DNA methylation as a biomarker for cerebral creatine deficiency syndromes

Abstract

The invention relates to methods for diagnosing, monitoring the efficacy of treatment, monitoring the evolution of a disease, or screening compounds for treating a cerebral creatine deficiency syndrome, the methods comprising the steps measuring a nucleic acid methylation level in a biological sample and comparing the measured level to a control nucleic acid methylation level. The invention relates also to folic acid and derivatives thereof for treating a cerebral creatine deficiency syndrome.

Description

[TITLE]DNA METHYLATION AS A BIOMARKER FOR CEREBRAL CREATINE DEFICIENCY SYNDROMES[TECHNICAL FIELD]

[0001] The present disclosure relates to method for diagnosing, monitoring the efficacy of treatment, monitoring the evolution of a disease, or screening compounds for treating a cerebral creatine deficiency syndrome, as well as to compounds and combination of compounds for treating a cerebral creatine deficiency syndrome.[TECHNICAL BACKGROUND]

[0002] Cerebral creatine deficiency syndrome (CCDS) is a group of rare genetic disorders that affect the way the body produces or transports creatine. CCDS can cause a variety of neurological problems, including intellectual disability, delayed speech and language development, seizures, and movement disorders. CCDS is caused by mutations in genes coding for proteins involved in cerebral transport of creatine (SLC6A8) or in producing creatine from amino acids (guanidinoacetate methyltransferase (GAMT) or arginine:glycine amidinotransferase (AGAT)).

[0003] Creatine T ransporter Deficiency (CTD) is an inborn error of creatine metabolism in which creatine (Cr) is not properly transported to the brain and muscles due to defective creatine transporters (CrT). Patients with CTD express speech and behavior abnormalities, intellectual disabilities, development delay, seizures, and autistic behavior due to lack of brain creatine as energy buffer (Braissant, Henry et al. 201 1 ). There is no pharmacological treatment to alleviate behavioral symptoms of CTD (Fernandes-Pires and Braissant 2022). Consequently, there is an urgent need to develop new therapeutic strategies and new relevant tools such as translational models and markers to evaluate therapeutic efficacy and speed up drug development.

[0004] Creatine plays a central role in brain energetics (Fernandes-Pires and Braissant 2022). Cerebral creatine originates from diet after transport across the blood brain barrier and cerebral cells membranes or from de novo synthesis. In CTD, the peripheral creatine is not distributed to the brain cells due to the non-functional CrT. Consequently, the creatine store of CTD patient brain cells only rely on de novo cerebral synthesis. CTD brain cells may try to increase their Cr synthesis to compensate the energy unbalance even if cerebral cells rarely express both enzymes glycine amidinotransferase (AGAT) and guanidinoacetate N-methyltransferase (GAMT) needed for Cr de novo synthesis (Braissant and Henry 2008, Braissant, Beard et al. 2010). Indeed, this upregulation of Cr biosynthesis was previously demonstrated in the muscle of CrT knockout (KO) mice (Russell, Ghobrial et al. 2014, Stockebrand, Sasani et al. 2018).

[0005] Creatine de novo synthesis needs S-adenosylmethionine (SAM) as a methyl donor, this group being transferred to guanidinoacetate by GAMT in the second step (Curt, Voicu et al. 2015). This last step consumes approximately 40% of methyl groups supplied by SAM and consequently places a significant burden on the labile methyl groups store (Stead, Au et al. 2001 , Brosnan, da Silva et al. 201 1 ).

[0006] Cerebral creatine deficiency syndromes such as cerebral creatine transporter deficiency can be difficult to diagnose. A proper diagnosis typically uses a combination of clinical evaluation, laboratory tests, and imaging studies.

[0007] Therefore, there is a need for reliable methods able to easily confirm the diagnosis of a cerebral creatine deficiency syndrome or a cerebral creatine transporter deficiency.

[0008] There is a need for methods which can be easily implemented, and which are reliable for monitoring the efficacy of a treatment in an individual having a cerebral creatine deficiency syndrome.

[0009] There is a need for methods which can be easily implemented, and which are reliable for monitoring the evolution of a cerebral creatine deficiency syndrome.

[0010] There is a need for methods which can be easily implemented, and which are reliable for screening new compounds for treating a cerebral creatine deficiency syndrome.

[0011] There is also a need for new compounds for treating a cerebral creatine deficiency syndrome.

[0012] The purpose of the disclosure is to meet all or part those needs.[SUMMARY]

[0013] According to one of its objects, the present disclosure relates to a method for monitoring an efficacy of a therapeutic agent proposed for treating a cerebral creatine deficiency syndrome in an individual in need thereof, said method comprising the steps of:

[0014] a) measuring a nucleic acid methylation level in an isolated biological sample obtained from said individual after administration to said individual of said therapeutic agent, and

[0015] b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylationlevel in an isolated biological sample obtained from said individual before administration to said individual of said therapeutic agent, and

[0016] wherein a measured methylation level of at least 1.10 times above the control level is indicative of the efficacy of said therapeutic agent for treating said cerebral creatine deficiency syndrome.

[0017] As shown in the Example section, the inventors have observed altered DNA and RNA methylation in CrT knockout mouse model and brain organoids derived from patients diagnosed with a cerebral creatine deficiency syndrome. Further, it was observed that CrT knockout mice exposed to dodecyl creatine ester (DCE) were having their DNA methylation restored to a healthy level. These observations validated that the cerebral deficiency of creatine diverts the cerebral transmethylation cycle to the production of creatine resulting in a reduction of methylation level of nucleic acid. These observations validated the use of nucleic acid methylation as a biomarker of treatment efficacy and for confirming the diagnosis of a cerebral creatine deficiency syndrome. Therefore, these observations also validated the use of folic acid, or derivatives thereof, able to activate the transmethylation cycle involved in the synthesis of creatine allowing production of creatine, and indirectly restoration of the level of methylation of nucleic acid and treating a cerebral creatine deficiency syndrome.

[0018] In some embodiments, the measured level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about 1.25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about 1 .50 times, or at least about 1 .60 times, or at least about 1 .70 times, or at least about 1 .80 times, or at least about 1 .90 times, or at least about 1 .95 times, or at least about 2.00 times, or at least about 3.00 times, or at least about 3.50 times, or at least about 4.00 times above the control level.

[0019] In some embodiments, the measured level is up to about 10.0 times above the control level, or up to about 8.00 or up to about 5.00 times above the control level.

[0020] In some embodiments, the disclosure relates to a diagnostic method for confirming a diagnostic of a cerebral creatine deficiency syndrome in an individual presumed to have a cerebral creatine deficiency syndrome, the method comprising:

[0021] a) measuring, in an isolated biological sample obtained from said individual, a nucleic acid methylation level,

[0022] b) comparing said measured methylation level to a methylation level of reference,

[0023] c) wherein a deviation observed between the measured methylation level and the methylation level of reference is indicative of a confirmation of a cerebral creatine deficiency syndrome in said individual.

[0024] In some embodiments, the methylation level of reference is a mean level of nucleic acid methylation measured in a population of healthy individuals.

[0025] In some embodiments, the observed deviation is a decrease of the measured methylation level of at least about 1 .20 times below the methylation level of reference.

[0026] In some embodiments, the biological sample is selected among a blood sample, a serum sample, a cerebrospinal fluid sample, a brain tissue sample, a skin sample, and all or part of a brain organoid prepared by dedifferentiation and reprogramming of cells obtained from said individual.

[0027] In some embodiments, the disclosure relates to a method for identifying a candidate therapeutic agent useful for treating a cerebral creatine deficiency syndrome, said method comprising the steps of:

[0028] a) measuring a nucleic acid methylation level in an isolated biological sample obtained from a biological model of a cerebral creatine deficiency syndrome contacted with said candidate therapeutic agent, and

[0029] b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylation level in said biological model in absence of said candidate therapeutic agent, and

[0030] c) identifying said candidate therapeutic agent as being useful for treating a cerebral creatine deficiency syndrome when the measured nucleic acid methylation level is at least 1.10 times above the control level.

[0031] In some embodiments, the biological model is selected among a brain organoid prepared by dedifferentiation and reprogramming of cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome, a creatine transporter (CrT) knockout non-human animal model, and white blood cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome.

[0032] In some embodiments, the cerebral creatine deficiency syndrome is a creatine transporter (CRTR) deficiency.

[0033] In some embodiments, the methylation level is measured on DNA or on RNA extracted from the sample.

[0034] In some embodiments, the methylation level is measured on genomic DNA or on whole-cell RNA.

[0035] In some embodiments, the methylation level is measured by a method selected among methylated nucleic acid ELISA, methylated nucleic acid immunoprecipitation, DNA bisulfite sequencing, RNA bisulfite sequencing, bisulfite PCR, methylation-specific PCR (MSP), reverse phase high pressure liquid chromatography (HPLC), mass spectrometry, and pyrosequencing.

[0036] In some embodiments, the disclosure relates to a folic acid, or a derivative thereof, for use in a method for treating a cerebral creatine deficiency syndrome.

[0037] In some embodiments, the folic acid, or the derivative thereof, is for use in combination with a creatine fatty ester, or a salt thereof.

[0038] In some embodiments, the disclosure relates to a combination comprising folic acid, or a derivative thereof, and a creatine fatty ester, or a salt thereof, for use in a method for treating a cerebral creatine deficiency syndrome.

[0039] In some embodiments, the folic acid, or a derivative thereof, and the creatine fatty ester, or a salt thereof, are administered concurrently or separately.

[0040] In some embodiments, the disclosure relates to a pharmaceutical composition, comprising, as active principle, a combination of folic acid, or a derivative thereof, and of a creatine fatty ester, or a salt thereof, and comprising at least one pharmaceutically acceptable excipient.

