Nap peptide for treatment of mitochondrial diseases

Abstract

The present invention provides methods of treating mitochondrial diseases using an active fragment of Activity -Dependent Neurotrophic protein, i.e., NAP (davunetide).

Description

[0001] NAP PEPTIDE FOR TREATMENT OF MITOCHONDRIAL DISEASES

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel methods of treating mitochondrial diseases using the neuroprotective peptide site of ADNP - the NAP peptide.

[0004] BACKGROUND OF THE INVENTION

[0005] It has previously been reported that Activity -Dependent Neurotrophic protein (ADNP) is involved in multiple physiological processes.

[0006] Previous studies indicated that ADNP-deficient embryos exhibit dramatic increases in mRNA species associated with lipid metabolism, coupled with a reduction in organogenesis / neurogenesis-related transcripts. First, ADNP was identified and characterized from the pluripotent teratocarcinoma cell-line P19 induced to differentiate into neuro-glial like cells. Further, it was shown that ADNP interacts with specific chromatin regions in the neuro-differentiated state, also linked with direct binding to heterochromatin protein 1 (HP1).

[0007] In deciphering ADNP transcriptional activities, it was shown that ADNP is involved in the regulation of hundreds of genes, including steroid biogenesis genes (Mandel, S., Rechavi, G. & Gozes, I. Dev Biol 303, 814-824 (2007); Bassan, M. et al. Journal of Neurochemistry 72, 1283-1293 (1999); Mandel, S. & Gozes, I. J Biol Chem 282, 34448- 34456 (2007). https: / / doi.org: 10.1074 / jbc.M704756200; Zamostiano, R. et al. J Biol Chem 276, 708-714 (2001). https: / / doi.org: 10.1074 / jbc.M007416200; Oz, S. et al. Molecular Psychiatry 19, 1115-1124 (2014). https: / / doi.org: 10.1038 / mp.2014.97; Gozes, I. et al. Transl Psychiatry 7, el 166 (2017). https: / / doi.org: 10.1038 / tp.2017.128; Sun, X et al., Nature Communications 11, 2984 (2020). https: / / doi.org: 10.1038 / s41467-020-16799-0; Gozes, I. & Shazman, S. Eur J Neurosci 58, 2641-2652 (2023). https: / / doi.org: 10.1111 / ejn.15920; and Grigg, I. et al. Transl Psychiatry 10, 228 (2020). https: / / doi.org: 10.1038 / s41398-020-00904- 4).

[0008] Mutations and aberrations in ADNP expression are associated with stress, autism, intellectual disability (ADNP syndrome, also known as Helsmoortel Van Der Aa), cognitive dysfunction, Alzheimer’s disease (AD), schizophrenia, as well as Parkinson’s disease and muscle disorders, emphasizing the importance of identifying ADNP’s mechanism of action and interactions. NAP (NAPVSIPQ single amino acid letter code, also known as davunetide, AL-108, CP201 and sometimes referred to as NAP peptide) is the smallest neuroprotective peptide site of ADNP. NAP and pipeline products protect nerve cells by associating with microtubule end-binding proteins (EB1 / EB3), through the SxIP motif (NAPVSIPQ), thus enhancing microtubule dynamics and Tau-microtubule interaction, protecting the synapse. NAP further enhances ADNP-EB1 / EB3, interactions, protecting against ADNP deficits (Oz, S. et al. Molecular Psychiatry 19, 1115-1124 (2014). https: / / doi.org: 10.1038 / mp.2014.97; Hacohen-Kleiman, et al., The Journal of Clinical Investigation 2018; 128(11): 4956-4969; Gozes, I. & Shazman, S. Eur J Neurosci 58, 2641-2652 (2023). https: / / doi.org: 10.1111 / ejn. l5920). US2018344809 describes compositions comprising NAP.

[0009] Mitochondrial diseases are a group of rare genetic disorders that occur when mitochondria fail to produce enough energy for the body to function properly. They have clinically heterogeneous manifestations. Mitochondrial diseases may be caused by mutations (acquired or inherited), in mitochondrial DNA or in nuclear genes that code for mitochondrial components. These disorders can be present at birth or develop later in life. Many of these diseases can manifest with central nervous system (CNS) dysfunction.

[0010] Situated within the mitochondrial matrix, mitochondrial DNA (mtDNA) is in direct vicinity of the respiratory complexes, thus increasing its exposure to reactive oxygen species (ROS). These dangerous radicals react with and damage the DNA which may result in the accumulation of somatic mutation (Yan, C. et al., 2019 Cells, 8(4), 379; Sharma, P., & Sampath, H., 2019, Cells, 8(2), 100) These damages are heightened as a result of the mtDNA’ s histone-less structure as well as its lack of effective DNA repair mechanisms (Yan et al). Aging is associated with heightened susceptibility of mtDNA to damage, potentially due to increase of ROS production and decrease of DNA repair ability which declines with age (Sharma & Sampath). Given the established link between aging and the accumulation of DNA damage, it is not surprising that many pathologies attested to BER, a mechanism prevalent in mtDNA repair, manifest as age-associated diseases (Sharma & Sampath). Numerous diseases have been connected to mtDNA mutations, and the maternal inheritance pattern of mtDNA often leads to the transmission of these disorders to offspring (Yan et al). mtDNA mutations can be classified into four primary categories: alterations in proteincoding genes, mutations affecting rRNA and tRNA genes which disrupt protein synthesis, rearrangements of mtDNA, and mutations in the control region that interfere with mtDNA replication and transcription (Sharma & Sampath). The obvious feature of mtDNA diseases is characterized by the appearance of various neurological symptoms. Kearns-Sayre syndrome (KSS) and Leber’s hereditary optic neuropathy (LHON) were among the first syndromes linked to mtDNA mutations. These diseases, as well as Stroke-like episodes (MELAS), Pearson Syndrome, and Mitochondrial Encephalopathy are linked to mtDNA mutation (Yan et al). KSS is an early onset neuromuscular disorder that displays symptoms such as progressive myopathy, ophthalmoplegia, and cardiomyopathy and is caused by a single, large deletion, changes, scientists struggle to uncover the significance of such alterations in carcinogenesis. Nonetheless, several mutations and deletions were reported in different cancer types (Sharma & Sampath).

