Methods for inducing complete ablation of hematopoiesis

Abstract

We identified autosomal dominant (AD) missense mutations in the RAC2 gene (encoding Ras-associated C3 botulinum toxin substrate 2 (RAC2)) in three severe combined immunodeficiency (SCID) patients whose clinical symptoms overlap with those of the SCID form of RD but lack AK2 mutations and hearing loss. Using biochemical and in vitro differentiation assays, we demonstrated that RAC2 mutations are closely associated with impaired cell differentiation capacity and defects in cellular and mitochondrial networks. Overall, this data demonstrates that dominant gain-of-function (GOF) mutations in the GDP / GTP-binding site of the RAC2 protein inhibit HSPC differentiation, leading to a severe AD form of SCID with clinical symptoms of RD. Therefore, these results prompted us to consider that introduction of the identified RAC2 muteins in the hematopoietic lineage may be appropriate to induce complete ablation of hematopoiesis.

Description

[Technical field]

[0001] FIELD OF THEINVENTION

[0002] The present invention is in the field of hematology.

[0003] 2. Background of the Invention

[0004] Hematopoietic stem cell transplantation (HSCT) is a potentially curative therapeutic approach for a variety of malignant and non-malignant hematopoietic disorders. When HSCT is performed in patients with malignant disorders, a preparative or conditioning regimen is administered as part of the procedure to achieve three goals: create space in the bone marrow (myeloablative), provide sufficient immune destruction to prevent graft rejection, and reduce tumor burden. The intensity of conditioning regimens varies greatly, and selecting the optimal conditioning regimen for any given patient requires consideration of disease-related factors, such as diagnosis and remission status, as well as patient-related factors, including age, donor availability, and the presence of comorbidities. Although complete consensus has not been reached within the HCT community, conditioning regimens have been classified as high-dose (myeloablative), reduced intensity, and non-myeloablative. For example, myeloablative, or "high-dose," regimens consist of alkylating agents (single or multiple agents) with or without total body irradiation and are expected to abolish hematopoiesis and not allow for autologous hematologic recovery. The administration of conditioning regimens is associated with immediate and delayed toxicity. For example, nausea, vomiting, transient acute parotitis, xerostomia, mucositis, and diarrhea are commonly observed acute complications. Interstitial pneumonia, idiopathic pulmonary fibrosis, and reduced pulmonary function may be observed. The occurrence of sick sinus syndrome (also known as hepatic veno-occlusive disease) is more common in chemotherapy-based regimens. Long-term side effects include infertility, cataract formation, hypothyroidism and thyroiditis, and secondary malignant lesions. Therefore, there is a need for new methods that allow complete ablation of hematopoiesis with minimal occurrence of short-term and long-term severe side effects.

[0005] RAC2 belongs to the Rac subfamily of the Rho family of small GTPases. In the inactive GDP-bound state, RAC2 is localized in the cytoplasm. Upon stimulation and activation by guanine nucleotide exchange factors, the active RAC2-GTP-bound form translocates to the plasma membrane. There, RAC2 triggers various signaling pathways until the GTP is hydrolyzed or the GTPase is degraded by the cell. 7-9 Unlike other members of the Rac subfamily (RAC1 and RAC3), expression of RAC2 is restricted to the hematopoietic lineage. 10,11 .

[0006] Summary of the Invention

[0007] As defined by the claims, the present invention relates to a method for inducing complete ablation of hematopoiesis and its use in conditioning for bone marrow transplantation and in the treatment of hematopoietic cell malignancies.

[0008] Detailed Description of the Invention

[0009] We have identified autosomal dominant (AD) missense mutations in the RAC2 gene (encoding Ras-associated C3 botulinum toxin substrate 2 (RAC2)) in three severe combined immunodeficiency (SCID) patients whose clinical symptoms overlap with the RD SCID form, but lack AK2 mutations and hearing loss. Using biochemical and in vitro differentiation assays, we demonstrated that RAC2 mutations are closely associated with impairments in cell differentiation capacity and defects in cellular and mitochondrial networks. Overall, the data demonstrate that dominant gain-of-function (GOF) mutations in the GDP / GTP-binding site of the RAC2 protein inhibit HSPC differentiation, leading to a severe AD form of SCID with clinical symptoms of RD. Thus, the results prompted us to consider that the introduction of the identified RAC2 muteins in the hematopoietic lineage may be appropriate to induce complete ablation of hematopoiesis.

[0010] A first object of the present invention relates to a method for completely ablating hematopoiesis in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated.

[0011] A further object of the present invention relates to a method for inhibiting the proliferation and differentiation of a population of hematopoietic stem cells comprising contacting said population with an effective amount of i) a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated.