[0041] In some embodiments, the disclosure relates to a kit-of-parts comprising:

[0042] a) a first container containing folic acid, or a derivative thereof, optionally with at least one pharmaceutically acceptable excipient

[0043] b) a second container containing a creatine fatty ester, or a salt thereof, optionally with at least one pharmaceutically acceptable excipient.

[0044] In some embodiments, the pharmaceutical composition according to the disclosure or the kit-of-parts according to the disclosure are for use in a method for treating a cerebral creatine deficiency syndrome.

[0045] the folic acid, or the derivative thereof, for use according to the disclosure, the combination for use according to the disclosure, the pharmaceutical composition or the kit-of- parts for use according to disclosure, are for use with dodecyl creatine ester or hydrochloride salt of dodecyl creatine ester.

[0046] In some embodiments, the folic acid, or the derivative thereof, for use according to the disclosure, the combination for use according to the disclosure, the pharmaceutical composition or the kit-of-parts for use according to disclosure, are for use for a creatine transporter deficiency.

[0047] In some embodiments, a cerebral creatine deficiency syndrome concerned by the methods and uses of the disclosure may be selected among creatine transporter deficiency (CTD), guanidinoacetate methyltransferase deficiency (GAMT) and arginine:glycine amidinotransferase deficiency (AGAT).

[0048] In some embodiments, the cerebral creatine deficiency syndrome may be creatine transporter deficiency (CTD).[DESCRIPTION OF THE FIGURES]

[0049] Figure 1 : represents the DNA methylation level in brain tissue of wild-type (WT) and CrT KO mice with and without DCE treatment. N = 3-4 mice per group (WT = wild-type; veh = CrT KO mice treated with the vehicle; DCE 2% = CrT KO mice treated with the dodecyl creatine ester formulation 2% w / w, 0.48 mg / day intranasally for 30 days). Data are expressed as relative quantification to controls and analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons test. * P< 0.05.

[0050] Figure 2: represents the DNA and RNA methylation levels in CTD brain organoids (CTD 1 -4, 2-3 and 3-7) vs healthy brain organoids. A: 5-mC in DNA (n = 6). B: m6A in mRNA (n=6-14). Data express as relative quantification to controls. And analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons test. * P< 0.05; ** P< 0.01 ; *** P<0.001 .

[0051] Figure 3: is a schematic representation of the link between creatine intracerebral synthesis and DNA methylation in CTD patient brain. SAM = S- adenosylmethionine AGAT = glycine amidinotransferase; GAMT = guanidinoacetate methyltransferase; DNMT = DNA methyltransferase; METTL = Methyltransferase-like ; GAA = Guanidinoacetic acid.[DETAILED DESCRIPTION]Definitions

[0052] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, may provide one of skill in the art with a general dictionary of many of the terms used in this disclosure. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’sspecifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0053] Units, prefixes, and symbols are denoted in their International System Units (Systeme International des Unites (SI)) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0054] All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0055] Throughout this specification and embodiments, the words “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” will be understood to imply the inclusion of a stated item or group of items but not the exclusion of any other item or group of items. It is understood that whatever aspects described with the language "comprising", analogous aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0056] It is to be noted that the term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence," is understood to represent one or more nucleotide sequences. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0057] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] The term “approximately” or "about" is used herein to mean approximately, roughly, around, or in the regions of. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. In general, the term "about" can modify a numerical value above and below the stated value by a variance of, e.g., 10 percent, up or down (higher or lower). In some embodiments, the term indicates deviation from the indicated numerical value by ±10%,±5%, ±4%, ±3%, ±2%, ±1 %, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%.

[0059] The terms “measuring”, “detecting” and “determining” are used interchangeably and refer to quantifying the methylation level of a nucleic acid, e.g., methylation level of DNA or methylation level of total RNA, in a sample. Measuring can be accomplished by methods known in the art and those further described herein including, but not limited to, quantitative polymerase chain reaction (PCR).

[0060] The term "methylation" refers to cytosine methylation at positions C5 or N4 of cytosine, the N6 position of adenine or other types of nucleic acid methylation.

[0061] "Hypomethylation" or "decrease level of methylation" intend to refer to a decrease in methylation of a given nucleic acid that is considered significant over a control level.

[0062] The terms "methylation status" or "methylation level" refers to the presence, absence and / or quantity of methylation in a given nucleic acid. The methylation status of a nucleic acid can indicate the methylation state within the sequence without providing precise information of where in the sequence the methylation occurs. The methylation status can be represented or indicated by a "methylation value" or "methylation level." A methylation value or level can be generated, for example, by quantifying the total amount or the amount of a subset of methylated nucleotides in a nucleic acid sequence.

[0063] The term "comparing" refers to making an assessment of how the methylation status, proportion, or level in a biological sample relates to the methylation status, proportion, or level of methylation in a standard, reference, or control sample. For example, "comparing" may refer to assessing whether the methylation status, proportion, or level in a biological sample is the same as, more or less than, or different from the methylation status, proportion, or level in standard, reference, or control sample.

[0064] By "purified" and "isolated" it is meant, when referring to a biological sample, that the indicated element has been separated from other substances or components that were originally present in a mixture or from its natural environment. The term "purified" as used herein in particular means at least 75%, 85%, 95%, or 98% by weight, of elements of the same type are present.

[0065] As used herein, the terms “individual”, “subject” or “patient" are used interchangeably and refers to a mammal, in particular a human. An individual in need thereof is an individual known or presumed to have a cerebral creatine deficiency syndrome. In some embodiments, an individual in need thereof is an individual known or presumed to have a cerebral creatine transporter deficiency.

[0066] “Sample” or “biological sample” intends to refer to a biological material obtained (or isolated) from an individual or a biological model. A sample may be obtained from a healthy subject, a diseased patient, a biological model or a control counterpart of a biological model. A sample may be a blood sample, a serum sample, a cerebrospinal fluid sample, a brain tissue sample, a brain organoid sample. The definition also includes samples that have been manipulated in any way after their procurement, such as by centrifugation, filtration, precipitation, dialysis, chromatography, treatment with reagents, washed, or enriched for certain cell populations.

[0067] A “reference” or a “control” value or level intends to refer to a value or level, determined in a control or reference sample obtained from a healthy patient or from a diseased patient non-treated with a medicament presumed active against the disease (untreated diseased patient) or obtained from a control counterpart of a biological model or from a biological model non-treated with a medicament presumed active against the disorder modelled by the biological model (untreated biological model).

[0068] A “biological model of a cerebral creatine deficiency syndrome” intends to refer to a biological system representing the biological processes of a cerebral creatine deficiency syndrome. It can be used to study the development and progression of the disease, identify potential therapeutic targets, and develop and test new drugs and treatments. As example of biological model suitable for the present disclosure, one may cite brain organoids obtained from dedifferentiated and reprogrammed patient’s cells or CrT k.o. mouse.

[0069] "Administer" or "administering," as used herein refers to delivering to a subject a composition described herein. The composition can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route.

[0070] The terms “treat”, “treatment”, or “therapy” refers to the administration of a compound or a composition according to the disclosure with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a disorder, the symptoms of the condition, or to prevent or delay the onset of the symptoms, complications, or otherwise arrest or inhibit further development of the disorder in a statistically significant manner. More particularly, “treating” or “treatment” includes any approach for obtaining beneficial or desired results in a subject’s cerebral creatine deficiency syndrome condition. Beneficial or desired clinical results can include, but are not limited to, alleviation, amelioration, diminishment, reduction of the extent, delay or slowing of one symptom of the disease. In other words, "treatment" as used herein includes any cure, amelioration, or reduction of the disease or asymptom of which. A “reduction” of a symptom or a disease means decreasing of the severity or frequency of the disease or symptom, or elimination of the disease or symptom.

[0071] The expression "therapeutically effective amount' intends to mean an amount of the compound which is effective in treating the named disorder or condition.

[0072] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0073] The list of sources, ingredients, and components as described hereinafter are listed such that combinations and mixtures thereof are also contemplated and within the scope herein.

[0074] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0075] All lists of items, such as, for example, lists of ingredients, are intended to and should be interpreted as Markush groups. Thus, all lists can be read and interpreted as items “selected from the group consisting of’ the list of items “and combinations and mixtures thereof.”

[0076] Referenced herein may be trade names for components including various ingredients utilized in the present disclosure. The inventors herein do not intend to be limited by materials under any particular trade name. Equivalent materials (e.g., those obtained from a different source under a different name or reference number) to those referenced by trade name may be substituted and utilized in the descriptions herein.Detailed descriptionNucleic acid methylation level measurement

[0077] The methylation level or status of nucleic acid in a biological sample may be measured on DNA or on RNA extracted from the sample.

[0078] In some embodiments, the measure may be carried out on total extracted DNA or on total extracted RNA. The expression “total extracted” DNA or RNA (or total extract DNA or total extract RNA) are used according to the common meaning in the field and intend to refer to DNA or RNA extracted without targeting a specific DNA or RNA, i.e., genomic DNA, mRNA, etc. That does not mean that all and every DNA or RNA molecules contained in a sample are extracted.

[0079] In some embodiments, the measure may be carried out on total extract DNA, genomic DNA, mitochondrial DNA, or cell-free DNA.

[0080] In some embodiments, the measure may be carried out on genomic DNA.

[0081] In some embodiments, the measure may be carried out on whole-cell RNA (or total extract RNA), mRNA, or exome RNA.

[0082] In some embodiments, the measure may be carried out on whole-cell RNA.

[0083] In some embodiments, the measure may be carried out on genomic DNA or on whole-cell RNA.

[0084] A nucleic acid may be extracted from a biological sample by any suitable methods known in the art to allow extracting a nucleic acid without affecting its methylation status.