[0011] MT-ATP6 (or ATP6) is a mitochondrial gene with the full name 'mitochondrially encoded ATP synthase membrane subunit 6' that encodes the ATP synthase subunit 6 (or subunit / chain A). The MT-ATP6 protein forms one part (subunit) of a large enzyme called ATP synthase. This enzyme, which is also known as complex V, is responsible for the final step of oxidative phosphorylation. Specifically, one segment of ATP synthase allows protons to flow across a specialized membrane inside mitochondria. Another segment of the enzyme uses the energy created by this proton flow to convert ADP to ATP. Mutations in MT-ATP6 and other genes affecting oxidative phosphorylation in the mitochondria have been associated with a variety of neurodegenerative and cardiovascular disorders, including mitochondrial complex V deficiency, Leber's hereditary optic neuropathy (LHON), mitochondrial encephalomyopathy with stroke-like episodes (MELAS), Leigh syndrome, and NARP syndrome.

[0012] There is a constant need for novel and effective treatments of mitochondrial diseases.

[0013] SUMMARY OF THE INVENTION

[0014] This invention is based on the unexpected discovery of direct ADNP / NAP (davunetide) regulation of MT-ATP6, an essential mitochondrial gene. Mutations in MT- ATP6 are associated with numerous mitochondrial diseases such as Leigh syndrome, Mitochondrial complex V deficiency, Neuropathy, ataxia and retinitis pigmentosa (NARP), Charcot-Mari e-Tooth disease and Familial bilateral striatal necrosis. Therefore, NAP is a therapy for these and other conditions associated with mutations in MT-ATP6.

[0015] According to one aspect, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a mitochondrial disease or disorder associated with or caused by an alteration in one of more mitochondrial gene in a subject. According to some examples, the mitochondrial gene is selected from MT- ATP6, MT-ND4, MT-ND1, MT-ND6, MT-TK, and MT-TL1. According to some examples, the mitochondrial disease or disorder is selected from Pearson's syndrome and Kearns- Sayre Syndrome. According to some examples, the mitochondrial disease or disorder is associated with a modification in MT-ATP6 protein or in a gene encoding it. According to some examples, the disease or disorder associated with a modification in MT-ATP6 protein or in a gene encoding it is selected from Leigh syndrome, Mitochondrial complex V deficiency, Neuropathy, ataxia and retinitis pigmentosa (NARP), Charcot-Mari e-Tooth disease and Familial bilateral striatal necrosis. According to some examples, the use comprises sexspecific treatment. According to some examples, the subject is a male. According to some examples, the subject is a female. According to some examples, use comprises intranasal administration of the pharmaceutical composition. According to some examples, the disease or disorder is not a neurodegenerative disease or disorder.

[0016] BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figs. 1A-1C: Comparison of bromodeoxyuridine (BrdU, a marker of neurogenesis)- labeled cell concentrations in hippocampal sub -ventricular zone (SVZ) of two ADNP mouse models (Hacohen-Kleiman, G. et al. The Journal of clinical investigation 128, 4956-4969 (2018); Karmon, G. et al. Biol Psychiatry 92, 81-95 (2022)), Adnp haplo-insufficient (Adnp ~) mice on ICR background or mice CRSIPR / Cas9-edited to present the most prevalent neurodevel opmental ADNP syndrome mutation, p.Tyr718* (Tyr) Adnp+ / ' on C57BL6 / NJ background. Adnp+ / ' mice, (Fig. 1A) Tyr mice, (Fig. IB) wild type, ICR and C57BL6 / NJ comparisons (Fig. 1C). Group differences in BrdU positive cell s / mm (mean t. SEM) were compared using a one-way analysis of variance with Tukey’s post hoc test. Technical replicates (6 per animal) were used during statistical analysis. Outliers were excluded using Grubb’s test.

[0018] (Fig. 1A) For Adnp+ / ' males, a statistically significant difference for BrdU positive cells was discovered (F2,77 = 33.030, p < 0.001), with Tukey’s post hoc test revealing a significant reduction in BrdU positive cells in Adnp+ / ' placebo (termed DD) treated males (N=4, biological repeats) compared to WT (N=5) (***P<0.001), which was significantly corrected with NAP treatment (***P<0.001, N=5). No such effect was found in females (N=5 per group). Significant sex differences were discovered in the WT and the NAP treated Adnp+ / ' group (***P<0.001 for both comparisons). (Fig. IB) For Tyr females, a statistically significant difference for BrdU positive cells was discovered (F2,77 = 11.272, ***P<0.001), with Tukey’s post hoc test revealing a significant reduction in BrdU positive cells in NAP treated Tyr females (N=5) compared to WT (N=4) (***P<0.001), with no difference when compared to DD treated Tyr females (N=5). For Tyr males, a statistically significant difference for BrdU positive cells was discovered (F2,89 = 12.777, ***p< 0.001), with Tukey’s post hoc test revealing a significant reduction in BrdU positive cells in DD and NAP treated Tyr males (N=5 for both groups) compared to WT (N=5) (***P<0.001). Significant sex differences were discovered in the WT and the DD treated Tyr groups (P<0.05 for both comparisons).

[0019] (Fig. 1C) Significant differences were discovered between males of different tested strains (P<0.01).

[0020] Fig- 2 shows heterozygous hippocampal Adnp expression in Tyr mice. The figure shows a boxplot of total Adnp gene expression (normalized expression). Note that allelic expression is based solely on reads overlapping the Tyr718 mutation locus, while overall gene expression is based on all reads overlapping the gene, regardless of genotype.

[0021] Fig. 3A-3C: Adnp-specific regulated hippocampal expression in Tyr males. (Fig. 3A) Heatmap of differential expression colored according to log2FoldChange for each gene (row) at each comparison (column). Significant differential expression is (FDR<0.05) and the cell is marked with a star. If differential expression rates were only significant at the alternatively spliced transcript level, the gene name is marked with a circle suffix (arrows and ellipses are described in the text). (Fig. 3B) A graph of relationships between differentially expressed genes (small nodes) and terms they are associated with. (Figure 3C) GSEA plot of genes upregulated by the HSP-90 inhibitor Geldanamycin according to their differential expression in the Tyr male comparison. The position of the enriched genes is marked on a ranked list at the bottom, starting from the most downregulated to the most upregulated.

[0022] Fig. 4A-4C shows Adnp-specific regulated expression in Tyr females. (Fig. 4A) Heatmap of differential expression colored according to log2FoldChange for each gene (row) at each comparison (column). Significant differential expression is (FDR<0.05) the cell is marked with a star. If differential expression was only significant at the transcript level, the gene name is marked with a circle suffix. (Fig. 4B) A graph of relationships between differentially expressed genes (small nodes) and terms they are associated with. (Fig. 4C) Mouse neuroblastoma N1E-115 cell clones CRISPR / Cas9-edited full-length ADNP to include green fluorescent protein (GFP), heterozygous ADNP p.Tyr718 and heterozygous ADNP p.Tyr718 (including GFP as above) + 10'9M NAP (incubated for seven days) were endogenously immune-stained with mitotracker (red) specific antibodies. Statistical analysis of the co-localization rate calculated by Leica sp8 fluorescent microscope of the merged staining. One way ANOVA followed by Tukey post hoc analysis using PRISM Statistics software, version 24 (IBM, Armonk, NY, USA) are shown, *P< 0.05, **P<0.01.