[0012] A further object of the invention relates to a method of inducing cell death in a population of hematopoietic cells comprising contacting said population with an effective amount of i) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated.

[0013] A further object of the invention relates to a method of inducing cell death of a population of malignant hematopoietic cells, comprising contacting said population with an effective amount of i) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated.

[0014] A further object of the present invention relates to a method for treating a hematopoietic cell malignancy in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of i) a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated, or ii) a polynucleotide encoding a polypeptide comprising the amino acid sequence as shown in SEQ ID NO:1, wherein the amino acid residue at position 12 (G) is mutated.

[0015] As used herein, the term "hematopoietic cell" has its general meaning in the art and refers to any type of cell of the hematopoietic system, including, but not limited to, undifferentiated cells, such as hematopoietic stem and progenitor cells, and differentiated cells, such as white blood cells (e.g., granulocytes, monocytes, and lymphocytes), platelets, and red blood cells.

[0016] As used herein, the term "hematopoietic stem cells" or "HSC" refers to blood cells that have the capacity to self-renew and to differentiate into precursors of circulating mature blood cells. These progenitor cells are immature blood cells that are unable to self-renew and differentiate into circulating mature blood cells. Within the bone marrow microenvironment, stem cells self-renew and maintain a continuous production of hematopoietic cells that give rise to all mature blood cells throughout life. In some embodiments, hematopoietic progenitor cells or hematopoietic stem cells are isolated from peripheral blood cells.

[0017] As used herein, the term "hematopoiesis" refers to the formation and development of hematopoietic cells, including proliferation and / or differentiation from stem cells.

[0018] Typically, the patient is selected from the group consisting of children, young adults, middle-aged adults, and elderly adults.

[0019] In some embodiments, the method of the present invention is particularly suitable for preparing (i.e., conditioning) a patient for bone marrow transplantation. Thus, the method of the present invention is particularly suitable for avoiding the use of chemotherapy and radiation therapy. The method of the present invention can help bone marrow to grow new blood stem cells, but also prevent the patient's body from rejecting the transplanted cells and help kill any cancer cells that may be present in the body. Typically, administration of the polypeptide or polynucleotide is performed before bone marrow transplantation.

[0020] As used herein, the terms "bone marrow transplant" or "hematopoietic stem cell transplant" should be considered interchangeable and refer to the transplantation of hematopoietic stem cells in a particular form to a recipient. Hematopoietic stem cells do not necessarily have to be derived from bone marrow, but can also be derived from other sources, such as umbilical cord blood or mobilized PBMCs. As used herein, the term "hematopoietic stem cell transplant" or "HSCT" refers to a component of the treatment of a wide range of blood disorders. In general, there are two types of HSCT: autologous transplant and allogeneic transplant. As used herein, the term "allogeneic" refers to being derived from, originating from, or being members of the same species, where the members are genetically related or unrelated but genetically similar. "Allogeneic transplant" refers to the transfer of cells or organs from a donor to a recipient, where the recipient is of the same species as the donor. Allogeneic transplant typically involves the infusion of donor stem cells using a donor that is MHC-matched to the recipient. As used herein, the term "autologous" refers to derived or originating from the same patient. "Autologous transplant" refers to the collection and re-implantation of a patient's own cells or organs.

[0021] Examples of hematopoietic cell malignancies that are cancer include leukemia, lymphoma, and multiple myeloma. Examples of leukemia include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myelogenous leukemia (CML). Examples of lymphomas include Hodgkin's disease and its subtypes; non-Hodgkin's lymphoma and its subtypes, including chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), mantle cell lymphoma (MCL), hairy cell leukemia (HCL), marginal zone lymphoma (MZL), Burkitt's lymphoma (BL), post-transplant lymphoproliferative disorder (PTLD), T-cell prolymphocytic leukemia (T-PLL), B-cell prolymphocytic leukemia (B-PLL), Waldenstrom's macroglobulinemia / lymphoplasmatic cell lymphoma, and other natural killer cell (NK cell) or T-cell lymphomas. Examples of other malignant conditions that are hematopoietic cell malignancies include myelodysplastic syndromes (MDS); myeloproliferative disorders such as polycythemia vera (i.e., PV, PCV, or primary polycythemia vera (PRV)), essential thrombocythemia (ET), myelofibrosis; and disorders with features of both myelodysplastic syndromes and myeloproliferative disorders (e.g., chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), atypical chronic myelogenous leukemia (aCML), and myelodysplastic / myeloproliferative disorders).

[0022] In particular, acute myeloid leukemia (AML) hematopoietic cell malignancies are affected by the mutations described herein.

