNTP. The first and rate-limiting step of this pathway is irreversibly catalyzed by deoxynucleoside kinases. Thymidine kinase 1 (TK1) and deoxycytidine kinase (dCK) act in the cytosol, whereas thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) are localized within the mitochondrion. Mutations in the mitochondrial kinases involved in the salvage pathway cause severe MDDS, indicating the dependence of mitochondria on the utilization of dNTP metabolic waste products to maintain DNA integrity and repair. Additional nucleotide kinases complete the phosphorylation of all four dNMPs to the corresponding final dNTPs required for DNA synthesis. The cytosolic and mitochondrial matrices are independent compartments, but they are in active contact, bidirectionally exchanging pool components across the inner mitochondrial membrane via carriers that are poorly characterized to date. As a result of this cross-exchange, changes in the dNTP pool size are thought to occur in parallel in both compartments. As a consequence, mitochondria become more vulnerable to defects in their own dNTP salvage pathway in post-miotic cells, where the cytosolic pool is greatly reduced.

The size of the dNTP pool depends on the balance between the anabolic pathways mentioned earlier, the rate of incorporation into DNA, and the catabolic processes responsible for dNTP degradation.

dNTPs are required at the replication fork as substrates to be incorporated into DNA by mitochondrial polymerase gamma (POLG). Although the precise mode of mtDNA replication is currently debated, the core mitochondrial replisome formed by polymerase gamma (consisting of a catalytic subunit encoded by the POLG1 gene and two accessory subunits encoded by the POLG2 gene), Twinkle helicase, and single-stranded DNA binding protein (SSB protein) has been reconstituted in vitro [28]. Additional, completely uncharacterized actions are required for complete in vivo replication of the mtDNA molecule (e.g., primase, topoisomerases) and for initiation and regulation of the process in response to various stimuli and stress. Some examples are the MGME1 and DNA2 genes, encoding proteins involved at some level in the replication process (7S RNA maturation, helicase activity, respectively), mutations in which have recently been associated with MDDS.

According to one embodiment, treatment of the syndrome is achieved by increasing polymerase gamma activity.

According to one embodiment, one or more deoxyribonucleosides are canonical deoxyribonucleosides. Preferably, when using such deoxyribonucleosides, the onset of effect can be obtained earlier, since no additional processing of the metabolite is required. In addition, since canonical deoxyribonucleosides are essentially identical to endogenous deoxyribonucleosides, it is possible to reduce the risk of side effects associated with the treatment of a given disease.

According to one embodiment of the first aspect of the invention, the syndrome is caused by a defect in the mitochondrial DNA replication complex.

According to another embodiment of the first aspect of the invention, the defect is caused by one or more mutations in one or more proteins of the mitochondrial DNA replication complex.

According to another embodiment, the protein is selected from the group consisting of: DNA polymerase gamma-1 subunit (POLG1), DNA polymerase gamma-2 subunit (POLG2), Twinkle protein (PEO1), mitochondrial genome maintenance exonuclease 1 (MGME1), and human DNA helicase/nuclease protein 2. According to another embodiment, the protein is DNA polymerase gamma-1 subunit (POLG1) or Twinkle protein.

Polymerase gamma is a heterotrimer consisting of one catalytic subunit (encoded by the POLG1 gene) and two accessory subunits that act as processivity factors and DNA binding modulators (encoded by the POLG2 gene). Its NCBI accession number is NP_001119603.1 (also shown as Fig. 1 and SEQ ID NO: 1).

POLG-associated disorders represent a spectrum of broad and overlapping phenotypes spanning ages from early childhood to old age. Clinical phenotypes of POLG-associated disorders include autosomal recessive and autosomal dominant forms of acquired progressive external ophthalmoplegia (PEO), myoclonic epilepsy, myopathy, sensory ataxia syndrome (MEMSA), the ataxia-neuropathy spectrum including mitochondrial recessive ataxia syndrome (MIRAS) and sensory ataxia, neuropathy, dysarthria, ophthalmoplegia (SANDO) syndrome, and hepatocerebral MOS (Alpers-Huttenlocher syndrome). More recently, POLG mutations have been identified in individuals with clinical features of MNGIE but without leukoencephalopathy.