[0023] Fig- 5 shows string bioinformatics analysis of MT-ATP6 interacting proteins associated with mitochondrial diseases.

[0024] DETAILED DESCRIPTION OF THE INVENTION

[0025] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.

[0026] The present invention is based on an unexpected observation that the level of an RNA transcript of MT-ATP6, a key protein important for crucial cellular pathways of oxidative phosphorylation, is dramatically reduced in female mice with mutated ADNP and that this condition was corrected by NAP peptide treatment. Therefore, conditions involving MT- ATP6 dysregulation, such as certain mitochondrial diseases, are treatable by NAP.

[0027] According to one aspect, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a mitochondrial disease or disorder associated with or caused by an alteration in one or more mitochondrial gene.

[0028] The term “treating” as used herein refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, ameliorating, abrogating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially ameliorating or alleviating clinical or esthetical symptoms of a condition, substantially preventing the appearance of clinical or esthetical symptoms of a disease, condition, or disorder, and protecting from harmful or annoying symptoms. Treating further refers to accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder(s) being treated; (c) limiting worsening of symptoms characteristic of the disorder(s) being treated; (d) limiting recurrence of the disorder(s) in patients that have previously had the disorder(s); and / or (e) limiting recurrence of symptoms in patients that were previously asymptomatic for the disorder(s). The term "treating" comprises the term preventing. As used herein, the term “preventing” when used in relation to a condition, refers to the administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.

[0029] The composition of the present invention may be administered by any known method. The term "administering” or “administration of’ a substance, a compound or an agent to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound or an agent can be administered intravenously, intra-arterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, by inhalation, orally (by ingestion), intranasally, intraspinally, intracerebrally, and transdermally (by absorption, e.g., through a skin duct). A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods. According to some embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a day. According to other embodiments, the composition is administered 1, 2, 3, 4, 5 or 6 times a month. In some embodiments, the administration includes both direct administration, including selfadministration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who instructs a patient to self-administer a drug, or to have the drug administered by another and / or who provides a patient with a prescription for a drug is administering the drug to the patient. According to some embodiments, the formulation of NAP protein is as described in US2012208763 incorporated herein by reference. The pharmaceutical composition may be administered as described hereinabove. According to some embodiments, the pharmaceutical composition is formulated for and administered via intranasal, intravenous, subcutaneous, intramuscular, or sublingual administration. According to some embodiments, the pharmaceutical composition is administered intranasally.

[0030] According to the principles of the present invention, NAP can be formulated into various pharmaceutical forms for purposes of administration. The pharmaceutical composition of interest may comprise at least one additive selected from a disintegrating agent, binder, flavoring agent, preservative, colorant, and a mixture thereof, as detailed for example in "Handbook of Pharmaceutical Excipients"; Ed. A. H. Kibbe, 3rd Ed., American Pharmaceutical Association, USA. For example, a compound of the invention, or its salt form or a stereochemically isomeric form, can be combined with a pharmaceutically acceptable carrier.

[0031] A "carrier" as used herein refers to a non-toxic solid, semisolid or liquid filler, diluent, vehicle, excipient, solubilizing agent, encapsulating material or formulation auxiliary of any conventional type, and encompasses all of the components of the composition other than the active pharmaceutical ingredient. The term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" as used herein refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The carrier may contain additional agents such as wetting or emulsifying agents, or pH buffering agents. Other materials such as anti-oxidants, humectants, viscosity stabilizers, and similar agents may be added as necessary. For example, in preparing the compositions in oral form, media such as water, glycols, oils, and alcohols can be used in liquid preparations such as suspensions, syrups, elixirs, and solutions. Alternatively, solid carriers such as starches, sugars, kaolin, lubricants, binders, and disintegrating agents can be used, for example, in powders, pills, capsules, or tablets. For parenteral compositions, the carrier can comprise sterile water or aqueous solutions, e.g. in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer, glucose solution, or a mixture thereof. Additionally, suspensions of the active compound may be prepared as appropriate oily injection suspensions. In addition, solid preparations that are converted to liquid form shortly before use can be made. Other suitable natural or synthetic carriers are well-known in the art (Pillai et al., 2001, Curr. Opin. Chem. Biol. 5, 447).

[0032] The pharmaceutically acceptable excipient(s) useful in the composition of the present invention are selected from but not limited to a group of excipients generally known to persons skilled in the art, e.g., diluents such as lactose (Pharmatose DCL 21), starch, mannitol, sorbitol, dextrose, microcrystalline cellulose, dibasic calcium phosphate, sucrose- based diluents, confectioner's sugar, monobasic calcium sulfate monohydrate, calcium sulfate dihydrate, calcium lactate trihydrate, dextrates, inositol, hydrolyzed cereal solids, amylose, powdered cellulose, calcium carbonate, glycine, and bentonite; disintegrants; binders; fillers; bulking agent; organic acid(s); colorants; stabilizers; preservatives; lubricants; glidants / antiadherants; chelating agents; vehicles; bulking agents; stabilizers; preservatives; hydrophilic polymers; solubility enhancing agents such as glycerin, various grades of polyethylene oxides, transcutol and glycofiirol; tonicity adjusting agents; pH adjusting agents; antioxidants; osmotic agents; chelating agents; viscosifying agents; wetting agents; emulsifying agents; acids; sugar alcohol; reducing sugars; non-reducing sugars and the like, used either alone or in combination thereof.

[0033] The disintegrants useful in the present invention include, but not limited to, starch or its derivatives, partially pregelatinized maize starch (Starch 1500®), croscarmellose sodium, sodium starch glycollate, clays, celluloses, alginates, pregelatinized corn starch, crospovidone, gums and the like used either alone or in combination thereof.

[0034] The lubricants useful in the present invention include but not limited to talc, magnesium stearate, calcium stearate, sodium stearate, stearic acid, hydrogenated vegetable oil, glyceryl behenate, glyceryl behapate, waxes, Stearowet, boric acid, sodium benzoate, sodium acetate, sodium chloride, DL-leucine, polyethylene glycols, sodium oleate, sodium lauryl sulfate, magnesium lauryl sulfate and the like used either alone or in combination thereof.

[0035] The anti -adherents or glidants useful in the present invention are selected from, but not limited to, a group comprising talc, corn starch, DLleucine, sodium lauryl sulfate, and magnesium, calcium, and sodium stearates, and the like or mixtures thereof. In another embodiment of the present invention, the compositions may additionally comprise an antimicrobial preservative such as benzyl alcohol.

[0036] The wetting agents useful in the present invention are selected from, but not limited to, a group comprising oleic acid, glyceryl monostearate, sorbitan monooleate, sorbitan monolaurate, triethanolamine oleate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monolaurate, sodium oleate, sodium lauryl sulfate and the like, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises at least one complexing agent such as cyclodextrin selected from a group comprising but not limited to alpha-cyclodextrin, beta-cyclodextrin, betahydroxycyclodextrin, gammacyclodextrin, and hydroxypropyl beta-cyclodextrin, or the like.

[0037] In an embodiment of the present invention, the composition may additionally comprise a conventionally known antioxidant such as ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, propyl gallate, and / or tocopherol.

[0038] It can be advantageous to formulate the compositions of the invention in dosage unit form for ease of administration and uniformity of dosage. “Dosage unit form” refers to physically discrete units suitable as unitary dosages, each unit containing a pre-determined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the chosen carrier.

[0039] In another embodiment, the dosage form of the present invention additionally comprises at least one wetting agent(s) such as a surfactant selected from a group comprising anionic surfactants, cationic surfactants, non-ionic surfactants, zwitterionic surfactants, or mixtures thereof. In yet another embodiment, the dosage form of the present invention additionally comprises of lipid(s) selected from, but not limited to, glyceryl behenate such as Compritol® ATO888, Compritol® ATO 5, and the like; hydrogenated vegetable oil such as hydrogenated castor oil e.g. Lubritab®; glyceryl palmitostearate such as Precirol® ATO 5 and the like, or mixtures thereof used either alone or in combination thereof. It will be appreciated that any given excipient may serve more than one function in the compositions according to the present invention.

[0040] Pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, grinding, pulverizing, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0041] For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants, for example, polyethylene glycol are generally known in the art.

[0042] For intranasal administration, the variants for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the peptide and a suitable powder base such as lactose or starch. Additional examples of NAP formulations are described in W02017130190, incorporated herein by reference in its entirety. Additional methods for increasing bioavailability can be used. Additional methods to enhance bioavailability and enhance specifically BBB penetration of NAP include using ultrasound as described e.g. in Shin J, et al., Fluids Barriers CNS. 2025 Aug 25;22(1):87. doi: 10.1186 / sl2987-025-00695- 0 or Li W et al., Proc Natl Acad Sci U S A. 2025 Sep 2;122(35):e2421800122. doi: 10.1073 / pnas.2421800122. Epub 2025 Aug 27.PMID: 40864654. For sublingual administration, the compositions may take the form of tablets or lozenges formulated in a conventional manner.

[0043] The terms "NAP" and "NAP protein" are used herein interchangeably and refer to an 8-amino acid peptide consisting of amino acids NAPVSIPQ and known also as davunetide (AL-108, also known as CP201). In its broadest definition, the term "NAP peptide" refers to peptides comprising the amino acid sequence NAPVSIPQ and to any derivative or analog of the peptide comprising the amino acids NAPVSIPQ or related sequences and having the same biological activity, e.g., as peptides defined in W02008084483, W02006099739, US2012208763 and US20150141345 and incorporated herein by reference in their entirety. According to some embodiments, the term “NAP” refers to one of the derivatives of NAP having an amino acid sequence selected from sequences SEQ ID NO: 2-49 as disclosed in WO2023112025 and incorporated herein by reference in its entirety or to a NAP alphaaminoisobutyric acid analog, or SKIP, (Ivashko-Pachima et al., J Mol Neurosci. 2021 Aug;71(8): 1515-1524) or Ac-SKIP (Ivashko-Pachima and Gozes Front Cell Neurosci. 2019 Oct 1;13:435. doi: 10.3389 / fncel.2019.00435). According to some embodiments, the methods of treatment of the present invention include use of other compounds that have an activity that is similar to that of NAP, i.e. alternatives of NAP, regulating ADNP. Examples of such NAP alternatives, regulating ADNP are vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) described e.g. in Sragovich et al (Translational Psychiatry volume 9, Article number: 235 (2019); SNV described in Eger et al., (Front Pharmacol. 2021 May 5;12:638128. doi: 10.3389 / fphar.2021.638128), or ketamine (Brown et al., Neuroscience. 2015 Apr 2;290:31-40) as well as gene therapy (e.g. antisense oligodeoxy nucleotides modulating ADNP expression. According to some embodiments, the formulation of NAP protein is as described in US2012208763 and US2018344809, incorporated herein by reference. In some embodiments, NAP is modified by any known in the art method, such as chemical modification, conjugation or cyclization. In some embodiments, NAP is acylated, acetylated, amidated, lapidated, stearylated, pegylated, biotinylated or modified by any other way.

[0044] According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 50 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 30 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 20 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 15 amino acids. According to some embodiments, the peptide comprises the amino acid sequence NAPVSIPQ and consists of from 8 to 12 amino acids.

[0045] As defined herein above, the pharmaceutical composition of the present invention is for use in treating a mitochondrial disease or disorder associated with or caused by an alteration in one or more mitochondrial gene.

[0046] The term “mitochondrial dysfunction” is used in accordance with its ordinary meaning and refers to aberrant activity of function of mitochondria, including for example aberrant respiratory chain activity, reactive oxygen species levels, calcium homeostasis, programmed cell death mediated by the mitochondria, mitochondrial fusion, mitochondrial fission, mitophagy, lipid concentrations in the mitochondrial membrane, and / or mitochondrial permeability transition. As used herein, the term “mitochondrial disease” refers to a disease, disorder, or condition in which the function of a subject’s mitochondria becomes impaired or dysfunctional. According to the teaching of the present invention, mitochondrial disorders may be caused by acquired or inherited mutations in mitochondrial DNA (mtDNA). The term “mitochondrial gene” as used herein refers to a gene encoded by Mitochondrial DNA and the encoded protein involved in mitochondrial function. Exemplary mitochondrial genes for use in conjunction with the compositions and methods described herein include MT- ND4, MT-ND4L, MT-ATP6, MT-C02, MT-TK, MT-TL1 MT-ND1, MT-ND4, MT-ND5, MT-ND6, MT-TH, MT-TE, MT-TK, MT-TS1, MT-TV and MT-CYB. The term "mitochondrial DNA" or "mtDNA" as used herein refers to the genetic material located in the mitochondria of living organisms. It is a closed, circular, double-stranded molecule. In humans, its mitochondrial DNA encodes 37 genes: two ribosomal RNA, 22 transfer RNA and 13 proteins that participate in oxidative phosphorylation.

[0047] The term “MT-CYB” as used herein refers to the human Mitochondrially Encoded Cytochrome B gene.

[0048] The term “MT-ND4” as used herein refers to the human Mitochondrially Encoded NADH. Ubiquinone Oxidoreductase Core Subunit 4 gene having a GENBANK™ Accession No. AAR92518.1 and encoding a protein having the amino acid sequence having UNIPROT™ Accession No. P03905-1.

[0049] The term “MT-ND4L” as used herein refers to the human Mitochondrially Encoded NADH: Ubiquinone Oxidoreductase Core Subunit 4L genehaving RefSeq Accession No. NC 012920.1 and encoding a protein having the amino acid sequence having UNIPROT™ Accession No. P03901 -1. The term “MT-C02” as used herein refers to the human Mitochondrially Encoded Cytochrome C Oxidase II gene having European Nucleotide Archive Accession No. X55654.1 and encoding a protein having the amino acid sequence having UNIPROT™ Accession No. P00403-1.

[0050] The term “MT-CYB” as used herein refers to the Mitochondrially Encoded Cytochrome B gene, having a European Nucleotide Archive Accession No. M28016.1 and encoding a protein having the amino acid sequence having UNIPROT™ Accession No. P00156-1.

[0051] The term “MT-ATP6” as used herein refers to the human Mitochondrially Encoded ATP Synthase Membrane Subunit 6 gene having RefSeq Accession No. NC_012920.1 and encoding a protein having the amino acid sequence having UNIPROT™ Accession No. P00846-1.

[0052] The term "MT-TK" refers to human Mitochondrially Encoded tRNA lysine.

[0053] The term "MT-ND1" refers to human Mitochondrially Encoded ubiquinone oxidoreductase core subunit 1 (MT-ND1).

[0054] The term "MT-ND5" refers to human mitochondrially encoded NADH-ubiquinone oxidoreductase chain 5.

[0055] The term "MT-TE " refers to human mitochondrially encoded tRNA glutamic acid (MT-TE).

[0056] The term "MT-TH" refers to human mitochondrially encoded tRNA histidine (MT- TH).

[0057] The term "MT-TL1" refers to human mitochondrially encoded tRNA leucine 1.

[0058] The term "MT- TS1" refers to human mitochondrially encoded tRNA serine 1.

[0059] The term "MT-TV" refers to human mitochondrially encoded tRNA valine.

[0060] The term "MT-ND6" refers to human mitochondrially encoded NADH dehydrogenase 6.

[0061] The term "MT-ND1 " refers to human mitochondrially encoded NADH dehydrogenase 1.

[0062] According to some embodiments, the mitochondrial gene is MT-ATP6. Therefore, according to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a mitochondrial disease or disorder associated with or caused by an alteration in the MT-ATP6 gene. According to some embodiments, the present invention provides a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a mitochondrial disease or disorder associated with or caused by an alteration in a protein coded by the MT-ATP6 gene.

[0063] The term "alteration" in a gene refers to any modification that affects the expression level of a protein encoded by the gene. The alteration included modifications in the nucleic acid sequence, including mutations, such as deletion, substitution or insertion mutation. The alteration included a change in the expression level of the gene due to regulatory machinery, negative feedback etc. The term “mutation” as used herein refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. According to some embodiments, alteration comprises a mutation is a gene. According to some embodiments, alteration includes a reduction in gene expression. In some embodiments, reduction in gene expression is caused by or mediated by activity-dependent neuroprotective protein (ADNP) or by a mutation or alteration in ADNP. The term “activity-dependent neuroprotective protein" (ADNP) refers to a human protein factor (Bassan et al.; Zamostiano et al., J Biol Chem. 2001;276(l):708-14). The protein has neurotrophic / neuroprotective activity as measured e.g., with in vitro cortical neuron culture assays described by, e.g., Gozes et al., (Proc. Natl. Acad. Sci. USA 93, 427- 432, 1996; Bassan et al.) and reviewed in Gozes. Book Chapter 13, in Neuroprotection in Alzheimer’s Disease, 1st Edition - December 30, 2016, Gozes, Ed., Academic Press). ADNP regulates hundreds of genes. A non-limiting example is Hacel as shown in Example 5. Hacel (ELECT Domain And Ankyrin Repeat Containing E3 Ubiquitin Protein Ligase 1) associated with ataxia (Bellamy et al., PLoS One. 2022;17(l):e0261845), Huntington disease (Ehrnhoefer et al., Hum Mol Genet. 2018;27(2):239-253. With mutations resulting in severe neurodevelopmental disorder (OMIM: 616756), and with further involvement in depression (Ciuculete et al., Clin Epigenetics. 2020;12(l):99) and Alzheimer's disease (Ni et al., J Alzheimers Dis. 2018;64(4): 1149-1161). With respect to a protein, alteration may have a meaning of reduced activity, damaged structure, or damaged function of the protein.

[0064] According to some embodiments, mitochondrial disease or disorder is selected from Pearson's syndrome and Kearns-Sayre Syndrome. According to some embodiments, the mitochondrial disease or disorder is Spastic paraplegia.

[0065] According to some embodiments, the mitochondrial disease or disorder is associated with a modification in the MT-ATP6 protein or in a gene encoding it or in its expression. According to some embodiments, the disease or disorder is selected from Leigh syndrome, Mitochondrial complex V deficiency, Neuropathy, ataxia and retinitis pigmentosa (NARP), Mitochondrial spastic paraplegia, Charcot-Mari e-Tooth disease, MT-ATP6-associated peripheral neuropathy, Adult-onset spinocerebellar ataxia (MT-ATP6 related) and Familial bilateral striatal necrosis.

[0066] According to some embodiments, the disease is caused by an alteration of a gene or protein occurring naturally in complex or interaction with the MT-ATP6 protein.

[0067] According to some embodiments, the use comprises a sex-specific treatment. According to some embodiments, the subject is a male subject. According to some embodiments, the subject is a female subject.

[0068] According to some embodiments, the pharmaceutical is administered intranasally.

[0069] According to any one of the above embodiments, the disease or disorder is not a neurodegenerative disease or disorder.

[0070] According to another aspect, the present invention provides a method for restoring activity or function of the MT-ATP6 protein by the method comprising administering a pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier to a subject.

[0071] According to another aspect, the present invention provides a use of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1 ; NAP peptide) in preparing a medicament for treating a mitochondrial disease or disorder associated with or caused by an alteration in one or more mitochondrial gene in a subject in need thereof.

[0072] According to another aspect, the present invention provides a method for treating a mitochondrial disease or disorder associated with or caused by an alteration in one or more mitochondrial gene in a subject in need thereof, the method comprising administering a therapeutically effective amount of a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) to the subject.

[0073] The invention will now be illustrated by the following non-limiting Examples.

[0074] Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.

[0075] The terms “a,” “an,” and “the” ” are used herein interchangeably and mean one or more. The term “and / or” is used to indicate one or both stated cases may occur, for example A and / or B includes, (A and B) and (A or B).

[0076] The term “or” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive.

[0077] The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term "substantially consisting of or "essentially consisting of is a partially open term, which does not exclude the presence of one or more element (s), ingredient (s) or additional step (s) as far as this (these) element (s), ingredient (s) or additional step (s) do not materially affect the basic properties of the invention.

[0078] As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of + / -10%, or + / -5%, + / -1%, or even + / -0.1% from the specified value.

[0079] EXAMPLES

[0080] Example 1.

[0081] Methods

[0082] Adult mouse neurosenesis: BrdU incorporation

[0083] Two and half month-old Adnp+ / ~ mice on an ICR background and Tyr mice (heterozygous for Adnp p.Tyr718*, also referred as "our Tyr model") on a C57BL6 / NJ background, were handled as described in Hacohen -Kleiman, G. et al. The Journal of clinical investigation 128, 4956-4969 (2018); Karmon, G. et al. Biol Psychiatry 92, 81-95 (2022)). Mice were habituated during the same time under the exact same conditions. At the age of 4 weeks, mice were treated intranasally with 0.5 pg NAP or vehicle (DD, in which each milliliter included 7.5 mg of NaCl, 1.7 mg of citric acid monohydrate, 3 mg of disodium phosphate dihydrate and 0.2 mg of a 50% benzalkonium chloride solution) for 5-6 weeks (Hacohen-Kleiman; Karmon et al). The mice were then injected (i.p.) with BrdU (80mg / kg), 4 injections at 2 h intervals, as previously described in Velazco-Mendoza et al., Neuroscience 396, 166-174 (2019). Mice were then euthanized -one hour from the last injection. Tissues were processed as previously described in Hacohen-Kleiman and in Karmon et al. At least three mice were used per experimental group.

[0084] BrdU immunohistochemistry

[0085] Immunofluorescence for BrdU detection was performed. In short, fixed tissue sections were incubated overnight with a primary antibody against rat BrdU (ab6326, Abeam, Cambridge, UK, 1 : 800), followed by a 2-hour incubation with a secondary antibody (goat anti-rat Alexa Fluor 555, a21434 Invitrogen, Waltham, MA, USA, 1 :500). All cell counting (cells / mm2) was executed by two different investigators, blind to the experiment, on six areas per section (near the lateral ventricles) spaced at least 50 pm apart. Only DAPI+ and BrdU+ nuclei were considered. Images were captured using an Axioplan- 2 optical / widefield fluorescent (Zeiss, Oberkochen, Germany) while confocal images were captured with Nikon Eclipse Ti and were assembled in Imaged (Fiji) software. Statistical analysis was performed using Sigmaplot (Grafiti LLC, CA, USA) as detailed in the figure legend (Figs. 1A-1C).

[0086] Hippocampal RNA-seq

[0087] RNA was extracted from 2.5-month-old Tyr mouse hippocampi, treated, handled, sequenced on NextSeq500 (Illumina, CA, USA), and analyzed as previously described in Karmon, G. et al. Originally, six groups of mice were used separated by sex, males and females (wild type, Tyr and Tyr treated with NAP), each including at least three biological replicates.

[0088] Mitochondrial immunocytochemistry

[0089] Neuronal-like differentiated mouse neuroblastoma N1E-115 cell clones CRISPR / Cas9-edited to express green fluorescent protein (GFP)-labeled full-length ADNP and GFP-labeled ADNP p.Tyr718 (Ganaiem M et al., Cells. 2022 Sep 26; 11(19):2994 ; Ganaiem M et al., Cells. 2023 Sep 11;12(18):2251) were immunostained with antibodies recognizing mitotracker (Thermo Fisher Scientific, Waltham, MA, USA) in a working concentration of 250nM as described in the manual protocol (https: / / tools.thermofisher.com / content / sfs / manuals / mp07510.pdf). Co-localization of two fluorophores was quantified as before. Bioinformatic analysis

[0090] Raw sequencing data were trimmed and filtered using fastp, followed by transcript quantification with Salmon according to the GRCm38 reference genome, length and positional corrections enabled. Batch effect was corrected using surrogate variables derived from the sva package, used as model covariates to DESeq2, or applied to the count data using limma. Gene-level analysis was performed using DESeq2, including normalization of expression levels, differential expression tests and test statistic correction. Differential transcript expression levels were further tested with the Swish package, controlling for uncertainty by generating 20 inferential replicates via Gibbs sampling. Heatmaps were created using the complexHeatmaps package, with colors restricted to a reduced log2- foldchange range, due to dynamic range considerations. To make the log2FoldChange values more representative of the magnitude of differential gene expression, we used a transformed value, produced by the empirical Bayesian adaptive shrinkage approach.

[0091] Enrichment testing was performed using the set-overlap and pre-ranked functions of the GSEApy package, with gene-sets from Enrichr and rummagene. All gene-sets mentioned in this work had significant enrichment results in one or more comparisons. Genotyping was performed according to the GATK best practices workflow for RNAseq short variant discovery. In short, processed reads were aligned to the GRCm38 reference genome using STAR (two pass alignment), followed by post-processing by picard and variant calling with GATK.

[0092] Results

[0093] Higher male / female neurogenesis regulated by the major neurodevelopmental / intellectual disability / autism-linked ADNP

[0094] Twice-higher BrdU labeling in the hippocampal sub -ventricular zone (SVZ) of Adnp+ / +male mice, compared \oAdnp+ / +female mice (wild type, WT, ICR background) was identified as depicted in Fig. 1A (BrdU immunohistochemistry), densitometry results (4-5 different animals, each with left and right hippocampi / condition). Similarly, twice higher BrdU labeling was also apparent in WT males (ADNP -intact) compared to WT females of C57BL6 / NJ mice (Fig. IB). Interestingly, the mouse strain affects BrdU labeling as well with ICR males showing 1.53-fold higher incorporation compared to C57BL6 / NJ (**p<0 01, Fig. 1C). Adnp haploinsufficiency (Adnp+ / ', ICR background) or CRSIPR / Cas9 editing to carry the most prevalent neurodevel opmental ADNP syndrome mutation, mouse equivalent p.Tyr718* (heterozygous Tyr mice, C57BL6 / NJ background) showed dramatic reductions in BrdU incorporation in males, resulting in mutated females presenting higher BrdU labeling than males (*P<0.05, Tyr mice, Fig. IB). NAP treatment resulted in significantly increased Adnp+ / ~ male mice BrdU incorporation, while in the Tyr mice only a trend was observed, coupled with a significant decrease in NAP -treated Tyr females compared to WT C57BL6 / NJ (Fig. 1A-1C).