[0023] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatments as well as curative or disease-modifying treatments, including treatment of patients at risk of or suspected of having a disease as well as patients diagnosed as ill or suffering from a disease or medical condition, including suppression of clinical recurrence. The treatment may be administered to a patient who has a medical disorder or may eventually acquire a disorder, to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurrent disorder, or to extend the survival of the patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant a pattern of treatment of a disease, e.g., a pattern of dosing used during treatment. 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 a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (in part or in whole) a "loading regimen", which may involve administering a larger dose of the drug than the physician may use during a maintenance regimen, administering the drug more frequently than the physician may administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used for the maintenance of a patient during disease treatment, e.g., to keep the patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous treatment (e.g., administering a drug at regular intervals, such as weekly, monthly, yearly, etc.) or intermittent treatment (e.g., interrupted treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of a certain predetermined criterion (e.g., disease manifestation, etc.).

[0024] As used herein, the term "Rac2" has its general meaning in the art and refers to Ras-related C3 botulinum toxin substrate 2. An exemplary amino acid sequence for human Rac2 is represented by SEQ ID NO:1. SEQ ID NO:1 >sp|P15153|Rac2_human Ras-related C3 botulinum toxin substrate 2 OS=Homo sapiens OX=9606 GN=Rac2 PE=1 SV=1 MQAIKCVVVGD G AVGKTCLLISYTTNAFPGEYIPTVFDNYSANVMVDSKPVNLGLWDTAGQEDYDRLRPLSYPQTDVFLICFSLVSPASYENVRAKWFPEVRHHCPSTPIILVGTKLDLRDDKDTIEKLKEKKLAPITYPQGLALAKEIDSVKYLECSALTQRGLKTVFDEAIRAVLCPQPTRQQKRACSLL

[0025] As used herein, the term "mutation" has its general meaning in the art and refers to substitution, deletion, or insertion. The term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. The term "deletion" means that a specific amino acid residue is removed. The term "insertion" means that one or more amino acid residues are inserted before or after a specific amino acid residue, more specifically, that one or more, preferably one or several, amino acid residues are attached to the a-carboxyl or a-amino group of the specific amino acid residue.

[0026] In some embodiments, the amino acid residue at position 12 (G) is substituted. In some embodiments, the amino acid residue at position 12 (G) is substituted with an amino acid residue (R).

[0027] As used herein, the term "polypeptide" has its general meaning in the art and refers to a polymer of amino acids of any length. A polymer can include modified amino acids. The term also includes amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides that include, for example, one or more analogs of amino acids (including, for example, unnatural amino acids, such as homocysteine, ornithine, p-acetylphenylalanine, D amino acids, and creatine, etc.), as well as other modifications known in the art.

[0028] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogs, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-, double-, and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double-, and single-stranded ribonucleic acid ("RNA"). It also includes modified, e.g., by alkylation and / or by capping, and unmodified forms of polynucleotides. In particular, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose) (including tRNA, rRNA, hRNA, siRNA, and mRNA) (whether spliced ​​or unspliced), any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base, and other polymers that contain normucleotidic backbones, such as polyamides (e.g., peptide nucleic acids, "PNAs") and polymorpholino polymers, as well as other synthetic sequence-specific nucleic acid polymers (provided that the polymer contains nucleobases in an arrangement that allows for base pairing and base stacking as found in DNA and RNA). In some embodiments, the polynucleotide includes an mRNA. In other aspects, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA contains at least one unnatural nucleobase. In some embodiments, all nucleobases of a particular class are substituted with non-natural nucleobases (e.g., all uridines in the polynucleotides disclosed herein can be substituted with non-natural nucleobases, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., synthetic RNA or DNA) contains only natural nucleobases, i.e., A, C, T, and G in the case of synthetic DNA, or A, C, T, and U in the case of synthetic RNA.

[0029] In some embodiments, a polynucleotide of the invention is messenger RNA (mRNA).

[0030] In some embodiments, the polynucleotide is inserted into a vector (such as a plasmid, cosmid, episome, artificial chromosome, phage, or viral vector). Typically, the vector is a viral vector that is an adeno-associated virus (AAV), retrovirus, bovine papilloma virus, adenovirus vector, lentivirus vector, vaccinia virus, polyoma virus, or infectious virus. Typically, the vector of the present invention includes "control sequences", which collectively refer to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, etc., which collectively provide for the replication, transcription, and translation of the coding sequence in the recipient cell. Not all of these control sequences need always be present, so long as the selected coding sequence is capable of being replicated, transcribed, and translated in a suitable host cell. Another nucleic acid sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region that contains DNA regulatory sequences, where the regulatory sequences are derived from a gene that are capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding sequence. Transcriptional promoters can include "inducible promoters" (wherein expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (wherein expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters."

[0031] In some embodiments, the polypeptide or polynucleotide of the present invention can be conjugated to at least one other molecule. Typically, the molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, sugars, vitamins, cofactors, and drugs. In some embodiments, the polypeptide or polynucleotide of the present invention is conjugated to a molecule having a specific affinity for hematopoietic cells, such as an antibody or peptide having binding affinity for a protein expressed on the surface of hematopoietic stem cells. The conjugation can provide increased stability and / or half-life, which can be particularly useful in targeting the polypeptide or polynucleotide to hematopoietic cells.

[0032] By "therapeutically effective amount" is meant a sufficient amount of active ingredient to treat or reduce symptoms, at a 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 determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed, the age, 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 treatment; drugs used in combination with the active ingredient; and similar factors well known in the medical art. 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 gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product 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 active ingredient for symptomatic adjustment of dosage to the subject being treated. Medicaments typically contain from about 0.01 mg to about 500 mg of active ingredient, typically from 1 mg to about 100 mg of active ingredient. An effective amount of the drug is usually supplied at a dosage level of from about 0.0002 mg / kg to about 20 mg / kg of body weight per day, particularly from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0033] Typically, the active ingredient (i.e., polypeptide or polynucleotide) of the present invention is combined with a pharma- ceutically acceptable excipient and, optionally, a sustained release matrix, such as a biodegradable polymer, to form a pharmaceutical composition. The term "pharmaceutical" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other unpleasant reactions when administered appropriately to a mammal, particularly a human. A pharma- ceutically acceptable carrier or excipient refers to any type of non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, or formulation auxiliary.

[0034] In some embodiments, the polypeptide or polynucleotide of the present invention is formulated with a lipidoid, the synthesis of which has been extensively described (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci US A. 2011 108:12996-3001). While these lipidoids have been used to effectively deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010), the present disclosure describes their formulation and use in the delivery of polynucleotides.

[0035] In some embodiments, a polypeptide or polynucleotide of the invention is formulated using one or more liposomes, lipoplexes, or lipid nanoparticles.

[0036] Liposomes are artificially prepared vesicles that may be composed primarily of lipid bilayers and can be used as delivery vehicles for administration of pharmaceutical formulations. Liposomes can be of different sizes, including but not limited to multilamellar vesicles (MLVs), which may include a series of concentric bilayers separated by narrow aqueous compartments, which may be hundreds of nanometers in diameter, small unilamellar vesicles (SUVs), which may be smaller than 50 nm in diameter, and large unilamellar vesicles (LUVs), which may be between 50 and 500 nm in diameter. Liposome designs can include opsonins or ligands to improve attachment of liposomes to unhealthy tissues or to activate events, such as, but not limited to, endocytosis. Liposomes can include low or high pH to improve delivery of pharmaceutical formulations. As non-limiting examples, liposomes, such as synthetic membrane vesicles, are prepared by the methods, apparatus, and devices described in U.S. Patent Publication Nos. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373, and US20130183372. In some embodiments, liposomes are formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (described in US20100324120) and liposomes capable of delivering small molecule drugs (such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA)).The polypeptides of the polynucleotides of the invention can be encapsulated by liposomes and / or can be contained in an aqueous core which can then be encapsulated by liposomes (see International Publication Nos. WO2012031046, WO2012031043, WO2012030901, and WO2012006378 and U.S. Patent Publication Nos. US20130189351, US20130195969, and US20130202684).

[0037] In some embodiments, the polynucleotides of the invention are formulated using stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (Wheeler et al. Gene Therapy. 1999 6:271-281; ​​Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114; Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 441:111-114). 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; US Patent Publication No. US20130122104). The original manufacturing method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as a spontaneous vesicle formation method. The liposome formulation is composed of 3-4 lipid components in addition to the polynucleotide. As an example, the liposomes can contain, but are not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleoxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, a particular liposome formulation includes, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, which can be 1,2-distearoyl-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinoleoxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

[0038] In some embodiments, the polynucleotides of the invention are formulated in lipid nanoparticles, such as those described in International Publication No. WO2012170930. Lipid nanoparticle formulations typically include lipids, particularly ionizable cationic lipids, and further include neutral lipids, sterols, and molecules capable of reducing particle aggregation, such as PEG or PEG-modified lipids. The lipids can be selected from, but are not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and aminoalcohol lipids. In some embodiments, the lipids are cationic lipids, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipids. The aminoalcohol cationic lipids can be lipids described in U.S. Patent Publication No. US20130150625 and / or can be made by the methods described therein. As non-limiting examples, cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in USC20130150625); -[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 in US20130150625); or any pharma- ceutically acceptable salt or stereoisomer thereof.The nanoparticle formulations of the present disclosure can be coated with surfactants or polymers to improve particle delivery. In some embodiments, the nanoparticles are coated with hydrophilic coatings, such as, but not limited to, PEG coatings and / or coatings with neutral surface charges. Hydrophilic coatings can help deliver nanoparticles with larger payloads, such as, but not limited to, polynucleotides, into the central nervous system. As a non-limiting example, nanoparticles with hydrophilic coatings and methods for making such nanoparticles are described in US Patent Publication No. US20130183244.

[0039] The present invention is further illustrated by the following figures and examples, which should not, however, be construed in any way as limiting the scope of the present invention. [Brief description of the drawings]

[0040] [Figure 1A]Identification of the p.G12R RAC2 mutation and its effect on the GTPase activity of RAC2. A. Pedigree of three patients from two unrelated kindreds. Closed boxes and circles represent affected males (P1) and females (P2, P3), respectively. Open boxes and circles represent unaffected individuals. The arrow represents the proband and the two horizontal bars represent relatives. B. Representative electropherograms of RAC2 DNA sequencing on control and patient cells showing the c.34G>A mutation. C. Representative immunoblots of RAC2 protein expression in lysates of control fibroblasts (C1, C2) and fibroblasts derived from affected individuals (P1, P2, and P3). Loading control corresponds to GADPH expression. D. HEK293T cells were either untransduced (NT, control) or transduced with a lentiviral empty vector (WPI) containing the wild-type (WT) form of RAC2 cDNA, the mutant form described herein (G12R), or (as a positive control) the constitutively activated RAC2 GTP form (G12V). Two days after transduction, cells were harvested for analysis using the G-LISA assay (15 μg total protein per well). Results are shown as the mean ± standard error of the mean (SEM) of three independent experiments (***p<0.001; ****p<0.0001). [Figure 1B]Identification of the p.G12R RAC2 mutation and its effect on the GTPase activity of RAC2. A. Pedigree of three patients from two unrelated kindreds. Closed boxes and circles represent affected males (P1) and females (P2, P3), respectively. Open boxes and circles represent unaffected individuals. The arrow represents the proband and the two horizontal bars represent relatives. B. Representative electropherograms of RAC2 DNA sequencing on control and patient cells showing the c.34G>A mutation. C. Representative immunoblots of RAC2 protein expression in lysates of control fibroblasts (C1, C2) and fibroblasts derived from affected individuals (P1, P2, and P3). Loading control corresponds to GADPH expression. D. HEK293T cells were either untransduced (NT, control) or transduced with a lentiviral empty vector (WPI) containing the wild-type (WT) form of RAC2 cDNA, the mutant form described herein (G12R), or (as a positive control) the constitutively activated RAC2 GTP form (G12V). Two days after transduction, cells were harvested for analysis using the G-LISA assay (15 μg total protein per well). Results are shown as the mean ± standard error of the mean (SEM) of three independent experiments (***p<0.001; ****p<0.0001). [Figure 1C]Identification of the p.G12R RAC2 mutation and its effect on the GTPase activity of RAC2. A. Pedigree of three patients from two unrelated kindreds. Closed boxes and circles represent affected males (P1) and females (P2, P3), respectively. Open boxes and circles represent unaffected individuals. The arrow represents the proband and the two horizontal bars represent relatives. B. Representative electropherograms of RAC2 DNA sequencing on control and patient cells showing the c.34G>A mutation. C. Representative immunoblots of RAC2 protein expression in lysates of control fibroblasts (C1, C2) and fibroblasts derived from affected individuals (P1, P2, and P3). Loading control corresponds to GADPH expression. D. HEK293T cells were either untransduced (NT, control) or transduced with a lentiviral empty vector (WPI) containing the wild-type (WT) form of RAC2 cDNA, the mutant form described herein (G12R), or (as a positive control) the constitutively activated RAC2 GTP form (G12V). Two days after transduction, cells were harvested for analysis using the G-LISA assay (15 μg total protein per well). Results are shown as the mean ± standard error of the mean (SEM) of three independent experiments (***p<0.001; ****p<0.0001). [Figure 1D]Identification of the p.G12R RAC2 mutation and its effect on the GTPase activity of RAC2. A. Pedigree of three patients from two unrelated kindreds. Closed boxes and circles represent affected males (P1) and females (P2, P3), respectively. Open boxes and circles represent unaffected individuals. The arrow represents the proband and the two horizontal bars represent relatives. B. Representative electropherograms of RAC2 DNA sequencing on control and patient cells showing the c.34G>A mutation. C. Representative immunoblots of RAC2 protein expression in lysates of control fibroblasts (C1, C2) and fibroblasts derived from affected individuals (P1, P2, and P3). Loading control corresponds to GADPH expression. D. HEK293T cells were either untransduced (NT, control) or transduced with a lentiviral empty vector (WPI) containing the wild-type (WT) form of RAC2 cDNA, the mutant form described herein (G12R), or (as a positive control) the constitutively activated RAC2 GTP form (G12V). Two days after transduction, cells were harvested for analysis using the G-LISA assay (15 μg total protein per well). Results are shown as the mean ± standard error of the mean (SEM) of three independent experiments (***p<0.001; ****p<0.0001). [Figure 2A]G12R mutation inhibits HSPC proliferation and differentiation. A. Proliferation of CD34 positive cells in 7-day culture. Flow cytometry was used to analyze the percentage of GFP+ expressing cells in the live cell (7-AAD negative) gate. The percentage of ROS-low, DilC1(5)-low, and Annexin V positive GFP+ live cells was determined on day 4. Results are shown as the mean ± SEM of 4 independent experiments. B. Neutrophil differentiation in 7-day culture. Flow cytometry was used to analyze the number of granulocytes (CD11b+CD15+) among GFP+ live cells. The percentage of ROS low, DilC1(5) low, and Annexin V+ cells among GFP+ live cells was analyzed on day 4. Results are shown as the mean ± SEM of 3 independent experiments. C. T cell differentiation in 7-day culture (n=3). Flow cytometry was used to analyze the percentage of GFP+ live cells. The number of T cell precursors (CD7+) was assessed in GFP+ live cells on day 7. Results are shown as the mean ± SEM of three independent experiments. For all experiments: *p<0.05; **p<0.01 and ***p<0.001. [Figure 2B] G12R mutation inhibits HSPC proliferation and differentiation. A. Proliferation of CD34 positive cells in 7-day culture. Flow cytometry was used to analyze the percentage of GFP+ expressing cells in the live cell (7-AAD negative) gate. The percentage of ROS-low, DilC1(5)-low, and Annexin V positive GFP+ live cells was determined on day 4. Results are shown as the mean ± SEM of 4 independent experiments. B. Neutrophil differentiation in 7-day culture. Flow cytometry was used to analyze the number of granulocytes (CD11b+CD15+) among GFP+ live cells. The percentage of ROS low, DilC1(5) low, and Annexin V+ cells among GFP+ live cells was analyzed on day 4. Results are shown as the mean ± SEM of 3 independent experiments. C. T cell differentiation in 7-day culture (n=3). Flow cytometry was used to analyze the percentage of GFP+ live cells. The number of T cell precursors (CD7+) was assessed in GFP+ live cells on day 7. Results are shown as the mean ± SEM of three independent experiments. For all experiments: *p<0.05; **p<0.01 and ***p<0.001. [Figure 2C] G12R mutation inhibits HSPC proliferation and differentiation. A. Proliferation of CD34 positive cells in 7-day culture. Flow cytometry was used to analyze the percentage of GFP+ expressing cells in the live cell (7-AAD negative) gate. The percentage of ROS-low, DilC1(5)-low, and Annexin V positive GFP+ live cells was determined on day 4. Results are shown as the mean ± SEM of 4 independent experiments. B. Neutrophil differentiation in 7-day culture. Flow cytometry was used to analyze the number of granulocytes (CD11b+CD15+) among GFP+ live cells. The percentage of ROS low, DilC1(5) low, and Annexin V+ cells among GFP+ live cells was analyzed on day 4. Results are shown as the mean ± SEM of 3 independent experiments. C. T cell differentiation in 7-day culture (n=3). Flow cytometry was used to analyze the percentage of GFP+ live cells. The number of T cell precursors (CD7+) was assessed in GFP+ live cells on day 7. Results are shown as the mean ± SEM of three independent experiments. For all experiments: *p<0.05; **p<0.01 and ***p<0.001. [Figure 3A] Testing of Molm-13 cells from acute myeloid leukemia (AML, translocation MLL-AF9). A. Molm-13 cells were transduced in the presence of an empty vector (WPI) or a lentiviral construct containing a vector with a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). All transduced cells express the GFP reporter gene, making it possible to detect by flow cytometry the % of GFP+ cells and the number of GFP+ transduced cells during 7 days of culture. B. On day 4, flow cytometric analysis was performed to evaluate the percentage of GFP+ transduced cells entering apoptosis (% Annexin V+), mitochondrial membrane polarization (% Δμm), and ROS production. Results are representative of 4 independent experiments (**p<0.01 and ***p<0.001). [Figure 3B]Testing of Molm-13 cells from acute myeloid leukemia (AML, translocation MLL-AF9). A. Molm-13 cells were transduced in the presence of an empty vector (WPI) or a lentiviral construct containing a vector with a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). All transduced cells express the GFP reporter gene, making it possible to detect by flow cytometry the % of GFP+ cells and the number of GFP+ transduced cells during 7 days of culture. B. On day 4, flow cytometric analysis was performed to evaluate the percentage of GFP+ transduced cells entering apoptosis (% Annexin V+), mitochondrial membrane polarization (% Δμm), and ROS production. Results are representative of 4 independent experiments (**p<0.01 and ***p<0.001). [Figure 4] Testing of MV4-11 cells from AML (translocation MLL-AF4). MV4-11 cells were transduced in the presence of an empty vector (WPI) or a lentiviral construct containing a vector with a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). All transduced cells express the GFP reporter gene, allowing the detection by flow cytometry of the % of GFP+ cells and the number of GFP+ transduced cells during 7 days of culture. Results are representative of two independent experiments. [Figure 5A]Testing of HL60 cells from AML. HL-60 cells were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). Following the transduction step, the cells were cultured in the presence of retinoic acid to induce their differentiation towards the granulocytic lineage. AB. All transduced cells express the GFP reporter gene, making it possible to detect by flow cytometry the number of GFP+ transduced cells as well as the number of granulocytes (CD11b+CD15+) 2 days (J2) and 4 days (J4) after the start of the culture. Results are representative of three independent experiments (*p<0.05 ***p<0.001). CD. On day 4, flow cytometric analysis was performed to evaluate the percentage of cells with low levels of mitochondrial membrane polarization and low levels of ROS production. The percentage of cells entering apoptosis was 2% and 10% for WT and G12R conditions, respectively. Histograms are representative of three independent experiments. [Figure 5B] Testing of HL60 cells from AML. HL-60 cells were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). Following the transduction step, the cells were cultured in the presence of retinoic acid to induce their differentiation towards the granulocytic lineage. AB. All transduced cells express the GFP reporter gene, making it possible to detect by flow cytometry the number of GFP+ transduced cells as well as the number of granulocytes (CD11b+CD15+) 2 days (J2) and 4 days (J4) after the start of the culture. Results are representative of three independent experiments (*p<0.05 ***p<0.001). CD. On day 4, flow cytometric analysis was performed to evaluate the percentage of cells with low levels of mitochondrial membrane polarization and low levels of ROS production. The percentage of cells entering apoptosis was 2% and 10% for WT and G12R conditions, respectively. Histograms are representative of three independent experiments. [Figure 5C]Testing of HL60 cells from AML. HL-60 cells were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). Following the transduction step, the cells were cultured in the presence of retinoic acid to induce their differentiation towards the granulocytic lineage. AB. All transduced cells express the GFP reporter gene, making it possible to detect by flow cytometry the number of GFP+ transduced cells as well as the number of granulocytes (CD11b+CD15+) 2 days (J2) and 4 days (J4) after the start of the culture. Results are representative of three independent experiments (*p<0.05 ***p<0.001). CD. On day 4, flow cytometric analysis was performed to evaluate the percentage of cells with low levels of mitochondrial membrane polarization and low levels of ROS production. The percentage of cells entering apoptosis was 2% and 10% for WT and G12R conditions, respectively. Histograms are representative of three independent experiments. [Figure 5D] Testing of HL60 cells from AML. HL-60 cells were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a wild-type form of RAC2 (WT) or a vector containing a mutated form of RAC2 (G12R). Following the transduction step, the cells were cultured in the presence of retinoic acid to induce their differentiation towards the granulocytic lineage. AB. All transduced cells express the GFP reporter gene, making it possible to detect by flow cytometry the number of GFP+ transduced cells as well as the number of granulocytes (CD11b+CD15+) 2 days (J2) and 4 days (J4) after the start of the culture. Results are representative of three independent experiments (*p<0.05 ***p<0.001). CD. On day 4, flow cytometric analysis was performed to evaluate the percentage of cells with low levels of mitochondrial membrane polarization and low levels of ROS production. The percentage of cells entering apoptosis was 2% and 10% for WT and G12R conditions, respectively. Histograms are representative of three independent experiments. [Figure 6A]A. Mononuclear cells from peripheral blood of healthy donors were cultured in the presence of phytohemagglutinin A and transduced with lentiviral constructs containing an empty vector (WPI) or a vector containing a mutated form of RAC2 (G12R). All transduced cells express the GFP reporter gene, allowing to follow by flow cytometry the % of GFP+ cells, the number of GFP+ transduced cells, and the proportion of GFP+ cells entering apoptosis (% Annexin V+). Results are representative of two independent experiments. [Figure 6B] B. B-EBV strains from healthy donors were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a mutated form of RAC2 (G12R). All transduced cells express the GFP reporter gene, allowing the % of GFP+ cells to be tracked by flow cytometry. Results are representative of three independent experiments (***p<0.001).

[0041] Working Example

[0042] Materials and Methods

[0043] Patient and human cord blood samples

[0044] The study was carried out in accordance with French law and the principles of the Declaration of Helsinki. Informed consent was obtained from the patients' parents or legal guardians and the study protocol was approved by the local independent ethical committee and the French Ministry of Research (DC 2014-2272 / 2015 / DC-2008-329.) Primary fibroblast cell lines were obtained from skin biopsies.

[0045] Human umbilical cord blood (CB) samples eligible for research purposes were obtained from the cord blood bank of Saint Louis Hospital (Paris, France; accredited 23 September 2014). Mononuclear cells were isolated by density separation on Lymphoprep (Abcys). CD34+ HSPCs were magnetically sorted using an autoMACSpro separator (Miltenyi Biotec) and cell purity was checked using a MACSQuant analyzer (Miltenyi Biotec).

[0046] Genes, sequencing, and gene expression analysis

[0047] Genomic DNA was isolated from fibroblasts (P1, P2, P3) or peripheral blood mononuclear cells (PBMCs) (P3 father) by phenol / chloroform extraction. Whole-exome sequencing was performed using 100 bp paired-end reads with the Illumina TruSeq Exome Enrichment Kit (Illumina). 85% of the targeted regions were observed with >20× coverage. Variant calling was not based on a specific genetic model. The G12R RAC2 variant was not found in multiple in-house and public sequence databases. Mutations in P1, P2, and P3 were confirmed by Sanger sequencing (ABI Prism 3700 sequencer, Life Technologies).

[0048] For Western blot analysis, primary fibroblasts from controls or patients were lysed in Tris buffer (20 mM Tris, pH 7.9; 300 mM NaCl; 1% Nonidet P-40) supplemented with protease and phosphatase inhibitors. Cell extracts were separated by SDS-PAGE, blotted, and stained with anti-RAC2 (ab154711, Abcam) or anti-GAPDH (SC-32233, Santa Cruz) antibodies. After staining with HRP-conjugated secondary antibodies, immunoblots were developed using an ECL+ kit (Amersham). Protein levels were quantified using Fiji software.

[0049] Construction and production of lentiviral vectors. The replication-deficient, self-inactivating pWPI lentiviral vector backbone was provided by Addgene (https: / / www.addgene.org / ). All constructs (G12R, G12V, D57N, and E62K) were generated by GenScript (https: / / www.genscript.com). Lentiviral supernatants were produced by the vector facility of SFR BioSciences Gerland-Lyon Sud (Lyon, France). All procedures using genetically modified cells were approved by the French National Council for Biotechnology and the French Ministry of Research (reference: 4983 / 3).

[0050] RAC2 activation assay and immunoblotting in HEK293T cell line

[0051] HEK293T cells were cultured overnight before transduction with the appropriate lentiviral supernatant at a multiplicity of infection of 20. After 2 days of culture, cell pellets were frozen in liquid nitrogen. The levels of activated RAC2 were determined using the G-LISA® RAC Activation Assay Biochem Kit™ (#BK125, Cytoskeleton Inc.). The G-LISA® kit was performed according to the manufacturer's instructions, except that 10 μg / ml RAC2-specific antibody (AT2G11, sc-517424, Santa Cruz Biotechnology, Inc.) and 1 / 2000 HRP-conjugated anti-mouse antibody (#1721011, Bio-Rad) were used to detect the amount of captured active RAC2. The reaction was visualized by adding 100 μl of chromogenic substrate (1-step UltraTMB, 34028, Thermo Scientific) for 3 min and stopped with 50 μl of H2SO4 1N. Absorbance at 450 nm was measured using a FLUOstar OPTIMA microplate reader. Total cell extracts were obtained by lysing cells in G-LISA lysis buffer supplemented with a protease inhibitor cocktail. Proteins were separated by SDS-PAGE and immunoblotted with anti-RAC2 antibody (AT2G11, sc-517424, Santa Cruz Biotechnology, Inc.) or anti-GAPDH antibody (14C10, