The use of NME1 / 2 inhibitors in methods of treating ß-hemoglobinopathies

Abstract

BCL11A plays a critical role in the regulation of hemoglobin production, specifically in the switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA) during development. BCL11A represents a target to develop therapies for β-hemoglobinopathies by reactivating HbF. The inventors leading to a better yield for the proteomic analysis of histidine phosphorylations in a whole proteome. They identify a phosphorylation on histidine of BCL11A by immunoprecipitation of BCL11A and by the detection of its phosphorylations on histidines by Western blot. Furthermore, the knock down of NME1 and NME2 by CRISPR / Cas9 in HUDEP2 erythroid cell line induces the expression of fetal hemoglobin, suggesting that NME1 / 2 could be involved in fetal to adult globin switch probably through the regulation of histidine phosphorylation of BCL11A. HUDEP-2 cell line is still able to differentiate until the orthochromaic erythroblast stage and do not show any increase in cell death.

Description

[0001] THE USE OF NME1 / 2 INHIBITORS IN METHODS OF TREATING P- HEMOGLOBINOPATHIES

[0002] FIELD OF THE INVENTION:

[0003] The present invention is in the field of medicine, in particular haematology and genetic disorders.

[0004] BACKGROUND OF THE INVENTION:

[0005] The P-hemoglobinopathies P-thalassemia (BT) and sickle cell disease (SCD) are the most frequent genetic disorders worldwide. These diseases are caused by mutations causing reduced or abnormal synthesis of the P-globin chain of the adult hemoglobin (Hb) tetramer. P- thalassemia (BT) is a genetic disorder with an estimated annual incidence of 1 : 100,000 worldwide and 1 : 10,000 in Europe. This disease is caused by more than 200 mutations (mainly point mutations) localized in functionally important regions of the P-globin (HBB) gene. The total absence of the P-globin chain (P0) is usually associated with the most severe clinical phenotype. Reduced or absent P-globin chain production is responsible for precipitation of uncoupled a -globin chains, which in turn leads to erythroid precursor apoptosis and impairment in erythroid differentiation (i.e. ineffective erythropoiesis), and hemolytic anemia. Sickle cell disease (SCD) is a severe genetic disorder affecting -312,000 newborns worldwide annually. A single point mutation in the HBB gene causes a Glu>Val amino acid substitution in the P- globin chain (ps-globin). The sickle hemoglobin (HbS, 0^2) has the propensity to polymerize under deoxygenated conditions, resulting in the production of sickle-shaped red blood cells (RBCs) that cause occlusions of small blood vessels, leading to impaired oxygen delivery to tissues, multiple organ damage, severe pain and early mortality. Symptomatic treatment of P- hemoglobinopathies (e.g., RBC transfusions and supportive care) are associated with high costs, reduced life expectancy and poor quality of life. The only curative option is allogeneic transplantation of hematopoietic stem cells (HSC), which, however, is severely limited by the availability of compatible donors.

[0006] Several experimental and clinical treatments have been used to increase HbF levels by direct targeting of fetal globin repressors including BAF Chromatin Remodeling Complex Subunit BCL11 A (BCL11 A). BCL11 A is a transcription factor that plays a critical role in the regulation of hemoglobin production, specifically in the switch from fetal hemoglobin (HbF) to adult hemoglobin (HbA) during development. Thus, BCL11A represents an interesting target to develop genome editing therapies for P-hemoglobinopathies by reactivating HbF. This approach aims at disrupting a BCL11 A erythroid-specific enhancer to limit BCL11 A silencing to the erythroid compartment and avoid side effects in HSCs (i.e., reduced engraftment capability) and other non-erythroid lineages (e.g., B-cells) (Canver, Matthew C., et al. "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis. " Nature 527. 7577 (2015): 192-197.).

[0007] Phosphorylations are among the most common and the most studied post translational modifications of proteins. Commonly studied phosphorylations are on serine, threonine, and tyrosine because of their lower lability level compared to other phosphorylated amino acids. In eukaryotes, 9 amino acids can be phosphorylated, forming in total four types of phosphates- protein link, including histidine phosphorylations, first reported in 1962 (Boyer, P. D., DeLuca, M., Ebner, K. E., Hultquist, D. E., & Peter, J. B. (1962). Identification of phosphohistidine in digests from a probable intermediate of oxidative phosphorylation. Journal of Biological Chemistry, 237(10), PC3306-PC3308.). This phosphorylation is estimated to represent 6% of the total phosphorylations in eukaryotes. However, until recently, there has been little to no records of research on this phosphorylation, in eukaryotes. In particular, the impact of said phosphorylations on the activity of BCL11 A has never been investigated.

[0008] SUMMARY OF THE INVENTION:

[0009] The present invention is defined by the claims. In particular, the present invention relates to the use of NME1 / 2 inhibitors for the treatment of P-hemoglobinopathies.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] The first object of the present invention relates to a method of treating a P-hemoglobinopathy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a NME1 / 2 inhibitor.