[0085] A suitable method for extracting nucleic acid may comprise the steps of:

[0086] - lysing the cells from a biological sample in a buffer preserving the methylation status of the nucleic acid, such as by chemical, enzymatic, or mechanical lysis,

[0087] - separating the nucleic acid from the proteins and cellular debris from the lysate, for example by centrifugation, precipitation, filtration or binding of the nucleic acid to beads.

[0088] As examples of methods known to extract nucleic acids without affecting methylation status, one may cite phenol-chloroform extraction, silica-based column purification, and magnetic bead-based methods.

[0089] In a phenol-chloroform extraction, a biological sample is lysed, and cellular components such as proteins and lipids are separated from nucleic acids. The aqueous phase containing the nucleic acids is then mixed with phenol-chloroform, which partitions the nucleic acids into the aqueous phase while removing contaminants. After centrifugation, the aqueous phase is separated and subjected to precipitation with ethanol or isopropanol to obtain total DNA or total RNA.

[0090] Silica-based column purification involves binding nucleic acids to a silica matrix in the presence of chaotropic salts, followed by washing to remove impurities and eluting the nucleic acids with a low-salt buffer.

[0091] Magnetic bead-based methods use magnetic beads coated with nucleic acidbinding substances. These beads selectively bind to DNA or RNA, allowing efficient separation from contaminants. After binding, the beads are washed, and the nucleic acids are eluted in a suitable buffer.

[0092] The methylation level of a nucleic acid obtained from a biological sample may be determined with any suitable methods known in the art.

[0093] Three types of natural methylation have been reported in nucleic acid. Cytosine can be modified either on the 5thcarbon of the ring to form 5-methylcytosine (5mC) or on the exocyclic amino group to form N4-methylcytosine (4mC). Adenine may be modified to form N6- methyladenine (6mA). In human DNA, methylation of the fifth carbon of cytosine can occur, while in human RNA, methylation of the adenine forming 6mA or methylation of the cytosine forming m5C can occur.

[0094] In some embodiments, the methylation level of a nucleic acid may be measured by a method selected among methylated nucleic acid ELISA (methylated RNA ELISA (MeRIP- ELISA) or methylated DNA ELISA (MeDIP-ELISA), methylated nucleic acid immunoprecipitation, DNA bisulfite sequencing, RNA bisulfite sequencing, bisulfite PCR, methylation-specific PCR (MSP), reverse phase high pressure liquid chromatography (HPLC), mass spectrometry, and pyrosequencing.

[0095] Methylated nucleic acid ELISA, i.e., methylated RNA ELISA (MeRIP-ELISA) or methylated DNA ELISA (MeDIP-ELISA), is a variation of the methylated nucleic acid immunoprecipitation, utilizing ELISA-based detection for quantifying methylated nucleic acid levels. The method may comprise the steps of:

[0096] a) nucleic acid (DNA or RNA) immunoprecipitation (DIP or RIP) using specific antibodies that recognize the methylated nucleotide(s) of interest. For example, antibodies targeting 5-methylcytosine (5mC) or 6-methyladenosine (6mA) may be used.

[0097] b) binding the immunoprecipitated nucleic acid to a microplate coated with capture antibodies specific to the nucleic acid, i.e., DNA or RNA,

[0098] c) binding a specific enzyme-linked or fluorescent probe labelled antibody to the antibodies bound to the methylated nucleotides. For example, an anti-m6A or anti-m5C antibody labeled with an enzyme (e.g., horseradish peroxidase) may be used,

[0099] d) measuring a detectable colorimetric, chemiluminescent of fluorescent signal either by adding a suitable substrate which can react with the enzyme label to yield a colorimetric or chemiluminescent signal or by exciting the fluorescent probe with a suitable excitation wavelength and measuring the signal at the emission wavelength.

[0100] Methylated nucleic acid immunoprecipitation (methylated DNA Immunoprecipitation (MeDIP) or methylated RNA Immunoprecipitation (MeRIP). This immunoprecipitation-based technique allows enriching and analyzing methylated nucleic acid fragments. Methylated nucleic acid fragments are immunoprecipitated using antimethylcytosine or anti-6-methyladenosine antibodies. The enriched methylated nucleic acid can then be analyzed by various downstream methods, such as qPCR or next-generation sequencing.

[0101] In DNA bisulfite sequencing (or whole genome bisulfite sequencing (WGBS)), the DNA is treated with sodium bisulfite, which converts unmethylated cytosines into uracils while leaving methylated cytosines unchanged. Subsequent DNA sequencing allows differentiation between methylated and unmethylated cytosines based on the presence of cytosine or thymine in the sequence data. Next-generation sequencing (NGS) platforms cand be used for the DNA sequencing.

[0102] In RNA bisulfite sequencing (RNA-Seq), RNA is treated with bisulfite, similar to DNA bisulfite sequencing, to convert unmethylated adenosines to inosines while leaving methylated adenosines unaffected. Next-generation sequencing may be then used to analyze the modified RNA and identify methylated sites.

[0103] Bisulfite PCR may be used for measuring nucleic acid methylation levels by combining bisulfite treatment with PCR. Bisulfite treatment converts unmethylated cytosines to uracils, while methylated cytosines remain unchanged. PCR is then used to amplify the bisulfite-converted nucleic acid.

[0104] Methylation-specific PCR (MSP) amplifies methylated or unmethylated DNA sequences. It employs primers designed to target regions with known methylation sites. Depending on the primer design, MSP can distinguish between methylated and unmethylated DNA in a given locus, providing qualitative information about methylation status.

[0105] Reverse phase high pressure liquid chromatography (HPLC) can be used to measure nucleic acid methylation levels by separating and quantifying the different methylated and unmethylated forms of the nucleosides. The methylated and unmethylated forms of a nucleoside have different hydrophobicity, so they will elute from the HPLC column at different times.

[0106] Mass spectrometry can be used to detect and quantify RNA modifications, including methylation. Total RNA can be hydrolyzed to nucleosides, and mass spectrometry is employed to analyze these nucleosides, allowing the quantification of methylated RNA bases.

[0107] Pyrosequencing is a sequencing-by-synthesis technique that allows detecting single-nucleotide polymorphisms (SNPs), which can be artificially created through bisulfitemodification. Comparing light emissions from C and T incorporation at a site measures methylation. Pyrosequencing is valuable for assessing global methylation.

[0108] In some embodiments, the methylation level of a nucleic acid may be measured by methylated nucleic acid ELISA.

[0109] In some embodiments, the methylation level of RNA may be measured by methylated RNA ELISA (MeRIP-ELISA).

[0110] In some embodiments, the methylation level of DNA may be measured by or methylated DNA ELISA (MeDIP-ELISA).Diagnosis and monitoring of patients

[0111] The disclosure relates to methods for diagnosing or monitoring patients having a cerebral creatine deficiency syndrome.

[0112] In some embodiments, the disclosure relates to methods for selecting a therapeutic agent for treating a cerebral creatine deficiency syndrome in an individual in need thereof, or for monitoring an efficacy of a therapeutic agent proposed for treating a cerebral creatine deficiency syndrome in an individual in need thereof.

[0113] In some embodiments, the cerebral creatine deficiency syndrome may be selected among creatine transporter deficiency (CTD), guanidinoacetate methyltransferase deficiency (GAMT) and arginine:glycine amidinotransferase deficiency (AGAT).

[0114] In some embodiments, the cerebral creatine deficiency syndrome may be creatine transporter deficiency (CTD).

[0115] In some embodiments, the disclosure relates to a method for monitoring an efficacy of a therapeutic agent proposed for treating a cerebral creatine deficiency syndrome in an individual in need thereof.

[0116] A method of the disclosure may comprise the steps of:

[0117] a) measuring a nucleic acid methylation level in an isolated biological sample obtained from said individual after administration to said individual of said therapeutic agent, and

[0118] b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylation level in an isolated biological sample obtained from said individual before administration to said individual of said therapeutic agent,

[0119] wherein a measured methylation level of at least 1.10 times above the control level is indicative of the efficacy of said therapeutic agent for treating said cerebral creatine deficiency syndrome.

[0120] In some embodiments, the control level may be obtained from a first isolated biological sample and the measured level obtained after administration of the therapeutic agent may be obtained from a second isolated biological sample.

[0121] In some embodiments, the disclosure relates to a method for monitoring an efficacy of a therapeutic agent proposed for treating a cerebral creatine deficiency syndrome in an individual in need thereof, said method comprising the steps of:

[0122] a) measuring, in a first isolated biological sample obtained from said individual before administration to said individual of said therapeutic agent, a first nucleic acid methylation level,

[0123] b) measuring, in a second isolated biological sample obtained from said individual after administration to said individual of said therapeutic agent, a second nucleic acid methylation level,

[0124] c) comparing the first and second measured levels,

[0125] wherein a measured second level of at least 1.10 times above the measured first level is indicative of the efficacy of said therapeutic agent for treating said cerebral creatine deficiency syndrome.

[0126] In some embodiments, the measured nucleic acid methylation level relative to the control level or the second measured nucleic acid methylation level relative to the first measured nucleic acid methylation level is at least about 1.10-fold, or at least about 1.15-fold, or at least about 1.20-fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1 .40-fold, or at least about 1 .50-fold, or at least about 1 .60-fold, or at least about 1 .70- fold, or at least about 1.80-fold, or at least about 1.90-fold, or at least about 1.95-fold, or at least about 2.00-fold, or at least about 3.00-fold, or at least about 3.50-fold, or at least about 4.00-fold.

[0127] A second given level value relative to a first given level value being x-fold intends to mean that the second level value corresponds to the first level value multiplied by x. Otherwise said the second level value is x-times the first level value.

[0128] In some embodiments, the measured nucleic acid methylation level relative to the control level or the second measured nucleic acid methylation level relative to the first measured nucleic acid methylation level is at least about 1.10-fold, or at least about 1.15-fold,or at least about 1.20-fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1 .40-fold, or at least about 1 .50-fold, or at least about 1 .60-fold, or at least about 1 .70- fold, or at least about 1.80-fold, or at least about 1.90-fold, or at least about 1.95-fold, or at least about 2.00-fold above the control level.