The prevalence of Alpers-Huttenlocher syndrome is estimated at approximately 1:50,000. It is the most severe phenotype associated with POLG mutations and is characterized by progressive encephalopathy with intractable epilepsy and psychomotor impairment, neuropathy, and liver failure. The disease usually affects individuals between 2 and 4 years of age and is characterized by epileptic seizures (focal, generalized, myoclonic, partial continuous epilepsies, or status epilepticus), headaches, usually accompanied by visual sensations or visual auras, hypotonia, and psychomotor regression. Areflexia and hypotonia are present in the early stages of the disease, followed by spastic paraparesis, which develops over months to years, eventually leading to psychomotor regression. Affected individuals develop liver dysfunction with elevated transaminases, hypoalbuminemia, coagulopathy, hypoglycemia, and hyperammonemia. Liver lesions may progress rapidly to end-stage liver failure within months. CSF protein is usually elevated. Neuroimaging may show gliosis and generalized brain atrophy. Liver histology may demonstrate macro- and microvesicular steatosis, centrilobular necrosis, fibrosis, cirrhosis, biliary proliferation, and mitochondrial proliferation. Hepatic mtDNA is decreased. Disease progression is variable, and life expectancy from symptom onset ranges from 3 months to 12 years.

According to one embodiment, the mitochondrial DNA depletion and/or deletion syndrome is caused by a defect in the mitochondrial replication pathway, wherein said defect is due to one or more of the following mutations in the POLG1 protein: a mutation at position 309 with an arginine substitution for cysteine [29], a mutation at position 748 with a tryptophan residue substitution for serine (rs113994097), a mutation at position 848 with a glycine substitution for serine (rs113994098), a mutation at position 1143 with a glutamic acid substitution for glycine (rs2307441), and a mutation at position 1177 with a valine substitution for leucine.

Alternatively, according to another embodiment of the first aspect of the invention, the defect is caused by one or more mutations in one or more proteins selected from the group consisting of: ANT1, MPV17, SUCLA2, FBXL4, ABAT, SUCLG1, MFN2 and OPA1. According to another embodiment of the first aspect, the defect is caused by one or more mutations in a protein selected from the group consisting of: OPA1, SUCLA2 and SUCLG1.

According to another embodiment, the one or more deoxyribonucleosides are canonical deoxyribonucleosides. According to another embodiment, the one or more deoxyribonucleosides are selected from the group consisting of: deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine. According to another embodiment, the composition comprises four canonical deoxyribonucleosides. According to another embodiment, the composition comprises four canonical deoxyribonucleosides and does not comprise any additional nucleosides (this means that the composition as a “deoxyribonucleoside component” comprises only these four deoxyribonucleosides, but may also include fillers, carriers, etc.) According to another embodiment, the composition comprises deoxyadenosine, deoxythymidine, deoxycytidine and deoxyguanosine. According to another embodiment, the composition comprises deoxyadenosine, deoxythymidine, deoxycytidine and deoxyguanosine and does not contain any additional nucleosides (this means that the composition as a “nucleoside component” contains only these four nucleosides, but may also include fillers, carriers, etc.).

A person skilled in the art is able to determine the amount of each of the deoxyribonucleosides necessary to restore mtDNA levels (i.e., a therapeutically effective amount to improve the manifestations and symptoms of mitochondrial disorders). According to another embodiment, when the composition contains more than one canonical deoxyribonucleoside, the nucleosides are present in an equimolar ratio.

According to one embodiment, the composition comprises a combination consisting of deoxyadenosine (dAdo), deoxycytidine (dCtd), deoxyguanosine (dGuo) and deoxythymidine (commonly referred to as thymidine, dThd), wherein all nucleosides are present in an equimolar ratio.

According to another embodiment, the composition also comprises one or more pharmaceutically acceptable nucleoside degradation inhibitors.

Inhibitors of nucleoside degradation are well known in the art. Illustrative, non-limiting examples are: immucillin H or forodesine as dGuo degradation inhibitors, tetrahydrouridine as a dCtd degradation inhibitor, 5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride (TPI) as a dThd degradation inhibitor, and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) as a dAdo degradation inhibitor. Fig. 1 shows the mechanism of action of these inhibitors.

One skilled in the art is able to determine the amount of inhibitor(s) required to ensure the bioavailability of the nucleoside(s) to restore mtDNA levels.

According to one embodiment, the composition also comprises one pharmaceutically acceptable nucleoside degradation inhibitor. According to another embodiment, the pharmaceutically acceptable inhibitor is a deoxyadenosine degradation inhibitor. According to another embodiment, the inhibitor is erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA).

The active ingredients described for use in this context may be combined with pharmaceutically acceptable excipients or carriers selected to make such compositions suitable for delivery orally, rectally, parenterally (e.g. intravenously, intramuscularly, intraarterially, intraperitoneally, etc.) or by inhalation, via an osmotic pump, topically, ophthalmologically, etc.

Ointments are semi-solid preparations consisting of an active ingredient incorporated into a fatty, waxy or synthetic base.