[0012] As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. In particular, the patient suffers from a P-hemoglobinopathy. In some embodiments, the patient is an infant. In some embodiments, the patient is a child. In some embodiments, the patient is an adult. As used herein, the term "P-hemoglobinopathy" has its general meaning in the art and refers to any defect in the structure or function of any hemoglobin of an individual, and includes defects in the primary, secondary, tertiary or quaternary structure of hemoglobin caused by any mutation, such as deletion mutations or substitution mutations in the coding regions of the HBB gene, or mutations in, or deletions of, the promoters or enhancers of such gene that cause a reduction in the amount of hemoglobin produced as compared to a normal or standard condition.

[0013] In some embodiments, the method of the present invention is particularly suitable for the treatment of sickle cell disease.

[0014] As used herein, the term "sickle cell disease" has its general meaning in the art and refers to a group of autosomal recessive genetic blood disorders, which results from mutations in a globin gene and which is characterized by red blood cells that assume an abnormal, rigid, sickle shape. They are defined by the presence of pS-globin gene coding for a P-globin chain variant in which glutamic acid is substituted by valine at amino acid position 6 of the peptide: incorporation of the PS-globin in the Hb tetramers (HbS, sickle Hb) leads to Hb polymerization and to a clinical phenotype. The term includes sickle cell anemia (HbSS), sickle-hemoglobin C disease (HbSC), sickle P-plus- thalassaemia (HbS / p+), or sickle P-zerothalassaemia (HbS / pO).

[0015] In some embodiments, the method of the present invention is particularly suitable for the treatment of P-thalassemia.

[0016] As used herein, the term "P-thalassemia" refers to a hemoglobinopathy that results from an altered ratio of a-globin to P-like globin polypeptide chains resulting in the underproduction of normal hemoglobin tetrameric proteins and the precipitation of free, unpaired a-globin chains.

[0017] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0018] In particular, the inhibitor of the present is particularly suitable for increasing the fetal hemoglobin content in the red blood cells of the patient.

[0019] As used herein, the term “red blood cells” or “RBCs” has its general meaning in the art and refers to highly-specialized cells responsible for delivery of oxygen to, and removal of carbon dioxide from, metabolically-active cells via the capillary network. They are shaped as biconcave discs and average about 8-10 microns in diameter.

[0020] As used herein, the term “hemoglobin” or “Hb” has its general meaning in the art and refers to a hemeprotein of about 64,500 Da molecular weight, the main biological function of which is the transport of oxygen (02). Adult Hb (HbA) consists of 4 polypeptides (two a- and two [3- chains) and one heme group. Fetal Hb (HbF) contains two a-chains and two y-chains. In the present invention, the term “fetal hemoglobin” thus denotes the tetrameric forms of HbF as well as heme-free HbF. As used herein, the term “alpha globin” or “a-globin” has its general meaning in the art and refers to protein that is encoded in human by the HBA1 and HBA2 genes. The human alpha globin gene cluster located on chromosome 16 spans about 30 kb and includes seven loci: 5'- zeta - pseudozeta - mu - pseudoalpha-1 - alpha-2 - alpha-1 - theta - 3'. The alpha- 2 (HBA2) and alpha- 1 (HBAB) coding sequences are identical. These genes differ slightly over the 5' untranslated regions and the introns, but they differ significantly over the 3' untranslated regions. The ENSEMBL IDs (i.e. the gene identifier number from the Ensembl Genome Browser database) for HBA1 and HBA2 are ENSG00000206172 and ENSG00000188536 respectively. As used herein, the term “beta globin” or “P-globin”” has its general meaning in the art and refers to a globin protein, which along with alpha globin (HBA makes up the most common form of haemoglobin (Hb) in adult humans. Normal adult human Hb is a heterotetramer consisting of two alpha chains and two beta chains. The p-globin is encoded by the HBB gene on human chromosome 11. It is 146 amino acids long and has a molecular weight of 15,867 Da. As used herein, the term “gamma globin” or “y-globin” has its general meaning in the art and refers to protein that is encoded in human by the HBG1 and HBG2 genes. The HBG1 and HBG2 genes are normally expressed in the fetal liver, spleen and bone marrow. Two y-globin chains together with two a-globin chains constitute fetal hemoglobin (HbF) which is normally replaced by adult hemoglobin (HbA) in the year following birth. The ENSEMBL IDs (i.e. the gene identifier number from the Ensembl Genome Browser database) for HBG1 and HBG2 are ENSG00000213934 and ENSG00000196565 respectively.

[0021] As used herein, the expression "increasing the fetal hemoglobin content” indicates that fetal hemoglobin is at least 5% higher in the red blood cells of the patient treated with the inhibitor of the present invention, than in a comparable patient who was not treated with the inhibitor of the present invention. In some embodiments, the fetal hemoglobin content in the red blood cells of the patient is at least 10% higher, at least 20% higher, at least 30% higher, at least 40% higher, at least 50% higher, at least 60% higher, at least 70% higher, at least 80% higher, at least 90% higher, at least 1-fold higher, at least 2-fold higher, at least 5-fold higher, at least 10- fold higher, at least 100 fold higher, at least 1000-fold higher, or more than in red blood cells of a patient who was not treated with the inhibitor of the present invention. Any method known in the art can be used to measure the expression of fetal hemoglobin (e. g. HPLC analysis of protein and RT-qPCR analysis of mRNA).