[0129] In some embodiments, the measured nucleic acid methylation level relative to the control level or the second measured nucleic acid methylation level relative to the first measured nucleic acid methylation level is at least about 1 .10-fold§, or at least about 1.15-fold, or at least about 1.20-fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1 .40-fold, or at least about 1 .50-fold above the control level.

[0130] In some embodiments, the measured level is up to about 10.0 times above the control level, or up to about 8.00 or up to about 5.00 times above the control level.

[0131] In some embodiments, the measured level is up to about 5.00 times above the control level.

[0132] A method for monitoring the efficacy of a therapeutic agent may be carried out in an individual in need thereof at least once every 6 months, or once every 2 months, or once every month, or once every 2 weeks, or once every week.

[0133] In some embodiments, the disclosure relates to a diagnostic method for confirming a diagnostic of a cerebral creatine deficiency syndrome in an individual presumed to have a cerebral creatine deficiency syndrome, the method comprising:

[0134] a) measuring, in an isolated biological sample obtained from said individual, a nucleic acid methylation level,

[0135] b) comparing said measured methylation level to a methylation level of reference,

[0136] c) wherein a deviation observed between the measured methylation level and the methylation level of reference is indicative of a confirmation of a cerebral creatine deficiency syndrome in said individual.

[0137] A diagnosis method of the disclosure may be used in addition of conventional methods used for diagnosing a cerebral creatine deficiency syndrome or a cerebral creatine transporter deficiency.

[0138] Conventional methods for diagnosing a cerebral creatine deficiency syndrome or a cerebral creatine transporter deficiency may include clinical evaluations, laboratory tests (such as dosing the creatine level in blood or urine), and imaging studies (magnetic resonance imaging (MRI) and spectroscopy (MRS)).

[0139] In some embodiments, a methylation level of reference may be obtained from a healthy individual, that is an individual presumed not having a cerebral creatine deficiency syndrome, of from a set of such individuals.

[0140] In some embodiments, the methylation level of reference may be a mean level of nucleic acid methylation measured in a population of healthy individuals.

[0141] In embodiments where the methylation level of reference is obtained from healthy individual(s), the observed deviation may be a decrease of the measured methylation level relative to the methylation level of reference. A decrease of the measured methylation level relative to the methylation level of reference obtained from healthy individuals may be indicative of a confirmation of a cerebral creatine deficiency syndrome.

[0142] In some embodiments, the disclosure relates to a method for monitoring an evolution of a cerebral creatine deficiency syndrome in an individual in need thereof, the method comprising:

[0143] a) measuring, in an isolated biological sample obtained from said individual, a nucleic acid methylation level,

[0144] b) comparing said measured methylation level to a methylation level of reference,

[0145] c) wherein a deviation observed between the measured methylation level and the methylation level of reference is indicative of an aggravation or of an improvement of said cerebral creatine deficiency syndrome in said individual.

[0146] In some embodiments, a methylation level of reference may be obtained from a healthy individual, that is an individual presumed not having a cerebral creatine deficiency syndrome, of from a set of such individuals.

[0147] In some embodiments, the methylation level of reference may be a mean level of nucleic acid methylation measured in a population of healthy individuals.

[0148] In embodiments where the methylation level of reference is obtained from healthy individual(s), the observed deviation may be a decrease of the measured methylation level relative to the methylation level of reference. A decrease of the measured methylation level relative to the methylation level of reference obtained from healthy individuals may be indicative of an aggravation of a cerebral creatine deficiency syndrome.

[0149] In some embodiments, when a level of reference is obtained from healthy individual(s), an observed deviation may be a decrease of the measured methylation level of at least about 1 .20 times below the methylation level of reference.

[0150] In some embodiments, an observed deviation may be a decrease of the measured methylation level relative to the methylation level of reference of at least about 1 .20- fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1.40-fold, or at least about 1 .50-fold, or at least about 1 .60-fold, or at least about 1 .70-fold, or at least about 1 .80-fold, or at least about 1 .90-fold, or at least about 1 .95-fold, or at least about 2.00-fold, or at least about 3.00-fold, or at least about 3.50-fold, or at least about 4.00-fold.

[0151] In some embodiments, an observed deviation may be a decrease of the measured methylation level relative to the methylation level of reference of at least about 1 .20- fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1.40-fold, or at least about 1 .50-fold, or at least about 1 .60-fold, or at least about 1 .70-fold, or at least about 1.80-fold, or at least about 1.90-fold, or at least about 1.95-fold, or at least about 2.00-fold above the control level.

[0152] In some embodiments, an observed deviation may be a decrease of the measured methylation level relative to the methylation level of reference of at least about 1 .20- fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1.40-fold, or at least about 1 .50-fold below the control level.

[0153] In some embodiments, the measured level may be a decrease of up to about 8.0 times below the control level, or up to about 6.00 or up to about 4.00 times below the control level.

[0154] In some embodiments, a methylation level of reference may be obtained from an individual known to have a cerebral creatine deficiency syndrome, of from a set of such individuals.

[0155] In some embodiments, the methylation level of reference may be a mean level of nucleic acid methylation measured in a population of individuals known to have a cerebral creatine deficiency syndrome.

[0156] In embodiments where the methylation level of reference is obtained from individual(s) known to have a cerebral creatine deficiency syndrome, the observed deviation may be an increase of the measured methylation level relative to the methylation level of reference. An increase of the measured methylation level relative to the methylation level of reference obtained from individuals known to have a cerebral creatine deficiency syndrome may be indicative of an improvement of a cerebral creatine deficiency syndrome.

[0157] In some embodiments, an observed deviation may be an increase of the measured methylation level of at least about 1.20 times above the methylation level of reference.

[0158] In some embodiments, an observed deviation may be an increase of the measured methylation level relative to the methylation level of reference of at least about 1 .20- fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1.40-fold, or at least about 1 .50-fold, or at least about 1 .60-fold, or at least about 1 .70-fold, or at least about 1 .80-fold, or at least about 1 .90-fold, or at least about 1 .95-fold, or at least about 2.00-fold, or at least about 3.00-fold, or at least about 3.50-fold, or at least about 4.00-fold.

[0159] In some embodiments, an observed deviation may be an increase of the measured methylation level relative to the methylation level of reference of at least about 1 .20- fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1.40-fold, or at least about 1 .50-fold, or at least about 1 .60-fold, or at least about 1 .70-fold, or at least about 1.80-fold, or at least about 1.90-fold, or at least about 1.95-fold, or at least about 2.00-fold above the control level.

[0160] In some embodiments, an observed deviation may be an increase of the measured methylation level relative to the methylation level of reference of at least about 1 .20- fold, or at least about 1.25-fold, or at least about 1.30-fold, or at least about 1.40-fold, or at least about 1 .50-fold above the control level.

[0161] According to another of its objects, the disclosure relates to a method for monitoring an evolution of a cerebral creatine deficiency syndrome in an individual in need thereof, said method comprising the steps of:

[0162] a) measuring, in an isolated biological sample obtained from said individual, a nucleic acid methylation level at a first point of time,

[0163] b) measuring, in an isolated biological sample obtained from said individual, a nucleic acid methylation level at a second point of time,

[0164] c) comparing the amount measured at step a) with the amount measured at step b),

[0165] wherein a difference between the measured amounts may be indicative of an improvement or an aggravation of the cerebral creatine deficiency syndrome in said individual.

[0166] A decrease of the methylation level measured at the second of point of time relative to the methylation level measured at a first of point of time may be indicative of an aggravation of a cerebral creatine deficiency syndrome.

[0167] An increase of the methylation level measured at the second of point of time relative to the methylation level measured at a first of point of time may be indicative of an improvement of a cerebral creatine deficiency syndrome.

[0168] A method for monitoring an evolution of a cerebral creatine deficiency syndrome may comprise in addition to the 1stand 2ndmeasures further subsequent measures taken at subsequent times. For example, a method may comprise a 3rd, a 4th, a 5th, a 6th, a 7th, an 8th, a 9th, a 10thor more measure. The method may comprise at least one subsequent measure. The at least one subsequent measure may be compared to the first measure or to a preceding measure.

[0169] In a method disclosed herein the measures may be repeated at regular or irregular intervals. For example, the measures may be carried every day, once a week every week, or every 2, 3, o r4 weeks, once a month every month, or every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or once year every year, or every 2, 3, or 4 years. The measures may be carried several times a week, a month or a year, for example, twice, or three, four, five or six time a week, or more in month or in a year. The measures may be carried out over a period of time ranging from one week to one year, or more.

[0170] In some embodiments, a biological sample may be selected among a blood sample, a serum sample, a cerebrospinal fluid sample, a brain tissue sample, a skin sample, and all or part of a brain organoid prepared by dedifferentiation and reprogramming of cells obtained from said individual.

[0171] A suitable blood sample or serum sample is a sample containing white blood cells. In some embodiments, a suitable sample may be the total white blood cells from a blood or serum sample obtained from an individual presumed to be diagnosed with a cerebral creatine deficiency syndrome.

[0172] In some embodiments, a brain organoid may be prepared by dedifferentiation and reprogramming of fibroblast cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome. Dedifferentiation of fibroblast cells may be carried out as disclosed in Broca-Brisson, Harati et al., eLife Sciences Publications, 2023. Brain organoids may be prepared as disclosed in Lancaster and Knoblich, Nat Protoc, 2014, and Nassor, Jarray et al., Front Cell Neurosci, 2020. Only a part or the whole brain organoid can be used as a sample in the diagnosing method.

[0173] In some embodiments, a skin sample contain fibroblasts.