Examples of suitable creams include, but are not limited to, water-in-oil and oil-in-water emulsions. Water-in-oil creams can be prepared using a suitable emulsifier with properties similar to, but not limited to, fatty alcohols such as cetyl alcohol or cetostearyl alcohol and an emulsifying wax. Oil-in-water creams can be prepared using an emulsifier such as cetomacrogol emulsifying wax. Suitable properties include the ability to modify the viscosity of the emulsion, as well as physical and chemical stability over a wide range of pH values. The water-soluble or water-miscible cream base may contain a mixture of preservatives and may also contain a buffer to maintain an acceptable physiological pH value.

In addition to the topical route described above, there are various methods for administering the compounds of the present invention systemically. One such means would include an aerosol suspension of respirable particles consisting of the active compound, which is inhaled by the subject. The active compound will be absorbed by the bloodstream through the lungs and contact the general circulation in a pharmaceutically effective amount. The respirable particles may be liquid or solid, with a particle size small enough to pass through the oral cavity and larynx when inhaled.

Another means of systemic administration of the active compounds to a subject will be the administration of a liquid/liquid suspension in the form of nasal drops of a liquid preparation or in the form of a nasal spray of inhalable particles that the patient inhales. Liquid pharmaceutical compositions of the active compound for the preparation of a nasal spray or nasal drops can be prepared by combining the active compound with a suitable carrier, such as sterile pyrogen-free water or sterile saline, using methods known to those skilled in the art.

Other methods of systemic administration of the active compound will include oral administration, wherein the pharmaceutical compositions containing the compounds of Formula I are in the form of a solid, solution, emulsion, dispersion, micelle, liposome and the like, wherein the final preparation contains the active compounds intended for use in this context, in admixture with an organic or inorganic carrier or excipient suitable for nasal, enteral or parenteral applications. The active ingredients can be mixed, for example, with conventional non-toxic, pharmaceutically or physiologically acceptable carriers for tablets, pellets, capsules, lozenges, pastilles, aqueous or oily suspensions, dispersible powders or granules, suppositories, solutions, emulsions, suspensions, hard or soft capsules, caplets or syrups or elixirs and any other forms suitable for use. Carriers that may be used include acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, corn starch, keratin, colloidal silicon dioxide, potato starch, urea, medium chain triglycerides, dextrans and other carriers suitable for use in the preparation of preparations in solid, semi-solid or liquid form. In addition, auxiliary, stabilizing, thickening agents and coloring agents may be used. The active compounds contemplated for use in this context are included in the pharmaceutical preparation in an amount sufficient to achieve the desired effect upon administration (i.e., in a therapeutically effective amount).

The powder, solution, suspension or tablet contain the active compound in a physiologically compatible carrier, which can be selected by those skilled in the art of developing oral delivery systems based on generally accepted criteria. For example, such preparations can contain one or more agents selected from flavor additives (such as peppermint, wintergreen oil or cherry oil), coloring agents, preservatives and the like, in order to provide pharmaceutically elegant and palatable preparations. Tablets containing active ingredients in admixture with non-toxic pharmaceutically acceptable excipients can also be prepared by known methods. The excipients used can be, for example, (1) inert diluents such as calcium carbonate, lactose, calcium phosphate, sodium phosphate and the like; (2) granulating agents and disintegration improvers such as corn starch, potato starch, alginic acid and the like; (3) binding agents such as gum tragacanth, corn starch, gelatin, acacia, and the like; and (4) lubricating agents such as magnesium stearate, stearic acid, talc, and the like. The tablets may be uncoated or they may be coated by known methods to delay disintegration and absorption in the gastrointestinal tract, thereby providing a prolonged action over a longer period. For example, a delayed-release material such as glyceryl monostearate or glyceryl distearate may be used.

If the preparations for oral use are in the form of hard gelatin capsules, the active ingredients therein may be mixed with a solid inert diluent, such as calcium carbonate, calcium phosphate, kaolin, or the like. The preparations may also be in the form of soft gelatin capsules, in which the active ingredients are mixed with an aqueous or oily medium, such as peanut oil, liquid paraffin, olive oil, and the like.

Additional means for systemic administration of the active compound to a subject may include a suppository form of the active compound such that a therapeutically effective amount of the compound reaches the systemic circulation.

Depending on the solubility of the specific active compound containing drug being administered, the daily dose for improvement of the manifestations and symptoms of mitochondrial disorders may be divided into one or more single doses.

In the description and claims, the word "comprises" and its various forms are not intended to exclude other technical features, additives, components or steps. Furthermore, the word "comprises" also includes the variant "consisting of". Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon review of the description or can be learned by practicing the invention. The following examples are given by way of illustration and are not intended to limit the present invention. Furthermore, the present invention covers all possible combinations of the specific and preferred embodiments described in this context.