[0022] In particular, the inhibitor of the present invention inhibits the phosphorylation on histidine 412 of BCL11 A, thereby increasing the expression of y-globin. As used herein, the term “BCL11A” has its general meaning in the art and refers to the gene encoding for BAF chromatin remodeling complex subunit BCL11A (Gene ID: 53335). The term is also known as EVI9; CTIP1; DILOS; ZNF856; HBFQTL5; BCL11A-L; BCL11A-S; BCL1 la-M; or BCL11 A-XL. Five alternatively spliced transcript variants of this gene, which encode distinct isoforms, have been reported. The protein associates with the SWI / SNF complex that regulates gene expression via chromatin remodeling. BCL11 A is highly expressed in several hematopoietic lineages, and plays a role in the switch from y- to P-globin expression during the fetal to adult erythropoiesis transition (Sankaran VJ et al. "Human fetal hemoglobin expression is regulated by the developmental stage-specific repressor BCL11 A ”, Science Science. 2008 Dec 19;322(5909): 1839-42). An exemplary amino acid sequence is represented by SEQ ID NO: 1.

[0023] SEQ ID NO : 1 >sp | Q9H165 | BC11A_HUMAN B-cell lymphoma / leukemia 11A 0S=Homo sapiens OX=9606 GN=BCL11A PE=1 SV=2 MSRRKQGKPQHLSKREFSPEPLEAILTDDEPDHGPLGAPEGDHDLLTCGQCQMNFPLGDI LI FIEHKRKQCNGSLCLEKAVDKPPSPSPIEMKKASNPVEVGIQVTPEDDDCLSTSSRGI CPKQEHIADKLLHWRGLSSPRSAHGALI PTPGMSAEYAPQGICKDEPSSYTCTTCKQPFT SAWFLLQHAQNTHGLRIYLESEHGSPLTPRVGI PSGLGAECPSQPPLHGIHIADNNPFNL LRI PGSVSREASGLAEGRFPPTPPLFSPPPRHHLDPHRIERLGAEEMALATHHPSAFDRV LRLNPMAMEPPAMDFSRRLRELAGNTSSPPLSPGRPSPMQRLLQPFQPGSKPPFLATPPL PPLQSAPPPSQPPVKSKSCEFCGKTFKFQSNLWHRRSHTGEKPYKCNLCDHACTQASKL KRHMKTHMHKSSPMTVKSDDGLSTASSPEPGTSDLVGSASSALKSWAKFKSENDPNLI P ENGDEEEEEDDEEEEEEEEEEEEELTESERVDYGFGLSLEAARHHENSSRGAWGVGDES RALPDVMQGMVLSSMQHFSEAFHQVLGEKHKRGHLAEAEGHRDTCDEDSVAGESDRIDDG TVNGRGCSPGESASGGLSKKLLLGSPSSLSPFSKRIKLEKEFDLPPAAMPNTENVYSQWL AGYAASRQLKDPFLSFGDSRQSPFASSSEHSSENGSLRFSTPPGELDGGI SGRSGTGSGG STPHI SGPGPGRPSSKEGRRSDTCEYCGKVFKNCSNLTVHRRSHTGERPYKCELCNYACA QSSKLTRHMKTHGQVGKDVYKCEICKMPFSVYSTLEKHMKKWHSDRVLNNDIKTE

[0024] As used herein, the term “NME1” has its general meaning in the art and refers to the nucleoside diphosphate kinase A that is an enzyme that in humans is encoded by the NME1 gene (Gene ID = 4830). The term is also known as NDKA or NM23-H1. As used herein, the term “NME2” has its general meaning in the art and refers to the nucleoside diphosphate kinase B that is an enzyme that in humans is encoded by the NME2 gene (Gene ID = 4831). The term is also known as NDKB or NM23-H2. The NME protein family consists of 10 members in human cells, and NME family members have been shown to have a variety of diverse activities, including nucleoside diphosphate kinase (NDPK) activity, geranyl / farnesyl pyrophosphate kinase activity, and exonuclease activity. The gene for human NME2 is adjacent to the NME1 gene in the amplified chromosome 17q region, and human NME1 and NME2 share 88% sequence homology and, thus, have similar structural and functional attributes. Both NME 1 and NME2 have been found to have histidine kinase activity, catalyzing transfer of the activated phosphate from the autophosphorylated histidine 118 residue (Hl 18) onto target proteins (Lecroisey, Anne, et al. "Phosphorylation mechanism of nucleoside diphosphate kinase: 3 IP- nuclear magnetic resonance studies. "Biochemistry 34.38 (1995): 12445-12450).