[0174] In some embodiments, a biological sample may be selected among a blood sample, a serum sample, a cerebrospinal fluid sample, a brain tissue sample, and a skin sample.

[0175] In some embodiments, the present disclosure relates to a method for treating an individual susceptible to suffer from a cerebral creatine deficiency syndrome, the method comprising at least the step of diagnosing or confirming a diagnostic of a cerebral creatine deficiency syndrome in said individual and a step of administering to said individual diagnosed with, or confirmed with a diagnosis of, a cerebral creatine deficiency syndrome a therapeutic treatment proposed for treating a cerebral creatine deficiency syndrome.

[0176] The step of diagnosing or confirming a diagnostic of a cerebral creatine deficiency syndrome may be carried according to the methods previously described.

[0177] In some embodiments, the present disclosure relates to a method for treating an individual susceptible to suffer from a cerebral creatine deficiency syndrome, the method comprising at least the step of monitoring a cerebral creatine deficiency syndrome in said individual and a step of administering to said individual monitored with an aggravation of a cerebral creatine deficiency syndrome a therapeutic treatment proposed for preventing and / or treating a cerebral creatine deficiency syndrome.

[0178] The step of monitoring a cerebral creatine deficiency syndrome may be carried according to the methods previously described.Screening of therapeutic candidate agents

[0179] The disclosure relates to methods for screening therapeutic candidate agents suitable for the treatment of a cerebral creatine deficiency syndrome. A therapeutic candidate agent suitable for the treatment of a cerebral creatine deficiency syndrome is agent capable of increasing the nucleic acid methylation level from a control or reference hypomethylated state, by at least about 10% or about 1.10 times.

[0180] In some embodiments, the cerebral creatine deficiency syndrome may be selected among creatine transporter deficiency (CTD), guanidinoacetate methyltransferase deficiency (GAMT) and arginine:glycine amidinotransferase deficiency (AGAT).

[0181] In some embodiments, the cerebral creatine deficiency syndrome may be creatine transporter deficiency (CTD).

[0182] In some embodiments, the disclosure relates to a method for selecting a therapeutic agent presumed suitable for treating a cerebral creatine deficiency syndrome in an individual in need thereof.

[0183] A method of the disclosure may comprise the steps of:

[0184] a) measuring a nucleic acid methylation level in an isolated biological sample obtained from said individual after administration to said individual of said therapeutic agent, and

[0185] b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylation level in an isolated biological sample obtained from said individual before administration to said individual of said therapeutic agent, and

[0186] c) selecting said therapeutic agent as suitable for treating said cerebral creatine deficiency syndrome in said individual when the measured methylation level is of at least 1.10 times above the control level.

[0187] In some embodiments, the disclosure relates to a method for selecting a therapeutic agent presumed suitable for treating a cerebral creatine deficiency syndrome in an individual in need thereof, said method comprising the steps of:

[0188] a) measuring, in a first isolated biological sample obtained from said individual before administration to said individual of said therapeutic agent, a first nucleic acid methylation level,

[0189] b) measuring, in a second isolated biological sample obtained from said individual after administration to said individual of said therapeutic agent, a second nucleic acid methylation level,

[0190] c) comparing the first and second measured levels, and

[0191] d) selecting said therapeutic agent as suitable for treating said cerebral creatine deficiency syndrome in said individual when the measured methylation level is of at least 1.10 times above the control level.

[0192] In some embodiments, the disclosure relates to a method for identifying a candidate therapeutic agent useful for treating a cerebral creatine deficiency syndrome, said method comprising the steps of:

[0193] a) measuring a nucleic acid methylation level in an isolated biological sample obtained from a biological model of a cerebral creatine deficiency syndrome contacted with said candidate therapeutic agent,

[0194] b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylation level in said biological model in absence of said candidate therapeutic agent, and

[0195] c) identifying said candidate therapeutic agent as being useful for treating a cerebral creatine deficiency syndrome when the measured nucleic acid methylation level is at least 1.10 times above the control level.

[0196] A measured nucleic acid methylation level being at least 1.1 times above the control level is indicative of a candidate therapeutic suitable for treating a cerebral creatine deficiency syndrome.

[0197] In some embodiments, the measured nucleic acid methylation level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about1 .25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about 1 .50 times, or at least about 1 .60 times, or at least about 1 .70 times, or at least about 1 .80 times, or at least about 1 .90 times, or at least about 1 .95 times, or at least about 2.00 times, or at least about 3.00 times, or at least about 3.50 times, or at least about 4.00 times above the control level.

[0198] In some embodiments, the measured nucleic acid methylation level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about1 .25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about 1 .50 times, or at least about 1 .60 times, or at least about 1 .70 times, or at least about 1 .80 times, or at least about 1 .90 times, or at least about 1 .95 times, or at least about 2.00 times above the control level.

[0199] In some embodiments, the measured nucleic acid methylation level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about1 .25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about 1 .50 times above the control level.

[0200] In some embodiments, the measured level is up to about 10.0 times above the control level, or up to about 8.00 or up to about 5.00 times above the control level.

[0201] In some embodiments, the measured level is up to about 5.00 times above the control level.

[0202] The control level may be measured on a sample isolated from the same biological model of step a), but before contacting the biological model with the candidate therapeutic agent. Alternatively, the control level may be measured on sample isolated from a separate biological model, but of same nature than the one used at step a).

[0203] In some embodiments, the disclosure relates to a method for selecting a candidate therapeutic agent for treating a cerebral creatine deficiency syndrome, said method comprising the steps of:

[0204] a) measuring, in a first isolated biological sample obtained from a biological model of a cerebral creatine deficiency syndrome before contacting said biological model with said candidate therapeutic agent, a first nucleic acid methylation level,

[0205] b) measuring, in a second isolated biological sample obtained from said biological model after contacting said biological model with said candidate therapeutic agent, a second nucleic acid methylation level,

[0206] c) comparing the first and second measured nucleic acid methylation levels, and

[0207] d) selecting the candidate therapeutic agent as being suitable for treating a cerebral creatine deficiency syndrome when the measured second level is at least 1.10 times above the measured first level.

[0208] A second measured nucleic acid methylation level being at least 10% above the first measured nucleic acid methylation level is indicative of a candidate therapeutic suitable for treating a cerebral creatine deficiency syndrome.

[0209] In some embodiments, the second measured nucleic acid methylation level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about 1 .25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about1 .50 times, or at least about 1 .60 times, or at least about 1 .70 times, or at least about 1 .80 times, or at least about 1 .90 times, or at least about 1 .95 times, or at least about 2.00 times, or at least about 3.00 times, or at least about 3.50 times, or at least about 4.00 times above the first measured nucleic acid methylation level.

[0210] In some embodiments, the second measured nucleic acid methylation level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about 1 .25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about1 .50 times, or at least about 1 .60 times, or at least about 1 .70 times, or at least about 1 .80 times, or at least about 1 .90 times, or at least about 1 .95 times, or at least about 2.00 times above the first measured nucleic acid methylation level.

[0211] In some embodiments, the second measured nucleic acid methylation level is at least about 1.10 times, or at least about 1.15 times, or at least about 1.20 times, or at least about 1 .25 times, or at least about 1 .30 times, or at least about 1 .40 times, or at least about1 .50 times above the first measured nucleic acid methylation level.

[0212] The biological model used at steps a) and b) may be a same entity, e.g., a same brain organoid or a same mouse, that is to say the first and second biological samples are obtained from said entity, e.g., brain organoid or mouse, before (first) and after (second) contacting it with a candidate therapeutic agent.

[0213] The biological model used at steps a) and b) may two (a first and a second) entities, e.g., a first and second brain organoids or a first and a second mice, that is to say a first biological sample is obtained from a first entity, e.g., a first brain organoid or a first mouse, in absence of the candidate therapeutic agent, and a second biological sample is obtained from a second entity, e.g., a second brain organoid or a second mouse.

[0214] A biological model of creatine deficiency syndrome suitable for the methods of the disclosure may be selected among a brain organoid prepared by dedifferentiation and reprogramming of cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome, a creatine transporter (CrT) knock-out non-human animal model, and white blood cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome.

[0215] In some embodiments, a brain organoid may be prepared by dedifferentiation and reprogramming of fibroblast cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome. Dedifferentiation of fibroblast cells may be carried out as disclosed in Broca-Brisson, Harati et al., eLife Sciences Publications, 2023. Brain organoids may be prepared as disclosed in Lancaster and Knoblich, Nat Protoc, 2014, and Nassor, Jarray et al., Front Cell Neurosci, 2020.

[0216] In some embodiments, a creatine transporter (CrT) knock-out non-human animal model may be a CrT ko mouse. A Crt ko mouse may be obtained as disclosed in Skelton et al., Pios One, 2011.

[0217] In some embodiments, white blood cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome may be the total white blood cells from a blood or serum sample obtained from an individual diagnosed with a cerebral creatine deficiency syndrome.

[0218] A sample may be obtained from the biological model by any suitable method known in the art. For example, in case of brain organoid, few cells may be taken from the brain organoid using a scalpel and suitable dissection conditions or the whole brain organoid may be used as a sample.

[0219] The candidate therapeutic agent is contacted with a biological model of creatine deficiency syndrome in conditions suitable for the candidate agent to exert its putative pharmacological action on the methylation of the nucleic acid. Such conditions depend on the used biological models, e.g., brain organoid or a mouse, and may easily be adapted by a person skilled in the art. For example, and in a non-limitative way, suitable conditions may involve suitable solutions, buffers, pH, temperature, culture media, etc.Therapeutic agents for the treatment of cerebral creatine deficiency syndrome

[0220] In some embodiments, the disclosure relates to the folic acid, or a derivative thereof, for use in a method for treating a cerebral creatine deficiency syndrome.