Description of examples of implementation of the invention

1. Methods

Patients

The study included cells from three patients suffering from POLG deficiency. All subjects gave informed consent in accordance with the internal Institutional Review Board and the Declaration of Helsinki. All three patients were found to have mutations in POLG using Sanger sequencing. (RefSeq NP_001119603.1). Total DNA was isolated from fibroblasts using QiaAMP Mini (Qiagen), and approximately 500 bp fragments including the mutation site of interest were amplified using conventional PCR (polymerase chain reaction) with the following primer pairs listed below and rTaq (Takara).

The following primers were used:

Primers 1 (R309C, 925 c->t)

Forward primer: GTCCACACCACCAAGCAGT (SEQ ID NO: 2)

Reverse primer: GGTCCCAAGCACTATGCTCC (SEQ ID NO: 3)

Primers 2 (W748S c. 2243G->C)

Forward primer: CCTTGCTGAA TGCAGGTGCT (SEQ ID NO: 4)

Reverse primer: TGTGCCTGAAA TCACACTCTGT (SEQ ID NO: 5)

Primers 3 (G848S c. 2542G->A)

Forward primer: ATGGTCTGCTGAGTGGTTGT (SEQ ID NO: 6)

Reverse primer: CCCTCAGAGCCCAGTTTCTAC (SEQ ID NO: 7)

Primers 3 (E1143G c. 3428A->G)

Forward primer: CCCAGTTTATGACCAGCCGT (SEQ ID NO: 8)

Reverse primer: CAAGGAACGCTCACCCAAAG (SEQ ID NO: 9)

Primers 4 (V1177L c.3529G->C)

Forward primer: AGGGGAAGCCCTGCTCTAAG (SEQ ID NO: 10)

Reverse primer: ACAAATGTGTTGTGCTCACCC (SEQ ID NO: 11).

Sequencing reactions were performed with the same primers and the BigDye v3.1 Sequencing Kit (Life Technologies), purified with the BigDye X-Terminator Purification Kit (Life Technologies), and sequenced on an ABI 3130 sequencer (Applied Biosystems). Patient 1 (homozygous for the p.R309C mutation) suffered from a severe neurological phenotype (neuropathy, encephalopathy, MNGIE), which leads to death at the age of 20; patient 2 (compound heterozygous for the p.W748S and p.G848S mutations) suffered from a less severe phenotype, but also predominantly neurological (neuropathy, psychiatric symptoms, MNGIE); Patient 3 (heterozygous for two dominant amino acid substitutions in cis p.V1177L and p.E1143G) showed a familial pattern of dominant inheritance of PEO (progressive external ophthalmoplegia), psychiatric symptoms and proximal muscle weakness. Skeletal muscles showed accumulation of mtDNA deletions but did not show any significant wasting.

Cell culture

Primary cultured fibroblasts were obtained from skin biopsies of patients 1–3 and 4 healthy donors. All subjects gave informed consent in accordance with the internal Institutional Review Board and the Declaration of Helsinki.

Cells were seeded in 6-well plates, 9.5 ^cm2 , in Dulbecco's modified Eagle's medium (DMEM) supplemented with 4.5 g/L glucose, supplemented with 2 mM L-glutamine, 100 U/mL penicillin and streptomycin, and 10% dialyzed fetal bovine serum (FBS) (lnvitrogen), in a humidified chamber at 37°C and 5% _CO2 . After tight confluence was achieved, FBS was reduced to 0.1% to induce quiescence, and co-treatment with 5 ng/mL EtBr (ethidium bromide, Merck) was started (day 0, t0). Cell medium was replaced with the above treatments every 2-3 days for two weeks. On day 14 (t14), EtBr was removed from the cell medium and a set of all cells was treated with 200 μM of all four deoxynucleosides (dNs): dAdo (deoxyadenosine, Sigma), dCtd (deoxycytidine, Sigma), dGuo (deoxyguanosine, Sigma), dThd (deoxythymidine, Sigma) and 5 μM EHNA (Sigma). Some cells were left untreated and all parameters were monitored in parallel. The cell medium was replaced with the same treatment every 2-3 days for an additional 12 days. On day 16 or 17, the media were collected and stored at -20°C until further use. For DNA analysis, cells were harvested at days 0, 7, 14, 21, and 26 by trypsinization, washed with phosphate-buffered saline, pelleted by centrifugation, and stored at -20°C until DNA extraction (Fig. 2). Total DNA was isolated using the QiaAMP DNA mini kit (Qiagen).