[0025] As used herein, the term “NME1 / 2 inhibitor” refers to any compound natural or not which is capable of inhibiting the activity of NME1 and / or NME2, in particular NME1 and / or NME2 kinase activity. The term encompasses any NME1 and / or NME2 inhibitor that is currently known in the art or that will be identified in the future, and includes any chemical entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the NME1 and / or NME2. For instance, the term encompasses inhibitors of protein-protein interactions, such as inhibitors of BCL11A-NME1 / 2 interactions. The term also encompasses inhibitor of expression. In some embodiments, the NME1 / 2 inhibitor is selective over the other NME kinases such as NME3; NME4; NME5; NME6; NME7; NME8; NME9; or NME10. By “selective” it is meant that the inhibition of the selected compound is at least 10-fold, preferably 25-fold, more preferably 100-fold, and still preferably 300-fold higher than the inhibition of the other NME kinases. The NME1 / 2 inhibition of the compounds may be determined using various methods well known in the art.

[0026] In some embodiments, the NME1 / 2 inhibitor is a small organic molecule.

[0027] In some embodiments, the NME1 / 2 inhibitor is coenzyme A as described in Tossounian, Maria- Armineh, et al. "A Unique Mode of Coenzyme A Binding to the Nucleotide Binding Pocket of Human Metastasis Suppressor NME1." International Journal of Molecular Sciences 24.11 (2023): 9359. As used herein, the term “coenzyme A” has its general meaning in the art and refers to a coenzyme known for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle. The term is also known as CoA, SHCoA, CoASH. The IUPAC name of coenzyme A is [(2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-4-hydroxy- 3-(phosphonooxy)tetrahydro-2-furanyl]methyl (3R)-3-hydroxy-2,2-dimethyl-4-oxo-4-({3- oxo-3 -[(2-sulfanylethyl)amino]propyl}amino)butyl dihydrogen diphosphate. In some embodiments, the NME1 / 2 inhibitor is a derivative of coenzyme A. As used herein, the term “derivative” refers to a compound having a chemical structure that contains a common core chemical structure as a parent or reference compound (e.g. coenzyme A), but differs by having at least one structural difference, e.g., by having one or more substituents added and / or removed and / or substituted, and / or by having one or more atoms substituted with different atoms. For instance, coenzyme-A derivatives include but are not limited to acetyl-CoA, malonyl-CoA and HMG-Co A (3 -hydroxy-3 -methy Iglutaryl-Co A) .

[0028] In some embodiments, the NME1 / 2 inhibitor is an inhibitor of NME1 / 2 expression. An “inhibitor of expression” refers to a natural or synthetic compound that has a biological effect to inhibit the expression of a gene. In some embodiments, said inhibitor of gene expression is a siRNA, an antisense oligonucleotide or a ribozyme. For example, anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of NME1 and / or NME2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of NME1 and / or NME2, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding NME1 and / or NME2 can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small inhibitory RNAs (siRNAs) can also function as inhibitors of expression for use in the present invention. The NME1 and / or NME2 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that NME1 or NME2 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Antisense oligonucleotides, siRNAs, shRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically cells expressing NME1 or NME2. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0029] According to the invention, the NME1 / 2 inhibitor is administered to the patient in a therapeutically effective amount. By a "therapeutically effective amount" is meant a sufficient amount of the active ingredient for treating or reducing the symptoms at reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the active ingredients; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0030] Typically the active ingredient of the present invention (e.g. NME1 / 2 inhibitor) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms.

[0031] A further aspect of the invention relates to a method for screening a plurality of test substances useful for the treatment of a P-hemoglobinopathy in a patient in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit the activity or expression of NME1 or NME2 and (b) and positively selecting the test substances capable of said inhibition.

[0032] In some embodiments, the screening method of the present invention comprises the step of (i) providing a NME1 or NME2 protein; (ii) contacting the NME1 or NME2 protein with a test substance wherein the substance is expected to inhibit the kinase activity of the NME1 or NME2 protein; and (iii) selecting a test substance as a candidate that decreases the kinase activity of NME1 or NME2 in comparison to a negative control that is not contacted with a test substance.

[0033] In some embodiments, the screening method of the present invention comprises the step of (i) providing a NME1 or NME2 protein and a BCL11A protein; (ii) contacting the NME1 or NME2 protein with a test substance wherein the substance is expected to inhibit the interaction ofNMEl orNME2 with BCLUA; and (iii) selecting a test substance as a candidate that inhibits said interaction in comparison to a negative control that is not contacted with a test substance. Typically, NME1, NME2 protein and BCL11A come from various sources and sequences in the art may be used for the present disclosure as long as they remain functional.

[0034] In some embodiments, NME1 or NME2 protein is provided as a cell that endogenously or exogenously express the protein. For example, mammalian cells are prepared to express the protein of interest such as NME1 or NME2 through a transient or stable transfection or cells that endogenously express the protein of interest may be used. Cells endogenously expressing NME1 or NME2 may include but is not limited to endothelial cells. The cells obtained may be cultured in a cell culture dish and treated with a test substance for a certain period time in a suitable medium, from which the whole proteins are extracted and tested / detected for kinase activity of NME1 or NME2 protein. Alternatively established cell lines may be used, in which case the cells are transfected with a plasmid expressing NME1 or NME2. The example of such cells include but is not limited to 293, 293T or 293A (Graham F L, Smiley J, Russell W C, Nairn R (July 1977). “Characteristics of a human cell line transformed by DNA from human adenovirus type 5”. J. Gen. Virol. 36 (1): 59-74; and Louis N, Evelegh C, Graham F L (July 1997). “Cloning and sequencing of the cellular-viral junctions from the human adenovirus type 5 transformed 293 cell line”. Virology 233 (2): 423-9).