[0095] Distinct hippocampal gene and transcript differential expression induced by the ADNP Tyr mutation and NAP treatment

[0096] Analysis of RNA sequencing data from hippocampal samples revealed that about 50% of the Adnp transcripts from Tyr mice were mutated regardless of sex and treatment. Total Adnp gene expression was slightly downregulated in the mutated groups, but the difference was statistically significant only in NAP -treated Tyr females (Fig. 2), paralleling BrdU incorporation levels (Fig. IB).

[0097] Overall, Adnp-mutation-related differential expression was almost entirely distinct between males and females, with few genes commonly differentially expressed in both sexes and / or treatments (data not shown). In addition to differentially expressed genes (DEGs), we found many differentially expressed transcripts (DETs), suggesting an effect on RNA splicing, most prominent in the Tyr groups (data not shown). The male differential expression of mRNA splicing regulators, such as the X-chromosome-linked cold-response protein RNA Binding Motif 3 (Rbm3) and the cold-inducible RNA binding protein (Cirbp, Fig. 3 A, marked by arrows)), might underlie some of the extensive differential splicing.

[0098] Unfolded protein response (UPR) genes were downregulated in male Tyr hippocampus, but maintained expression when treated with NAP

[0099] In males, the Tyr mutation induced 34 DEGs and 23 DETs, while NAP treatment largely maintained expression, with just 10 DEGs and 6 DETs.

[0100] A large set of genes associated ER-driven cellular stress response, commonly referred to as unfolded protein response (UPR) genes, were downregulated in Tyr males exclusively, an effect that was either weakened or made insignificant upon NAP treatment (Fig. 3 A, 3B). Gene-set enrichment analysis indicates these genes are a part of a broader downregulated HSP-90-inhibition transcriptional signature of 26 genes. Genes induced by the HSP90 inhibitor Geldanamycin were strongly downregulated in male TYR mice (NES=-2.59; FDR<le-5) (Fig. 3C), including HSP90 co-chaperones Cdc3711 and Stipl. Hippocampal expression of UPR genes is known to be essential for long-term memory formation and impaired learning when disrupted. We further discovered that Hspa5 is downregulated in Tyr males (at the transcript level, Fig. 3 A, dashed arrow). It was previously shown that over-expressed Hspa5 reverses the dysregulation of Nr4a, which in turn induces downregulation of UPR genes. While we found no significant expression difference in genes of the Nr4a family, there was a significant enrichment of downregulated glucocorticoid- induced genes (FDR<l x lO'4, Fig. 3B), which are known to regulate NR4A. We also found that downregulation of neuroprotective genes such as Manf and Sipal 13 (Fig. 3A, ellipses), known to facilitate neurological benefit in the hippocampi of old mice, was reversed by NAP treatment. The genes Sunl and Ifnarl, associated with neuronal injury and potential disruption of synaptic plasticity, were upregulated in Tyr male mice and kept unchanged by NAP treatment.

[0101] NAP induced downregulation of various ER stress genes in males, including the pro- neuroinflammatory Nkd2, Hsphl (Hspl 10 / 105) and Faml07a (DRR1), which were shown to exacerbate cerebral ischemia, and Mertk, the mediator of alpha-synuclein fibril uptake. There were few male Tyr DEGs that were maintained regardless of the treatment, including upregulation of Pla2g4e, which is associated with cognitive resilience in late-onset AD models and downregulation of the glycosylation-related Man2b2.

[0102] Differential expression in Tyr-mutated female and the divergent NAP transcriptional signature

[0103] In females, the Tyr mutation induced 43 DETs and 4 additional DEGs, while NAP treatment had 27 DEGs and 9 DETs (Fig. 4A). The genes affected by DETs exclusive to Tyr females were enriched for various neurodegenerative diseases. Examples of such genes are Wdr26, associated with intellectual disability, Col6a2, associated with behavioral and cortical dysfunction, Hecw2 associated with a wide range of neurodevelopmental disorders and Reelin, a crucial corticogenesis gene.

[0104] Enrichment analysis comparing our new discovered results to known pathways in the literature found that differentially expressed genes and gene transcripts of female Tyr mice were previously disproportionately associated with schizophrenia, dysregulation of glial cell transcriptome in autism and GABAergic genes conferring neuropsychiatric disorder risk Differentially expressed ATP metabolism genes such as the mitochondrially encoded ATP synthase membrane subunit 6 (mt-ATP6), Atp2b2 and Abca5 are associated with neuropathologies / neurodegenerations like Alzheimer’s disease (AD) and autism. The surprising finding of the present invention is the dysregulation of the mitochondrial transcript MT-ATP6 as a result of the ADNP pathogenic mutation. Thus, the level of an RNA transcript of MT-ATP6, which is important for crucial cellular pathways of oxidative phosphorylation, is dramatically reduced in the Tyr female and further surprisingly corrected by NAP treatment. Therefore, conditions encompassing MT-ATP6 dysregulation are treatable by NAP.

[0105] Furthermore, NAP treatment either prevented or moderated nearly all Tyr-induced differential expression (DE) in females to statistical insignificance compared to controls, while inducing a distinct transcriptional signature. NAP treatment upregulated GABA vesicle transporters Slc32al, Slc6al3, and the anti-neuroinflammatory Prg4, neuroinflammation regulator downregulating the pro-apoptotic E2fl and the microglial inflammasome activator Inpp5d (Fig. 4A, 4B).

[0106] While genes from the previously mentioned UPR pathway were not differentially expressed in females, Uggt2, which functions as a misfolded protein sensor for UPR activation, is downregulated in female Tyr mice. The Tyr female genes were especially enriched in targets of the SUZ12 transcription factor (Fig. 4B) (FDR<0.05), which is a vital regulator of neurogenesis, both in the embryonic and adult stages.

[0107] Mechanistically, we further questioned whether the ADNP enters mitochondria to potentially regulate mitochondrial gene expression as indicated above for mt-ATP6. To answer this question, we utilized our genome edited GFP-ADNP and GFP-ADNP p.Tyr718 cells stained with mitotracker. We discovered extensive ADNP -mitochondrial localization at the protein level in association with the mitotracker staining, which was significantly reduced in the Tyr-mutated cells and corrected by NAP treatment (Fig. 4C), in agreement with a significant change in the alternative splicing of mt-ATP6 and correction by NAP treatment (Fig. 4A). Interestingly, modeling human ADNP and ADNP p.Tyr719 structure (homologous the mouse sequence above), identified an a helical basic amino acid enriched mitochondrial targeting sequence in ADNP which is shortened in the ADNP p.Tyr719 mutated molecule (data not shown), explaining the biological results (Fig. 4C).

[0108] We further performed a bioinformatics analysis of MT-ATP6 interacting proteins associated with mitochondrial disease. The results are presented in Fig. 5. As can be seen, MT-ATP6 is closely related and cross-reacts with MT-ND4, MT-ND6, MT-ND1 and TK2 genes / proteins. Misfunction in each one of these proteins leads to similar manifestations of the mitochondrial disease. Discussion

[0109] Our results suggest intact ADNP content as a key regulator of increased male neurogenesis through the unfolded protein response (UPR). In other words, male neurogenesis deficits induced by heterozygous Adnp Tyr mutation were mediated by mostly separate, sex-specific pathways, and partial alleviation by NAP was underlined by prevention of ADNP Tyr mutation-induced differential expression, as well as possible compensation by induced neuroprotective factors. We also discovered female-specific mitochondrial RNA alternative splicing regulation by ADNP, exemplified by mt-ATP6.

[0110] Our results indicated that the Tyr mutation in ADNP caused a significant downregulation of numerous hippocampal UPR genes exclusively in males, which was either moderated or prevented by NAP treatment. This downregulation of male UPR signaling might contribute to the substantial decrease in hippocampal neurogenesis, since it is a pivotal regulator of neurodevelopment, in a sex-dependent manner. The discovery of ADNP female-specifically regulating mt-ATP6 splicing / expression reveals ADNP mitochondrial activity that is associated with reduced Adnp pTyr718* mitochondrial bioavailability, with mt-ATP6 playing a crucial role in oxidative stress regulation. Importantly, global variability in gene expression and alternative splicing is modulated by mitochondrial content. With cytoskeletal health, playing a major role in mitochondrial function being regulated by NAP. With NAP correction of mt-ATP6 splicing / expression our results pave the path to NAP (davunetide) - sex-specific development accompanied by further in-depth evaluation of the mitochondrial function of ADNP / NAP.

[0111] Example 2

[0112] In order to test the efficacy of NAP peptide in the treatment of mitochondrial disease or disorder associated with or caused by an alteration in MT-ATP6 protein, several experiments are conducted.

[0113] Patient-Derived Fibroblasts

[0114] Skin fibroblasts from patients with the common m.8993T>G / C mutation (high heteroplasmy for MILS or moderate for NARP) are cultured.

[0115] The cells are grown as in known protocols. After acclimatization, the cells are supplemented with NAP or control (e.g. sham peptides) and the mitochondrial activity is measured. The cells provide a convenient vitro system to test if NAP (davunetide) improves cell viability, ATP levels, or reduces oxidative stress under energy-deficient conditions. iPSC-Derived Neurons

[0116] Induced pluripotent stem cells (iPSCs) made from patient fibroblasts (e.g., with m.8993T>G) are differentiated into neural cells as known in the art. These patient-specific neurons recapitulate the neuronal deficits of NARP / MILS - such as impaired bioenergetics and vulnerability to degeneration. iPSC-derived neural progenitors have been highlighted as effective models for drug discovery in mtDNA disorders

[0117] The cells are grown as in known protocols. After acclimatization, the cells are supplemented with NAP or control (e.g. sham peptides) and the mitochondrial activity is measured. Testing Davunetide on MT-ATP6 mutant neurons or brain organoids reveals neuroprotective effects (e.g. improved neurite outgrowth or survival under metabolic stress).

[0118] Animal Models

[0119] Mice with a pathogenic mtDNA mutation (m.8414A>G in mt-Atp6) is used to generate a mouse strain carrying the mutation.

[0120] Leigh syndrome mouse model is the Ndufs4 knockout (which causes complex I deficiency and Leigh-like neurodegeneration Int J Mol Sci. 2023 Jan 9;24(2): 1300. doi: 10.3390 / ijms24021300). Although these mice do not have an MT-ATP6 mutation, they manifest a similar phenotype (brainstem lesions, early death) and are used to test candidate therapies for Leigh syndrome. Davunetide (NAP) is evaluated in such mice for its ability to delay neurodegeneration or improve motor function.

[0121] ATP6 gene in its mitochondrial genome, and a mutant allele ATP 6^1 has been identified that causes a mitochondrial encephalomyopathy in the fly. This fly model exhibits adult-onset neuromuscular dysfunction and shortened lifespan, analogous to a milder NARP-like phenotype. NAP (davunetide) is tested for its effect on locomotor activity or lifespan of ATP6 mutant flies. An improvement suggests neuroprotective efficacy in vivo.

[0122] Yeast (S. cerevisiae) has been used to study the impact of human-equivalent ATP6 mutations on oxidative. Though yeasts lack a nervous system, they allow rapid testing of compounds on mitochondrial function. Additionally, cellular assays like neuronal co-culture or brain slice cultures from affected mice are used to examine NAP (davunetide) neuroprotective mechanisms in a controlled environment.

[0123] Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

CLAIMS1. A pharmaceutical composition comprising a peptide comprising the amino acid sequence NAPVSIPQ (SEQ ID NO: 1; NAP peptide) and a pharmaceutically acceptable carrier, for use in treating a mitochondrial disease or disorder associated with or caused by an alteration in one or more mitochondrial genes in a subject.

2. The pharmaceutical composition for use according to claim 1, wherein the mitochondrial gene is selected from MT-ATP6, MT-ND4, MT-ND1, MT-ND6, MT-TK, and MT-TL1.

3. The pharmaceutical composition for use according to claim 1, wherein the mitochondrial disease or disorder is selected from Pearson's syndrome, Kearns- Sayre Syndrome and Spastic paraplegia.

4. The pharmaceutical composition for use according to claim 2, wherein the mitochondrial disease or disorder is associated with a modification in MT-ATP6 protein or in a gene encoding it.

5. The pharmaceutical composition for use according to claim 4, wherein the disease or disorder is selected from Leigh syndrome, Mitochondrial complex V deficiency, Neuropathy, ataxia and retinitis pigmentosa (NARP), Charcot-Mari e-Tooth disease and Familial bilateral striatal necrosis.

6. The pharmaceutical composition for use according to claim 4 or 5, wherein the use comprises sex-specific treatment.

7. The pharmaceutical composition for use according to claim 6, wherein the subject is a male.

8. The pharmaceutical composition for use according to claim 6, wherein the subject is a female.

9. The pharmaceutical composition for use according to any one of claims 4 to 8, wherein the use comprises intranasal, subcutaneous or oral administration of the appropriate pharmaceutical composition.

10. The pharmaceutical composition for use according to any one of claims 1 to 9, wherein the disease or disorder is not a neurodegenerative disease or disorder.

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