Evaluation of the stability of deoxynucleosides in cell culture medium

dNs and some related metabolites were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Acquity UPLCMS/MS instrument (Acquity UPLC-Xevo TM TQ mass spectrometer, Waters, Milford, MA) as described previously [2]. Cellular media were deproteinized by ultrafiltration (Amicon Ultra 3 kDa millipore ultrafilter) at 14,000×g and 4°C for 30 min prior to injection into the LC/MS/MS system.

mtDNA research

The mtDNA copy number was assessed by quantitative PCR as described previously [2].

mtDNA deletions were investigated using long-fragment PCR according to the PCR conditions and primers described in Nishigaki et al. [30]:

Direct primer (F1142-1516):

ACCGCCCGTCACCCTCCTCAAGTATACTTCAAAGG (SEQ ID NO: 12)

Reverse primer (R1180-1146):

ACCGCCAGGTCCTTTGAGTTTTAAGCTGTGGCTCG (SEQ ID NO: 13)

2. Results

EtBr Exposure Induces Greater mtDNA Depletion in POLG-Deficient Quiescent Fibroblasts

An important limitation in testing potential treatments for mtDNA depletion is the fact that in many cases, cells obtained from a patient with MDDS do not show any mtDNA abnormalities in cell culture. For this reason, various models have been developed to demonstrate molecular defects affecting mtDNA replication. In the current state of the art, EtBr exposure is used periodically to initiate mtDNA depletion in cultured cells and to analyze their replicative capacity to restore normal mtDNA levels (Pontarin et al. "Mammalian ribonucleotide reductase subunit p53R2 is required for mitochondrial DNA replication and DNA repair in quiescent cells", 2012, PNAS, v. 109(33), pp. 13302-13307). mtDNA depletion was induced in both healthy control cells and POLG-deficient quiescent cells by exposure to 5 ng/ml EtBr for 14 days. Marked mtDNA depletion was observed in all cells. However, all POLG-deficient cells showed a higher degree of mtDNA depletion compared to that observed in fibroblast lines derived from healthy controls (mean percentage of residual mtDNA levels plus or minus standard error of the mean (SEM): 17.5 plus or minus 2.3%; in controls: 39.68 plus or minus 6.6%). These data suggest that the molecular defect caused by POLG mutations may enhance EtBr interference with the replication process.

POLG-deficient fibroblasts fully restore mtDNA levels after EtBr-induced exhaustion during treatment with a combination of dNs plus EHNA

After removal of EtBr, the effect of adding cell culture medium containing all four dNs on the recovery of mtDNA levels was studied. It has been previously reported that dAdo is particularly sensitive to extracellular and intracellular enzymatic cleavage, mainly by ADA (adenosine deaminase) [2]. Therefore, an excimolar concentration of all four canonical dNs (200 μM each dGuo, dAdo, dThd, and dCtd) plus 5 μM EHNA (Sigma) was added to the medium to provide partial inhibition of dAdo catabolism and improve its stability. On day 16-17, the concentration of added dNs and some of their derived metabolites was measured in 2-3 day old conditioned media according to the protocol described in Camara Y. et al., 2014 [2]. Despite partial degradation, the concentration of all dNs remained above 70% of the initially added concentration in all cases (Table 2). No significant differences in dNs stability were observed between conditioned media from control and patient-derived cells.

Table 2.
Concentration of compounds in media after 2-3 days of treatment Control Patients dCtd 168.9 ± 27.4 196 ± 24.0 dUrd 46.7 ± 27.6 6.2 ± 3.4 dThd 154.8 ± 9.1 153.9 ± 10.2 Timin 48.4 ± 17.3 36.4 ± 10.2 dGuo 171.6±17.9 173.3±16.5 Guanine 71.0 ± 27.5 57.9±12.1 dAdo 140.4±15.7 142.9±17.5 Dlno 50.7 ± 22.1 30.7 ± 10.7 Hypoxanthine 23.7 ± 8.8 22.7 ± 8.3

Results represent the mean plus/minus SD for three different POLG-deficient and four control cell lines. dUrd: deoxyuridine; dlno: deoxyinosine.

mtDNA recovery was monitored before and after EtBr removal in the presence or absence of dNs supplementation (following the same protocol as described in Camara Y. et al., 2014 [2]). The mtDNA copy number from control cells reached values greater than 100% of the initial levels 12 days after EtBr removal regardless of dNs treatment (average percentage of residual mtDNA levels plus/minus SEM: 129.8 plus/minus 35.2% without treatment; 153.9 plus/minus 40.6% with treatment).

In contrast, POLG-deficient cells were unable to restore normal mtDNA levels (26.6 plus/minus 1.5%) without being supplemented with dNs.

Table 3.
mtDNA levels in quiescent fibroblasts at different time points during the experiment A control 1 control 2 control 3 control 4 day without processing during processing without processing during processing without processing during processing Without processing during processing 0 100,0 100,0 100,0 100,0 7 69.6 53.9 45.4 44.6 14 28.4 58.8 35.3 36.0 21 63.7 65.0 244.1 261.3 74.2 73.9 71.3 124.3 26 92.7 92.5 235.4 257.4 92.0 85.9 99.0 179.9

B patient 1 patient 2 patient 3 without processing during processing without processing during processing without processing during processing 0 100,0 100,0 100,0 7 77.3 44.3 48.6 14 22.0 15.4 15.0 21 18.3 116.3 22.4 48.8 18.9 40.5 26 28.4 193.1 27.7 137.2 23.7 120.6

Values are expressed as percentage of mtDNA copy number relative to the value at day 0. After EtBr-induced depletion, cells were treated or not with dNs+EHNA from day 14 of the experiment.

The results presented in Table 3 allow us to conclude that administration of canonical nucleosides allows us to achieve mtDNA levels in patients with MDDS comparable to those in healthy subjects (150.3 plus/minus 21.9%, Table 3). Thus, our data indicate that nucleoside administration can restore mtDNA levels in patients with MDDS caused by a defect other than dNTP metabolism to levels corresponding to the “healthy” state, which indicates the therapeutic potential of the combination in the treatment of such diseases.

List of documents used in the application

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2. Camara Y, Gonzalez-Vioque E, Scarpelli M, Torres-Torronteras J, Caballero A, et al. (2014) Administration of deoxyribonucleosides or inhibition of their catabolism as a pharmacological approach for mitochondrial DNA depletion syndrome, Hum. Mol. Genet., 23: 2459–2467.

3. Camara Y, Gonzalez-Vioque E, Scarpelli M, Torres-Torronteras J, Marti R (2013) Feeding the deoxyribonucleoside salvage pathway to rescue mitochondrial DNA, Drug Discov. Today, 18: 950–957.

4. Suomalainen A, lsohanni P (2010) Mitochondrial DNA depletion syndromes - many genes, common mechanisms Neuromuscul Disord., 20: 429–437.

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7. Naviaux RK, Nyhan WL, Barshop BA, Poulton J, Markusic D, et al. (1999) Mitochondrial DNA polymerase gamma deficiency and mtDNA depletion in a child with Alpers' syndrome, Ann. Neurol., 45: 54-58.

8. Hakonen AH, lsohanni P, Paetau A, Herva R, Suomalainen A, et al. (2007) Recessive Twinkle mutations in early onset encephalopathy with mtDNA depletion, Brain, 130: 3032–3040.

9. Kornblum C, Nicholls TJ, Haack TB, Scholer S, Peeva V, et al. (2013) Loss-of-function mutations in MGME1 impair mtDNA replication and cause multisystemic mitochondrial disease, Nat. Genet.

10. Ronchi D, Di Fonzo A, Lin W, Bordoni A, Liu C, et al. (2013) Mutations in DNA2 Link Progressive Myopathy to Mitochondrial DNA Instability, Am. J. Hum. Genet.

11. Longley MJ, Clark S, Yu Wai Man C, Hudson G, Durham SE, et al. (2006) Mutant POLG2 disrupts DNA polymerase gamma subunits and causes progressive external ophthalmoplegia, Am. J. Hum. Genet., 78: 1026–1034.

12. Mandel H, Szargel R, Labay V, Elpeleg 0, Saada A, et al. (2001) The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA, Nat. Genet., 29: 337–341.

13. Saada A, Shaag A, Mandel H, Nevo Y, Eriksson S, et al. (2001) Mutant mitochondrial thymidine kinase in mitochondrial DNA depletion myopathy, Nat. Genet., 29: 342–344.

14. Bourdon A, Minai L, Serre V, Jais JP, Sarzi E, et al. (2007) Mutation of RRM2B, encoding p53-controlled ribonucleotide reductase (p53R2), causes severe mitochondrial DNA depletion, Nat. Genet., 39: 776–780.

15. Nishina I, Spinazzola A, Hirano M (1999) Thymidine phosphorylase gene mutations in MNGIE, a human mitochondrial disorder, Science, 283: 689–692.

16. Rouzier C, Bannwarth S, Chaussenot A, Chevrollier A, Verschueren A, et al. (2012) The MFN2 gene is responsible for mitochondrial DNA instability and optic atrophy 'plus' phenotype, Brain, 135: 23–34.

17. Renaldo F, Amati-Bonneau P, Slama A, Romana C, Forin V, et al. (2012) MFN2, a new gene responsible for mitochondrial DNA depletion, Brain.

18. Ostergaard E, Schwartz M, Batbayli M, Christensen E, Hjalmarson O, et al. (2005) A novel missense mutation in SUCLG1 associated with mitochondrial DNA depletion, encephalomyopathic form, with methylmalonic aciduria, Eur. J. Pediatr., 169: 201–205.

19. Amati-Bonneau P, Valentino ML, Reynier P, Gallardo ME, Bornstein B, et al. (2008) OPA1 mutations induce mitochondrial DNA instability and optic atrophy 'plus' phenotypes, Brain, 131: 338–351.

20. Blakely EL, Butterworth A, Hadden RD, Bodi I, He L, et al. (2012) MPV17 mutation causes neuropathy and leukoencephalopathy with multiple mtDNA deletions in muscle, Neuromuscul Disord., 22: 587–591.

21. Spinazzola A, Viscomi C, Fernandez-Vizarra E, Carrara F, D'Adamo P, et al. (2006) MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion, Nat. Genet., 38: 570–575.

22. Wedding IM, Koht J, Tran GT, Misceo D, Selmer KK, et al. (2014) Spastic paraplegia type 7 is associated with multiple mitochondrial DNA deletions, PLoS One 9: e86340.

23. Bonnen PE, Yarham JW, Besse A, Wu P, Faqeih EA, et al. (2013) Mutations in FBXL4 cause mitochondrial encephalopathy and a disorder of mitochondrial DNA maintenance, Am. J. Hum. Genet., 93: 471–481.

24. Gai X, Ghezzi D, Johnson MA, Biagosch CA, Shamseldin HE, et al. (2013) Mutations in FBXL4, encoding a mitochondrial protein, cause early-onset mitochondrial encephalomyopathy, Am. J. Hum. Genet., 93: 482–495.

25. Nogueira C, Almeida LS, Nesti C, Pezzini I, Videira A, et al. (2014) Syndromes associated with mitochondrial DNA depletion, Ital. J. Pediatr., 40: 34.

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27. Copeland WC (2014) Defects of mitochondrial DNA replication, J. Child. Neurol, 29: 1216–1224.

28. Korhonen JA, Pham XH, Pellegrini M, Falkenberg M (2004) Reconstitution of a minimal mtDNA replisome in vitro , Embo. J., 23: 2423–2429.

29. Amiot A, Tchikviladze M, Joly F, Slama A, Hatem DC, et al. (2009) Frequency of mitochondrial defects in patients with chronic intestinal pseudo-obstruction, Gastroenterology, 137: 101–109.

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SEQUENCE LIST

<110> FUNDACIO HOSPITAL UNIVERSITARI VALL D'HEBRON - INSTITUT DE

RECERCA CENTRO DE INVESTIGACION BIOMEDICA EN RED

<120> Treatment of mitochondrial diseases

<130> P3332EP00

<150> EP15170825

<151> 2015-06-05

<160>

<170> PatentIn version 3.5

<210> 1

<211> 1190

<212> PRT

<213> Homo sapiens

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Gly Pro Val Pro Ala Pro Gly Arg Trp Val Ser Ser Ser Val Pro Ala

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Ser Asp Pro Ser Asp Gly Gln Arg Arg Arg Gln Gln Gln Gln Gln Gln

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Gln Gln Gln Gln Gln Gln Gln Pro Gln Gln Pro Gln Val Leu Ser Ser

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Glu Gly Gly Gln Leu Arg His Asn Pro Leu Asp Ile Gln Met Leu Ser

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Arg Gly Leu His Glu Gln Ile Phe Gly Gln Gly Gly Glu Met Pro Gly

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Glu Ala Ala Val Arg Arg Ser Val Glu His Leu Gln Lys His Gly Leu

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Trp Gly Gln Pro Ala Val Pro Leu Pro Asp Val Glu Leu Arg Leu Pro

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Pro Leu Tyr Gly Asp Asn Leu Asp Gln His Phe Arg Leu Leu Ala Gln

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Lys Gln Ser Leu Pro Tyr Leu Glu Ala Ala Asn Leu Leu Leu Gln Ala

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Gln Leu Pro Pro Lys Pro Pro Ala Trp Ala Trp Ala Glu Gly Trp Thr

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Arg Tyr Gly Pro Glu Gly Glu Ala Val Pro Val Ala Ile Pro Glu Glu

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Arg Ala Leu Val Phe Asp Val Glu Val Cys Leu Ala Glu Gly Thr Cys

195 200 205

Pro Thr Leu Ala Val Ala Ile Ser Pro Ser Ala Trp Tyr Ser Trp Cys

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Ser Gln Arg Leu Val Glu Glu Arg Tyr Ser Trp Thr Ser Gln Leu Ser

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Pro Ala Asp Leu Ile Pro Leu Glu Val Pro Thr Gly Ala Ser Ser Pro

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Thr Gln Arg Asp Trp Gln Glu Gln Leu Val Val Gly His Asn Val Ser

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Phe Asp Arg Ala His Ile Arg Glu Gln Tyr Leu Ile Gln Gly Ser Arg

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Met Arg Phe Leu Asp Thr Met Ser Met His Met Ala Ile Ser Gly Leu

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Ser Ser Phe Gln Arg Ser Leu Trp Ile Ala Ala Lys Gln Gly Lys His

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Lys Val Gln Pro Pro Thr Lys Gln Gly Gln Lys Ser Gln Arg Lys Ala

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Arg Arg Gly Pro Ala Ile Ser Ser Trp Asp Trp Leu Asp Ile Ser Ser

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Val Asn Ser Leu Ala Glu Val His Arg Leu Tyr Val Gly Gly Pro Pro

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Leu Glu Lys Glu Pro Arg Glu Leu Phe Val Lys Gly Thr Met Lys Asp

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Ile Arg Glu Asn Phe Gln Asp Leu Met Gln Tyr Cys Ala Gln Asp Val

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Trp Ala Thr His Glu Val Phe Gln Gln Gln Leu Pro Leu Phe Leu Glu

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Arg Cys Pro His Pro Val Thr Leu Ala Gly Met Leu Glu Met Gly Val

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Ser Tyr Leu Pro Val Asn Gln Asn Trp Glu Arg Tyr Leu Ala Glu Ala

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Gln Gly Thr Tyr Glu Glu Leu Gln Arg Glu Met Lys Lys Ser Leu Met

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Asp Leu Ala Asn Asp Ala Cys Gln Leu Leu Ser Gly Glu Arg Tyr Lys

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Glu Asp Pro Trp Leu Trp Asp Leu Glu Trp Asp Leu Gln Glu Phe Lys

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Gln Lys Lys Ala Lys Lys Val Lys Lys Glu Pro Ala Thr Ala Ser Lys

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Asp Leu Gly Pro Cys Ser Glu Glu Glu Glu Phe Gln Gln Asp Val Met

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Cys Pro Arg Leu Asp Asp Pro Ala Trp Thr Pro Gly Pro Ser Leu Leu

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Ser Leu Gln Met Arg Val Thr Pro Lys Leu Met Ala Leu Thr Trp Asp

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Gly Phe Pro Leu His Tyr Ser Glu Arg His Gly Trp Gly Tyr Leu Val

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Pro Gly Arg Arg Asp Asn Leu Ala Lys Leu Pro Thr Gly Thr Thr Leu

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<---

Claims (16)

1. Use of a composition comprising deoxynucleosides in an amount of more than one selected from the group consisting of deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine for the treatment of mitochondrial DNA depletion and/or deletion syndrome caused by a defect in the DNA polymerase gamma-1 subunit (POLG1). 2. Use of a composition according to claim 1, wherein treatment of the syndrome is achieved by increasing the activity of polymerase gamma. 3. Use of a composition according to any of the preceding claims, wherein said defect is caused by one or more of the following mutations in POLG1: R309C, W748S, V1177L, G848S and/or E1143G, wherein the positions are indicated relative to SEQ ID NO: 1. 4. Use of a composition according to claim 1, wherein said composition does not contain other additional nucleosides. 5. The use of a composition according to any of the preceding claims, wherein said composition additionally comprises one or more pharmaceutically acceptable nucleoside degradation inhibitors. 6. Use of a composition according to claim 5, wherein said composition additionally contains one pharmaceutically acceptable nucleoside degradation inhibitor. 7. Use of a composition according to claim 6, wherein the pharmaceutically acceptable inhibitor is a deoxyadenosine breakdown inhibitor. 8. Use of a composition according to claim 6, wherein the pharmaceutically acceptable inhibitor is erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). 9. Use of a composition according to claim 1, wherein the defect is in the exonuclease domain of POLG1. 10. Use of a composition according to claim 1, wherein the defect is in the polymerase domain of POLG1. 11. Use of a composition according to claim 1, wherein the defect is in the linker region of POLG1. 12. Use of a composition according to claim 1, wherein said deoxynucleosides in an amount of more than one are present in an equimolar ratio. 13. Use of a composition according to claim 1, wherein said deoxynucleosides in an amount of more than one are deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine. 14. Use of a composition according to claim 1, wherein said deoxynucleosides in an amount of more than one are deoxyadenosine, deoxyguanosine, deoxycytidine and deoxythymidine, present in an equimolar ratio. 15. Use of a composition according to claim 1, wherein said deoxynucleosides in an amount of more than one are deoxycytidine and deoxythymidine. 16. Use of a composition according to claim 1, wherein said deoxynucleosides in an amount of more than one are deoxycytidine and deoxythymidine present in an equimolar ratio.