[0221] The folic acid, or derivatives thereof, are used to activate the transmethylation cycle involved in the cerebral production of creatine by increasing the source of methyl. The production of creatine with an extra source of methyl increases the creatine production and the methylation of nucleic acid level in individuals having a cerebral creatine deficiency syndrome and hypomethylated nucleic acids.

[0222] In some embodiments, the disclosure relates to the use of folic acid, or a derivative thereof, in the manufacture of a medicament for use in a method for treating a cerebral creatine deficiency syndrome.

[0223] In some embodiments, the disclosure relates to a method for treating a cerebral creatine deficiency syndrome in an individual in need thereof, the method comprising at least a step of administering to said individual a folic acid, or a derivative thereof.

[0224] The uses and methods of the disclosure are for individuals in need thereof. An individual in need thereof is an individual having a cerebral creatine deficiency syndrome.

[0225] The folic acid or derivative thereof is used in a therapeutically active amount. A therapeutically active amount may vary depending on the individual to be treated, its gender, age, weight, and stage of the disorder to be treated, as well as the formulation type and the route of administration. A person skilled in the art may rely on the general knowledge in the field to adjust the dosage accordingly.

[0226] As example of suitable dosage of folic acid or derivatives thereof, one may cite a dosage taken from a range of about 200 pg / day to about 6000 pg / day, or from about 400 pg / day to about 4000 pg / day, or from about 800 pg / day to about 2000 pg / day, or at about 1000 pg / day .

[0227] The term “derivative of folic acid” intends to refer to compounds having a homology of structure with folic acid and having the same functionality, that is compounds able to act as precursor of the S-adenosyl methionine for the transmethylation cycle. A derivative of folic acid is a pharmaceutically acceptable derivative of folic acid.

[0228] As examples of derivatives of folic acid suitable for the present disclosure, one may cite tetrahydrofolate, 5-methyltetrahydrofolate (5-MTHF), L-methylfolate, folinic acid ((5- formyltetrahydrofolate), salts of folic acid, and folic acid conjugates.

[0229] A salt of folic acid suitable for the disclosure is a pharmaceutically acceptable salt.

[0230] As examples of salts of folic acid suitable of the disclosure, one may cite salts of sodium, potassium, calcium, magnesium, ammonium, meglumine, choline, diethylamine, triethylamine, trimethylamine, benzylamine, decylamine, and cetyltrimethylammonium.

[0231] Pharmaceutically acceptable conjugates of folic acid are molecules that are formed by covalently linking folic acid to another molecule, such as a protein, peptide, or aminoacid. As examples of conjugates of folic acid suitable of the disclosure, one may cite conjugates of polyethylene glycol (PEG), albumin, transferrin, glycine and glutamate.

[0232] As examples of esters of folic acid suitable of the disclosure, one may cite 5- methyltetrahydrofolate (5-MTHF), L-methylfolate, folinic acid.

[0233] In some embodiments, the folic acid, or the derivative thereof, may be used in combination with a creatine fatty ester, or a salt thereof.

[0234] The expression “in combination with” intends to mean that the folic acid, or derivative thereof, is administered before, after, or concurrently with the creatine fatty ester, or salt thereof. In some embodiments, the term “in combination with” includes sequential, separate, or simultaneous administration. In some embodiments, “combination" intends to refer to compounds individually formulated and sequentially or separately administered. In some embodiments, “combination" intends to refer to compounds formulated together and simultaneously administered.

[0235] In a simultaneous administration, the compounds are administered by the same route.

[0236] In a separate administration, the compounds are administered by separate routes.

[0237] In a sequential administration, the compounds may be administered at different subsequent times.

[0238] A creatine fatty ester, or a salt thereof, suitable for the present disclosure may a creatine fatty ester as disclosed in WO 2020 / 221780.

[0239] In some embodiments, the creatine fatty ester may be represented by the formula (I):

[0240] (NH2)-C(NH)-N(CH3)-CH2-COOR (I)

[0241] in which R represents an alkyl radical with 4 to 30 carbon atoms, an alkenyl radical with 4 to 30 carbon atoms or an aryl radical with 6 to 30 carbon atoms.

[0242] Regarding R, by « alkyl radical with 4 to 30 carbon atoms » is meant a linear, branched or cyclic (hetero)alkyl group, optionally substituted, with 4 to 30 carbon atoms, notably with 4 to 25 carbon atoms and in particular, with 4 to 20 carbon atoms, the heteroatom(s) of the heteroalkyl group being N, O, P or S.

[0243] Regarding R, by « alkenyl radical with 4 to 30 carbon atoms » is meant a linear, branched or cyclic (hetero)alkenyl group, optionally substituted, with 4 to 30 carbon atoms, notably with 4 to 25 carbon atoms and in particular, with 4 to 20 carbon atoms, the heteroatom(s) of the heteroalkenyl group being N, O, P or S.

[0244] Regarding R, by « aryl radical with 6 to 30 carbon atoms », is meant a mono- or poly-cyclic (hetero)aromatic group, optionally substituted, having from 6 to 30 carbon atoms,notably from 6 to 25 carbon atoms, in particular, from 6 to 20 carbon atoms, the heteroatom(s) of the heteroaromatic group being N, O, P or S.

[0245] By « optionally substituted » is meant a radical which can be substituted with one or more groups selected from an alkyl group, an aryl group, an alkoxy group, a halogen, a hydroxy, a cyano, a trifluoromethyl or a nitro. For substitution groups, an « alkyl group » is a linear, branched or cyclic (hetero)alkyl group with 1 to 10 carbon atoms, the heteroatom(s) of the heteroalkyl group being N, O, P or S. For substitution groups, an « aryl group » is a mono- or poly-cyclic (hetero)aromatic group having from 6 to 15 carbon atoms, the heteroatom(s) of the heteroaromatic group being N, O, P or S. For substitution groups, an « alkoxy group » is an oxygen atom substituted by an alkyl group as above defined.

[0246] By « halogen » is meant a fluorine, chlorine, bromine or iodine.

[0247] In some embodiments, the radical R present in the creatine fatty ester of formula (I) is represented by the following formula (II):

[0248] -CH2-R' (II)

[0249] in which R' is chosen in the group consisting of an alkyl radical with 3 to 30 carbon atoms, an alkenyl radical with 3 to 30 carbon atoms and an aryl radical with 6 to 30 carbon atoms.

[0250] Regarding R', by « alkyl group with 3 to 30 carbon atoms » is meant a linear, branched or cyclic (hetero)alkyl group, optionally substituted, with 3 to 30 carbon atoms, notably with 3 to 25 carbon atoms and in particular, with 3 to 20 carbon atoms, the heteroatom(s) of the heteroalkyl group being N, O, P or S.

[0251] Regarding R', by « alkenyl group with 3 to 30 carbon atoms » is meant a linear, branched or cyclic (hetero)alkenyl group, optionally substituted, with 3 to 30 carbon atoms, notably with 3 to 25 carbon atoms and in particular, with 3 to 20 carbon atoms, the heteroatom(s) of the heteroalkenyl group being N, O, P or S.

[0252] In some embodiments, a salt of the creatine fatty ester may be a salt of an alkali metal, in particular sodium or potassium salt, or salt of alkaline earth metal, in particular magnesium or calcium, or even a salt with an organic amide, more particularly with an amino acid such as arginine or lysine.

[0253] A salt of the creatine fatty ester is a pharmaceutically acceptable salt.

[0254] In some embodiments, a salt of the creatine fatty ester may be a salt of inorganic acid such as, for example, hydrochloric acid, sulfuric acid, or hydrobromic acid, or in the form of an organic salt, such as, for example, acetic acid, formic acid, triflic acid, tartatic acid, oxalic acid, citric acid, trifluoroacetic acid or methanesulfonic acid.

[0255] In some embodiments, the creatine fatty ester or salt thereof is dodecyl creatine ester or hydrochloride salt of dodecyl creatine ester.

[0256] The creatine fatty ester or salt thereof is use at a therapeutically effective amount.

[0257] In some embodiments, a composition suitable for the present disclosure may comprise at least one creatine fatty ester or salt thereof in an amount of 0.01 % to 6% by weight and in particular of 0.02% to 5.5% by weight based on the total weight of the composition.

[0258] In some embodiments, the disclosure relates to a combination comprising folic acid, or a derivative thereof, and a creatine fatty ester, or salt thereof, for use in a method for treating a cerebral creatine deficiency syndrome.

[0259] In some embodiments, the folic acid, or a derivative thereof, and the creatine fatty ester, or salt thereof, are administered concurrently or separately.

[0260] In some embodiments, the present disclosure relates to a pharmaceutical composition, comprising, as active principle, a combination of folic acid, or a derivative thereof, and a creatine fatty ester, or a salt thereof, and comprising at least one pharmaceutically acceptable excipient.

[0261] The expression "pharmaceutical composition" indicates a mixture containing ingredients that are compatible when mixed and which may be administered. A pharmaceutical composition may include one or more medicinal drugs. Additionally, the pharmaceutical composition may include carriers, buffers, acidifying agents, alkalizing agents, solvents, adjuvants, tonicity adjusters, emollients, expanders, preservatives, physical and chemical stabilizers e.g., surfactants, antioxidants and other components, whether these are considered active or inactive ingredients. A “pharmaceutically acceptable excipient” is an excipient which is physiologically acceptable (e.g., physiologically acceptable pH) while retaining the therapeutic properties of the substance with which it is administered. A pharmaceutically acceptable excipient, diluent or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, or encapsulating material.

[0262] Standard acceptable pharmaceutical carriers and their formulations are known to one skilled in the art and described, for example, in Remington: The Science and Practice of Pharmacy, (20th ed.) ed. A. R. Gennaro A. R., 2000, Lippencott Williams & Wilkins and in R.C.Rowe et al. (Ed), Handbook of Pharmaceutical excipients, PhP, May 2013 update. One exemplary pharmaceutically acceptable carrier is physiological saline solution.