[0035] In some embodiments, the screening method of the present invention further comprises the steps of (a) testing each of the test substances for its ability to inhibit the phosphorylation on histidine 412 of BCL11A and (b) positively selecting the test substances capable of said inhibition. Typically, the detection of the phosphorylation on histidine 412 of BCL11A is performed as described in the EXAMPLE.

[0036] The term “test substance” refers generally to a material that is expected to decrease, reduce, suppress or inhibit the kinase activity or expression of NME1 or NME2, which include small molecules, high molecular weight molecules, mixture of compounds such as natural extracts or cell or tissue culture products, biological material such as proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamer, RNAzymes and DNAzymes, or glucose and lipids, but is not limited thereto. These materials are obtained from synthetic or natural compound libraries and the methods to obtain or construct libraries are known in the art. For example, synthetic chemical library may be obtained from Maybridge Chemical Co. (UK), Comgenex(USA), Brandon Asociates(USA), Microsource(USA) and Sigma-Aldrich(USA). The chemical library of natural origin may be obtained from Pan Laboratories (USA) and MycoSearch(USA). Further test substances may be obtained by various combinatorial library construction methods known in the art including for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries. Test substance of a library may be composed of peptides, peptoides, circular or liner oligomeric compounds, template based compounds such as benzodiazepine, hydantoin, biaryls, carbocyclic and polycyclic compounds such as naphthalene, phenothiazine, acridine, steroids and the like, carbohydrate and amino acid derivatives, dihydropyridine, benzhydryl and heterocyclic compounds such as triazine, indole, thiazolidine and the like, but does not limited thereto.

[0037] In some embodiments, the test substance is a coenzyme A derivative.

[0038] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0039] FIGURES:

[0040] Figure 1. Validation of BCL11A phosphorylation on histidine. BCL11A was immunoprecipitated in UT7epo cell line and its phosphorylation analyzed by western blot.

[0041] Figure 2. NME1 histidine kinase knock down induces the expression of fetal globin. NME1 and NME2 histidine kinases were knocked down by CRISPR / Cas9 in HUDEP2 erythroid cell line. The expression of fetal hemoglobin was analyzed two and four days after the induction of erythroid differentiation.

[0042] Figure 3. Control of NME1 and NME2 depletion in HUDEP-2 cell line. NME1 and NME2 were depleted in HUDEP-2 cell line using CRISPR / Cas-9 and cloned by FACS single cell sorting. (A) The invalidation was tested using Sanger sequencing by Eurofin Genomics© and analysis by ICE anlysis of Synthego©. (B) The invalidation was tested using Mass spectrometry approach with unique peptide quantification. Figure 4. HUDEP-2 cells depleted for NME1 or NME2 are able to differentiate until orthochromatic stage, as for control undepleted cells. NME1 and NME2 were depleted in HUDEP-2 cell line using CRISPR / Cas-9 and cloned by FACS single cell sorting. (A) CD49d and Band3 cell differentiation markers were followed by flow cytometry. (B) Cell differentiation was followed by May-Grunwald Giesma staining, n = 2.

[0043] Figure 5. Depletion of NME1 or NME2 by CRISPR / Cas9 does not induce an increase in cell death. NME1 and NME2 were depleted in HUDEP-2 cell line using CRISPR / Cas-9. Dead and alive cells are counted by trypan blue staining. The calculation of the percentage of blue stained cells is shown, n = 2.

[0044] EXAMPLE:

[0045] Material & methods:

[0046] Cell line Culture

[0047] HUDEP-2 Cell line culture

[0048] HUDEP-2 cells were cultured and amplified in IMDM medium with 15% BIT, EPO 3UI / mL, hSCF lOOng / mL, Doxycyclin Ipg / mL, Dexamethasone l,0xl0-eM, 0.5% Penicilin-Strepavidin and 0.5% L-Glutamin. 8 Million cells per condition were washed and put in differentiation in IMDM medium with 5% AB human serum, EPO 3UI / mL, Insulin lOpg / mL, Holo-transferrin 330pg / mL, hSCF lOOng / mL, heparin 2U / mL, Doxycyclin Ipg / mL, 1% Penicilin-Strepavidin, 1% L-Glutamin.

[0049] UT7epo cell line culture

[0050] UT7epo cells were cultured and amplified in MEMa medium with 5% SVF, EPO 2UI / mL, 0.5% Penicilin-Strepavidin, and 0.5% L-Glutamin.

[0051] Sample preparation for nLC-MS / MS proteomic analysis of histidine phosphorylations

[0052] UT7epo cell line was deprived overnight and restimulated by EPO 5 U / ml during 15 minutes. PBS was used as a control. Cells were then wash 2 times in cold PBS, frozen as a pellet at - 80°C. Cells (the equivalent of 2mg of proteins) were resuspended on ice in lysis buffer (lOOmM Tris-HCl pH8.5, 1% Triton X-100, 1% Sodium Deoxycholate, 7M Uree, 1 mM MgC12) with an extemporaneously addition of 5mM TCEP and 30 mM CAA for protein reduction and alkylation and ImM Sodium Orthovanadate, PhosSTOP phosphatase inhibitor, complete EDTA-free protease inhibitors and benzonase. Cell lysate were sonicated during 45min in a Bioruptor sonicator (Diagenode) 20 sec on, 40 sec off. Cell lysate were then centrifuged 25min at 25000 g at 4°C. Protein precipitation were performed by the sequential dilution of supernatant in 1 :4 Methanol, 1 :3 Chloroform and 1 :5 ultrapure water. After each dilution, samples were thoroughly vortexed. Samples were then centrifuged 15min a 3220 g at room temperature. The upper phase was discarded and precipitated proteins were washed twice in Methanol. Cell pellets were resuspended in 100 mM TrisHCl pH8.5 and digested overnight in 200pg trypsin (Worthington) for 2mg of proteins. Histidine phosphorylated peptides were then enriched on Strong Anion Exchange batch columns (Hypersep SAX Spe Column 200mg, Thermo Scientific) using a batch adapted protocol from Hardman et al., Meth Mol Biol., 2019 1,2. Briefly, Samples were diluted in Buffer A (Ammonium acetate, 20 mM, pH 6.8, 10% CAN), put on the cartridges and fractionated in 10 fractions using an increasing concentration of Buffer B (Trirthyammonium phosphate pH6.8, 10% ACN) from 0% to 100% in a linear way. Peptides were lyophilized, solubilized in ultrapure water and desalted on C18 reverse phase cartridges (Interchim) at 4°C and without acid. Loaded peptides were washed 2 times in 100 pl water and eluted in 50% ACN. nLC-MS / MS proteomic analysis of histidine phosphorylations

[0053] Peptides from each of these five fractions were solubilized in 5% acetonitrile (ACN) and loaded, concentrated, and washed for 3min on a C18 reverse phase column (5pm particle size, 100 A pore size, 300 pm inner diameter, 0.5 cm length, from Thermo Fisher Scientific) with loading solvent containing 5% ACN. Peptides were then separated by C18 rapid separation liquid chromatography (RSLC) Dionex U3000 on an Aurora C18 reverse phase column (1.6 pm particle size, 100 Angstrom pore size, 75pm inner diameter, 25cm length) with a 2h binary gradient from 99% solution A (0.1% formic acid in H2O) to 55% solution B (80% ACN, 0.085% formic acid) before injection into an Orbitrap Fusion mass spectrometer (from Thermo Scientific).

[0054] The Orbitrap Fusion mass spectrometer acquired data throughout the elution process and operated in a data-dependent scheme with a cycle time of 5 s with full mass spectrometry (MS) scans acquired with the orbitrap detector, followed by HCD fragmentation and Ion trap fragment detection of the most abundant ions detected in the MS scan. Mass spectrometer settings for full scan MS were: 2.0Ee AGC, 60,000 target resolution, 350-1500 mlz range, maximum injection time of 60 ms. HCD MS / MS fragmentation was allowed for 2+ to 7+ precursor ions reaching more than 5. OEs minimum intensity. Quadrupole-filtered precursors within 1.6 mlz isolation window were fragmented with a Normalised Collision Energy of 30. The limiting ions accumulation values were 2.0E4 AGC Target and 100 ms maximum injection time. The Ion-trap detector was used for its fast and sensitive detection capabilities. A 30 s dynamic exclusion time was set. nLC-MS / MS proteomic data analysis

[0055] Maxquant software version 1.6.17.0 (Cox J et al., Nat Protoc, 2009 3, Cox J et al., Mol Cell Prot, 2014 Q was used for the analysis of raw data from mass spectrometer. The database used was a concatenation of human sequences from the Uniprot-Swissprot database (release 2020- 03) and the list of contaminant sequences adapted from Maxquant. The enzyme specificity was trypsin / P. Carbamidomethylation of cysteins was set as constant modification and acetylation of protein N-terminus and oxidation of methionines were set as variable modifications as well as phosphorylation on serine, threonine, tyrosine, histidine, cysteine, lysine, arginine, aspartic acid, and glutamic acid. Second peptide search was allowed and minimal length of peptides was set at 6 amino acids. False discovery rate (FDR) was kept below 1% on both peptides and proteins and below 5% for PSM. Label-free protein quantification (LFQ) was done using both unique and razor peptides. At least 2 peptidic ratios were required for LFQ quantification. The “match between runs” (MBR) option was allowed with a match time window of 0.7 min and an alignment time window of 20 min.

[0056] BCL11A immunoprecipitation

[0057] Cell pellet of 10 million UT7epo cells were lysed by lysis buffer (lOOnM NaCl, 25mM Tris HC1 pH: 8.8, EDTA ImM, 10% Glycerol, phosphatase and protease inhibitors IX, 0.3% NP40), incubated on ice, and centrifuged 15min at 14000rpm. Supernatants were collected. Magnetic beads (G protein for immunoprecipitation Dynabeads™, ThermoFisher, 10003D) were used for immunoprecipitation. G protein beads were washed three time with cold PBS. For each condition 30pL of G protein were added to the supernatants and incubated Ihour at 4°C on a spinning wheel. Supernatants were collected and incubated with 2pg of primary antibodies for 1 hour on cold ice. 15pL of Beads were added and incubated for lh30 at 4°C. Supernatants were discarded and beads were washed four times with lysis buffer, and one time with PBS. 75pL of Triethylamine (TEA) and incubated 15min on ice. Beads were discarded. 5pL of the immunoprecipitation products were sampled and mixed with 5pL of Laemli 2X pH:8.8 for western blotting. Protein soluble fractions, and non-fixated fractions and immunoprecipitation product were also collected for western blotting.

[0058] Western blot

[0059] Western Blot for histidine phosphorylation analysis of immunoprecipitated BCL11A

[0060] Immunoprecipitation products were assessed using SDS 10% electrophoresis gel (10% Mini- PROTEAN® TGX Stain-Free™ Protein Gels, 15 well, 15 pl, 4568036) for migration Gel runned in IX migration buffer (lOx Tris / Glycine / SDS, 1610732), and were transferred overnight in liquid transfer system using transfer buffer (Tris 25mM, 0.1% SDS, 192mM glycine, 20% Ethanol) and nitrocellulose membrane (nitrocellulose membrane 0.2pM,30cmx3.5M, 1620112). Membranes were then blocked in PBS-tween 5% milk buffer for 1 hour and incubated overnight with primary antibody anti-BCLUA (Abeam, Ab 191401, 1 : 10000), and anti-l-pHIS (Merck, MABS1330, 0.3pg / mL), anti-3-pHIS (Merck, MABS1352, 0.52pg / mL), and anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1 : 1000). Membranes were incubated with secondary goat antibodies for BCL11 A, 1-pHIS and 3-pHIS (Southern biotech, 4050-05), and rat antibodies for HSC70. Signals were revealed using Substrat chimiluminescent SuperSignal™ West Pico PLUS (Pierce, XI352070).

[0061] Western blot for hemoglobin expression

[0062] Cells for each clone of the NME1 / 2 KO were differentiated and 500pL of medium was collected at day 2, 4, 6 and 8 of differentiation. Cells were lysed and proteins were quantified using BCA quantification. 12.5pg of protein were analyzed using a SDS 10% electrophoresis gel (10% Mini-PROTEAN® TGX Stain-Free™ Protein Gels, 15 well, 15 pl, 4568036) for migration Gel runned in IX migration buffer (lOx Tris / Glycine / SDS, 1610732), and were transferred overnight with liquid transfer system using transfer buffer (Tris 25mM, 0.1% SDS, 192mM glycine, 20% Ethanol) and nitrocellulose membrane (nitrocellulose membrane 0.2pM,30cmx3.5M, 1620112). Membranes were then blocked in PBS-tween 5% milk buffer for 1 hour and incubated overnight with primary antibody anti-HBB (Santa Cruz Biotechnology, Sc21757), anti-HBF (Santa Cruz Biotechnology, Sc21756), and anti-HSC70 (Enzo Life Sciences, ADLSPA-815, 1 : 1000). Membranes were incubated with secondary goat antibodies for anti-HBB and anti-HBF (Southern biotech, 4050-05), and rat antibodies for HSC70. Membranes bands were revealed using Substrat chimiluminescent SuperSignal™ West Pico PLUS (Pierce, XI352070). Results:

[0063] Example 1

[0064] Phosphorylations are among the most common and the most studied post translational modifications of proteins. Commonly studied phosphorylations are on serine, threonine, and tyrosine because of their lower lability level compared to other phosphorylated amino acids. In eukaryotes, 9 amino acids can be phosphorylated, forming in total four types of phosphates- protein link, including histidine phosphorylations, first reported in 1962 (Boyer, P. D., DeLuca, M., Ebner, K. E., Hultquist, D. E., & Peter, J. B. (1962). Identification of phosphohistidine in digests from a probable intermediate of oxidative phosphorylation. Journal of Biological Chemistry, 237(10), PC3306-PC3308). This phosphorylation is estimated to represent 6% of the total phosphorylations in eukaryotes. However, until recently, there has been little to no records of research on this phosphorylation, in eukaryotes. This is primarily due to the lack of phosphoprotective techniques, that would prevent the high lability of the phosphorylation. Yet, since 2015, new protocols and tools have been developed allowing histidine phosphorylation study (Fush et al., Cell, 2015). The first published studies show that histidine phosphorylations are involved in hepatocellular carcinoma (Hindupur S. et al., Nature, 2018), eosophageal squamous cell carcinoma (Zang J., et al., Oncogene, 2023) and in stress induces depressionlike behaviours (Lin D., et al, Mol Psychiatry, 2023).

[0065] On Proteom’IC platform of Institut Cochin, we set up a modified protocol, leading to a better yield for the proteomic analysis of histidine phosphorylations in a whole proteome. This protocol allowed us to identify a phosphorylation on histidine 412 of BCL11A with a localization probability of 0.86 and a Posterior Error Probability (PEP) of 0.070 (Table 1). This histidine is localized in its zinc finger domain involved in its binding to DNA and thus to the repression of fetal (Gamma) and the switch of Gamma to Alpha globin (Liu, N. et al. Cell, 2018). Histidine phosphorylation was confirmed by immunoprecipitation of BCL11A following by the detection of histidine phosphorylation by Western blot (Figure 1). Furthermore, the knock down of NME1 and NME2, the two histidine kinases identified in mammals, by CRISPR / Cas9 in HUDEP2 erythroid cell line induces the expression of fetal hemoglobin expression, suggesting that NME1 / 2 could be involved in fetal to adult globin switch (Figure 2) probably through the regulation of histidine phosphorylation of BCL11 A. Table 1. Characteristics of histidine phosphosites characterization.

[0066] Example 2

[0067] NME1 and NME2 were depleted in HUDEP-2 cell line using CRISPR / Cas-9 and cloned by FACS single cell sorting. After the validation of 3 different clones for NME1 and for NME2 depletion or decrease by Sanger sequencing and mass spectrometry protein LFQ quantification using unique peptides (figure 3), the impact of the depletions on cell differentiation (Figure 4) and cell survival (Figure 5) was analyzed by flow cytometry and MGG staining. Our data show that the HUDEP2 cell line CRISPR KO for NME1 or NME2 alone is still able to differentiate until the orthochromatic stage, as for undepleted control. Furthermore, we did not observe any increase in the number of dead cells.

[0068] REFERENCES:

[0069] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0070] 1. Hardman, G. et al. Strong anion exchange - mediated phosphoproteomics reveals extensive human non - canonical phosphorylation. EMBO J. 38, el 00847 (2019).

[0071] 2. Hardman, G. & Eyers, C. E. High-Throughput Characterization of Histidine Phosphorylation Sites Using UP AX and Tandem Mass Spectrometry, in Histidine Phosphorylation (ed. Eyers, C. E.) vol. 2077 225-235 (Springer US, 2020). 3. Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC -based quantitative proteomics. Nat. Protoc. 4, 698-705 (2009).

Claims

CLAIMS:

1. A method of treating a P-hemoglobinopathy in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a NME1 / 2 inhibitor.

2. The method of claim 1 wherein the patient suffers from sickle cell disease or P- thalassemia.

3. The method according to claim 1 or 2 wherein the NME1 / 2 inhibitor increased the fetal hemoglobin content in the red blood cells of the patient.

4. The method according to any one of claims 1 to 3 wherein the NME1 / 2 inhibitor inhibits the phosphorylation on histidine 412 of BCL11 A, thereby increasing the expression of y-globin.

5. The method according to any one of claims 1 to 4 wherein the NME1 / 2 inhibitor is coenzyme A or a coenzyme A derivative.

6. The method according to any one of claims 1 to 4 wherein the NME1 / 2 inhibitor is an inhibitor of expression that blocks the translation of NME1 and / or NME2 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the expression level of NME1 and / or NME2.

7. The method of claim 6 wherein the inhibitor of expression is siRNA, an antisense oligonucleotide or a ribozyme.

8. A method for screening a plurality of test substances useful for the treatment of a P- hemoglobinopathy in a patient in need thereof comprising the steps consisting of (a) testing each of the test substances for its ability to inhibit the activity or expression of NME1 or NME2 and (b) and positively selecting the test substances capable of said inhibition.

9. The screening method of claim 8 that comprises the step of (i) providing a NME1 or NME2 protein; (ii) contacting the NME1 or NME2 protein with a test substance wherein the substance is expected to inhibit the kinase activity of the NME1 or NME2 protein; and (iii) selecting a test substance as a candidate that decreases the kinase activity ofNME1 or NME2 in comparison to a negative control that is not contacted with a test substance.

10. The screening method of claim 8 that comprises the step of (i) providing a NME1 or NME2 protein and a BCL11 A protein; (ii) contacting the NME1 or NME2 protein with a test substance wherein the substance is expected to inhibit the interaction of NME1 orNME2 with BCL11 A; and (iii) selecting a test substance as a candidate that inhibits said interaction in comparison to a negative control that is not contacted with a test substance.

11. The screening method according to any one of claims 8 to 10 that further comprises the steps of (a) testing each of the test substances for its ability to inhibit the phosphorylation on histidine 412 of BCL11 A and (b) positively selecting the test substances capable of said inhibition.

12. The screening method according to any one of claims 8 to 11 wherein the test substance is a coenzyme A derivative.

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