[0263] A pharmaceutical composition may include one or more compounds of the disclosure and at least one pharmaceutically acceptable excipient.

[0264] In some embodiments, the pharmaceutical composition may comprise at least one excipient selected among omega-3 fatty acid, or a salt thereof, and glyceride.

[0265] An omega-3 fatty ester or salt thereof suitable for the present disclosure may be docosahexaenoic acid (DHA).

[0266] A glyceride suitable for the present disclosure may be a mixture of mono-, di- and triglycerides of linoleic and oleic acids.

[0267] In some embodiments, the present disclosure relates to a kit-of-parts comprising:

[0268] a) a first container containing folic acid, or a derivative thereof, optionally with at least one pharmaceutically acceptable excipient,

[0269] b) a second container containing a creatine fatty ester, or a salt thereof, optionally with at least one pharmaceutically acceptable excipient.

[0270] In some embodiments, the kit-of-parts may be for simultaneous, sequential or separate use as a medicament.

[0271] The kit of parts may comprise the active agents in dosage units containing a particular amount of the active agent, for example, one or more ampoules or capsules. The dosage units may comprise one or more of the active agents. Generally, a kit includes instructions for use, for example the nature of administration.The pharmaceutical composition or the kit-of-parts according to the disclosure may be for use in a method for treating a cerebral creatine deficiency syndrome.[EXAMPLES]

[0272] The following examples illustrate the embodiments of the invention that are presently best known. However, it is to be understood that the following are only exemplary or illustrative of the application of the principles of the present invention. Numerous modifications and alternative compositions, methods, and systems may be devised by those skilled in the art without departing from the spirit and scope of the present invention. Thus, while the present invention has been described above with particularity, the following examples provide further detail in connection with what are presently deemed to be the most practical and preferred embodiments of the invention.Example 1 : Materials & MethodsChemicals

[0273] DCE was synthesized as described previously (Trotier-Faurion, Dezard et al. 2013). kollisolv MCT70 was from BASF and Montanox 80 from SEPPICAnimals and Intranasal treatment

[0274] All procedures were in accordance with European directives on the protection and use of laboratory animals (Council Directive 2010 / 63 / UE, French decree 2013-118). The experimental protocol was evaluated and validated by a local ethic committee for animal use and approved by the French government (n° APAFIS#7466-20161 10417049220).

[0275] Male CrT- / y and CrT+ / y mice were generated on the C57BL / 6J background as previously described (Skelton et al., 201 1 ). The mice were housed at 22°C on a 12-12 h lightdark cycle and provided food and water ad libitum.

[0276] Dodecyl Creatine Ester (DCE) was synthesized as described previously (Trotier-Faurion et al., 2013). The emulsion formulation comprised 22.3% w / w Montanox 80 (SEPPIC), 19% w / w of kollisolv MCT70 (BASF) and 56.7% w / w of saline solution 0.9%Nacl. The specified amount of DCE (2%w / w) was first suspended in the mix of oil and surfactant with magnetic stirring at room temperature. The premix and the saline were preheated at 40°C. Finally, a fixed amount of preheated regular saline was added to the above mixture and stirred continuously for 10 min until a homogenous emulsion was obtained. The emulsion was kept under stirring during the cooling phase then store at 4°C until use. The vehicle was prepared with the same process with replacement of the 2% w / w of DCE by regular saline.

[0277] DCE was intra-nasally administered as previously reported (Mabondzo et al., 2023) to CrT KO mice for 30 days, while wild-type (WT) and vehicle-treated CrT KO mice were used as controls. Using a P20 micropipette, 6 pl of DCE formulation or vehicle was placed in the nostril. The intranasal treatment DCE (20 mg / g) or vehicle was given twice bilaterally (24 pl total volume) for 30 days. In the DCE treated group, each mouse received 0.48 mg of DCE daily.IPSC culture and Brain organoids generation

[0278] Healthy BJ iPSCs were a generous gift of Frank Yates (Pavoni et al., 2018). CTD iPSCs were obtained by reprogramming CTD patient’s fibroblasts in a previous study (Broca-Brisson, Harati et al. 2023). All the mutations were previously described by Valayannopoulos et al., 2013:

[0279] Patient 1 : c1006_1008delAAC (pAsn336del): 9 years old showing mild mental retardation, speech delay, learning difficulties, mild behavioral impairment and facial hypotonia.

[0280] Patient 2: c1497_1500delGAG (pGly499del): 10 years old showing mild psychomotor retardation, speech delay, seizures, impulsivity, facial and trunk hypotonia.

[0281] Patient 3: c1221_1223delTTC (pGly414del): 3 years old showing no speech, autistic behavior with hyperactivity and emotional instability.

[0282] All iPSC lines were maintained on hESC-qualified Matrigel (CORNING) in mTeSRTMl medium (STEMCELL Technologies) and passaged using ReLeSR™ (STEMCELL Technologies).

[0283] Brain organoids were generated as previously described (Broca-Brisson et al., 2023,) following the protocol published by Lancaster & Knoblich with minor modifications from Nassor et al (Lancaster and Knoblich 2014, Nassor, Jarray et al. 2020). Brain organoids were used at 2 month-old.

[0284] The methylation level measured from untreated brain organoids, (healthy and CTD patients.DNA extraction and 5-methylcytosine (5mC) methylation quantification

[0285] Total DNA was extracted from 20 to 25 mg of brain tissue or each brain organoids using the NucleoSpin Tissue Mini kit for DNA (MACHEREY-NAGEL, 740952.50), according to manufacturer's instructions. The purity and concentration of DNA were measuredusing the NanoDrop ND-1000 spectrophotometer at 230, 260 and 280 nm (NANODROP TECHNOLOGIES).

[0286] Global 5mC DNA content in brain tissue or brain organoids was quantified using the ELISA-based Methylated DNA Quantification Kit (Colorimetric) (ABCAM AB117128). The input DNA was diluted in TE buffer (Tris-EDTA buffer) to an optimum 100 ng per reaction. The assay was performed in duplicates according to the manufacturer’s instructions. Plates was read at 450 nm on a BioTek Epoch Microplate Spectrophotometer (AGILENT TECHNOLOGIES). The negative control absorbance was subtracted to all measurements for background absorbance correction. Levels of DNA-methylation were expressed as relative to the control group mean.RNA extraction and 6-methyladenosine (m6A) quantification

[0287] To check the methylation at the RNA level, total RNA was isolated from brain organoids with RNeasy Plus Universal Tissue Mini kit (Qiagen) and Precellys Evolution tissue homogenizer (Bertin). The concentration and purity of RNA samples were checked using the NanoDrop ND-1000 spectrophotometer at 260 and 280 nm (NanoDrop Technologies); the A260 / 280 ratio ranged from 1 .8 to 2.2.

[0288] Global m6A RNA content in brain organoids was quantified using the ELISA- based EpiQuik m6A RNA Methylation Quantification Kit (Colorimetric) (Epigentek P-9005). The input RNA was diluted in TE buffer (Tris-EDTA buffer) to an optimum 200 ng per reaction. The assay was performed in duplicates according to the manufacturer’s instructions. Plate was read at 450 nm on a BioTek Epoch Microplate Spectrophotometer (Agilent Technologies). The negative control absorbance was subtracted to all measurements for background absorbance correction. Level of RNA methylation were expressed as relative to the control group mean.Statistics

[0289] Statistical analysis was performed using the GraphPad Prism 10.1 program. Experimental comparisons with multiple groups were analyzed using Kruskal-Wallis test with Dunn’s multiple comparisons test for post hoc analysis.Example 2: Results

[0290] To investigate our hypothesis of deficient nucleic acid methylation in CTD, we first used CrT KO mice, a model that recapitulates cognitive impairments and majorpathophysiological aspects of CTD (Skelton, Schaefer et al. 2011 , Udobi, Kokenge et al. 2018, Udobi, Delcimmuto et al. 2019).

[0291] In the nucleus, the most abundant modification of DNA is methylation on the 5thcarbon of cytosines (5-mC), mainly in the context of CpG dinucleotides. The presence of methylated cytosine in the promoter sequence of a gene is generally associated with transcriptional repression because it directly inhibits the binding of certain transcription factors and can also recruit repressors from the MBP (methyl binding proteins) family. We demonstrated a hypo-methylation of DNA in CrT KO mice brain corresponding to a decrease of 5-methylcytosine (5mC) of about 20% compared to wild-type mice (Fig. 1). This hypomethylation of DNA could impact gene expression leading to cognitive dysfunctions observed in the CrT KO mice model.

[0292] DCE administered by the nasal route is a promising therapeutic option to replenish creatine level in CTD patients brain cells. We previously demonstrated the efficacy of this strategy in vivo by showing improvement of cognitive functions, restoration of the expression of keys molecular markers and increase cerebral creatine in a well-characterized CTD mice model (Ullio-Gamboa, Udobi et al. 2019, Mabondzo, et al. 2023). In this study, we evaluated the potential of the hypomethylation of DNA as a biomarker of therapeutic efficacy. 0.48 mg of intranasal DCE daily for 30 days tended to restore DNA methylation level in the brain of CrT KO mice (Fig. 1 ). This observation demonstrated that restauration of creatine store in the brain directly impacts DNA methylation and consequently a wide variety of gene expressions.

[0293] In a second step, to evaluate the potential translation of this marker in a human model, we used a CTD brain organoid model previously described and characterized (Broca- Brisson, Harati et al. 2023). Consistently, we highlighted a significant reduction of 5mC level in DNA of all CTD brain organoids compared to heathy organoids (Fig. 2). This hypomethylation corresponded to a 31 %, 43% and 36% decrease of 5mC in DNA in CTD 1 -4, 2-3 and 3-7 respectively (Fig. 2A).

[0294] Just like DNA, RNA can be the target of various modifications of its constituent nucleotides, allowing very fine control of protein synthesis. In mammals, the most frequent modification is the addition of a methyl group at position 6 of adenosine residues (m6A). We completed the analysis of methylation status in CTD brain organoids by investigating mRNA methylation. The profile of RNA methylation was similar to results obtained on DNA, with a decrease of m6A levels in organoids from CTD patient cells corresponding to about 30% to 54% decrease depending on patients (Fig. 2B).Example 3: Conclusion

[0295] Physiopathologic mechanisms implicated in CTD remain understudy while creatine plays a pivotal role in energy metabolism and is implicated in many cellular processes, especially in the brain (Fernandes-Pires and Braissant 2022). In this study, we used both CrT KO mice and CTD human brain organoids to investigate the epigenetic changes in CTD pathology. In addition, we evaluated how DCE, a promising creatine prodrug can ameliorate this marker in vivo.

[0296] Creatine de novo synthesis places a huge burden on methyl balance (Stead, Au et al. 2001 , Brosnan, da Silva et al. 2011 ). The implication of creatine synthesis on the methylation cycle and main findings are schematized on Figure 3.

[0297] In this study, we highlighted a DNA and mRNA hypo-methylation in CTD cerebral cells using both CrT KO mice model and CTD patient’s derived brain organoids.

[0298] Methylation of nucleic acids is one of several epigenetic mechanisms that cells can use to control gene expression which play key regulatory roles in organogenesis, homeostasis and pathological processes (Yao, Christian et al. 2016). In the central nervous system, DNA methylation is crucial for normal brain development and functioning (Younesian, Yousefi et al. 2022). Recent studies have pointed to the role of epigenetic changes such as DNA, RNA methylation or histone modifications in various neurological disorders (Younesian, Yousefi et al. 2022). As example, observations in patients suffering of autism spectrum disorders, that share similar symptomatology with CTD, highlight the importance of DNA methylation in this disease (Williams and LaSalle 2022). The DNA and RNA hypo-methylation observed in both CrT KO mice and CTD brain organoids validateed our hypothesis of methylation dysregulation and could be related to the CTD symptomatology.

[0299] Our hypothesis was that endogenous synthesis of creatine is stimulated in CTD cerebral cells in an attempt to restore creatine pool. The adaptative response may impact S- adenosylmethionine (SAM) abundance. It will be interesting to evaluate SAM levels and the other metabolites of the methylation cycle (e.g., homocysteine and S-adenosylhomocystein) to confirm this mechanism. Investigation of the expression and activity of the enzymes implicated in methylation process may also complete the description of the impact of the lack of creatine. In addition, it will be interesting to look at other epigenetic processes such as methylation and acetylation of histones .

[0300] We established previously that DCE, a prodrug of creatine improves creatine cerebral levels and cognitive functions of CrT KO mice after one month of intranasal administration (Ullio-Gamboa, Udobi et al. 2019, Mabondzo, Harati et al. 2023). Improvement of the methylation balance after treatment with DCE in CrT KO mice places this dysregulationas a potential biomarker of pharmacology efficacy for preclinical studies. This biomarker could complement the potential efficacy marker highlighted previously (Mabondzo et al. 2023).

[0301] In addition, this hypo-methylation of DNA is a potential target for adjunctive therapies. Indeed, the SAM supply relies on the one-carbon cycle in which enter folate, vitamin B12, vitamin B2, and other precursors such as serine, glycine, histidine, tryptophan and choline. Even after restoring creatine cerebral levels, CTD patients may benefit from dietary supplementation with known methyl donors such as betaine, methionine or precursors of SAM that will balance the available methyl donor levels.

[0302] Today, several CrT knockout rodent models are available and recapitulate many aspects of the human pathophysiology including cognitive disorders (Skelton, Schaefer et al. 201 1 , Baroncelli, Alessandri et al. 2014, Duran-Trio, Fernandes-Pires et al. 2022). Those models are used for pre-clinical investigations, but development of new therapeutic strategy is slow-down by the lack of easily quantifiable markers to monitor pharmacological efficacy. In addition to rodent models, human cell-based organoids were recently allowed to study humanspecific aspects of the physiopathology and may offer new opportunities to better understand molecular pathways involved in the disease, discover new markers, and potential targets (Broca-Brisson et al., 2023, Mabondzo et al. 2023). Consistency of the observations in in vivo models and CTD patient’s brain organoids confirms the translational potential of this in vitro model for preclinical pharmacology studies.

[0303] Overall, using mouse model of CTD and human cell based brain organoids, we demonstrated for the first time the implication of epigenetic alterations in the pathophysiology of CTD. Increase creatine in the brain of CrT KO mice after DCE treatment improves DNA and RNA methylation levels, which unveils the potential of using DNA methylation as a marker to monitor the drug efficacy.[REFERENCES]Baroncelli, L., M. G. Alessandri, J. Tola, E. Putignano, M. Migliore, E. Amendola, C. Gross, V. Leuzzi, G. Cioni and T. Pizzorusso (2014). "A novel mouse model of creatine transporter deficiency." FI OOORes 3: 228.Braissant, O., E. Beard, C. Torrent and H. Henry (2010). "Dissociation of AGAT, GAMT and SLC6A8 in CNS: Relevance to creatine deficiency syndromes." Neurobiology of Disease 37(2): 423-433.Braissant, O. and H. Henry (2008). "AGAT, GAMT and SLC6A8 distribution in the central nervous system, in relation to creatine deficiency syndromes: A review." 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Claims

[CLAIMS]1 . A method for monitoring an efficacy of a therapeutic agent proposed for treating a cerebral creatine deficiency syndrome in an individual in need thereof, said method comprising the steps of: a) measuring a nucleic acid methylation level in an isolated biological sample obtained from said individual after administration to said individual of said therapeutic agent, and b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylation level in an isolated biological sample obtained from said individual before administration to said individual of said therapeutic agent, wherein a measured methylation level of at least 1.10 times above the control level is indicative of the efficacy of said therapeutic agent for treating said cerebral creatine deficiency syndrome.

2. The method according to claim 1 , wherein the measured level is at least 1.10 times, or at least 1.15 times, or at least 1 .20 times, or at least 1 .25 times, or at least 1 .30 times, or at least 1 .40 times, or at least 1 .50 times, or at least 1 .60 times, or at least 1 .70 times, or at least 1 .80 times, or at least 1 .90 times, or at least 1 .95 times, or at least 2.00 times, or at least 3.00 times, or at least 3.50 times, or at least 4.00 times above the control level.

3. A diagnostic method for confirming a diagnostic of a cerebral creatine deficiency syndrome in an individual presumed to have a cerebral creatine deficiency syndrome, the method comprising: a) measuring, in an isolated biological sample obtained from said individual, a nucleic acid methylation level, b) comparing said measured methylation level to a methylation level of reference, c) wherein a deviation observed between the measured methylation level and the methylation level of reference is indicative of a confirmation of a cerebral creatine deficiency syndrome in said individual.

4. The method according to claim 3, wherein the methylation level of reference is a mean level of nucleic acid methylation measured in a population of healthy individuals.

5. The method according to claim 4, wherein said observed deviation is a decrease of the measured methylation level of at least 1.20 times below the methylation level of reference.

6. The method of any one claims 1 to 4, wherein the biological sample is selected among a blood sample, a serum sample, a cerebrospinal fluid sample, a brain tissue sample, a skin sample, and all or part of a brain organoid prepared by dedifferentiation and reprogramming of cells obtained from said individual.

7. A method for identifying a candidate therapeutic agent useful for treating a cerebral creatine deficiency syndrome, said method comprising the steps of: a) measuring a nucleic acid methylation level in an isolated biological sample obtained from a biological model of a cerebral creatine deficiency syndrome contacted with said candidate therapeutic agent, and b) comparing said nucleic acid methylation level measured at step a) to a control nucleic acid methylation level, said control level being a measure of a nucleic acid methylation level in said biological model in absence of said candidate therapeutic agent, and c) identifying said candidate therapeutic agent as being useful for treating a cerebral creatine deficiency syndrome when the measured nucleic acid methylation level is at least 1.10 times above the control level.

8. The method according to claim 1 , wherein the biological model is selected among a brain organoid prepared by dedifferentiation and reprogramming of cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome, a creatine transporter (CrT) knock-out non-human animal model, and white blood cells obtained from an individual diagnosed with a cerebral creatine deficiency syndrome.

9. The method according to any one of claims 1 to 8, wherein the cerebral creatine deficiency syndrome is a creatine transporter (CRTR) deficiency.

10. The method according to any one of claims 1 to 8, wherein the methylation level is measured by a method selected among methylated nucleic acid ELISA, methylated nucleic acid immunoprecipitation, DNA bisulfite sequencing, RNA bisulfite sequencing, bisulfite PCR,methylation-specific PCR (MSP), reverse phase high pressure liquid chromatography (HPLC), mass spectrometry, and pyrosequencing.11 . Folic acid, or a derivative thereof, in combination with a creatine fatty ester, or a salt thereof, for use in a method for treating a cerebral creatine deficiency syndrome.

12. A combination comprising folic acid, or a derivative thereof, and a creatine fatty ester, or a salt thereof, for use in a method for treating a cerebral creatine deficiency syndrome.

13. A pharmaceutical composition, comprising, as active principle, a combination of folic acid, or a derivative thereof, and of a creatine fatty ester, or a salt thereof, and comprising at least one pharmaceutically acceptable excipient.

14. A kit-of-parts comprising: a) a first container containing folic acid, or a derivative thereof, optionally with at least one pharmaceutically acceptable excipient b) a second container containing a creatine fatty ester, or a salt thereof, optionally with at least one pharmaceutically acceptable excipient.

15. The pharmaceutical composition according to claim 13 or the kit-of-parts according to claim 14, for use in a method for treating a cerebral creatine deficiency syndrome.

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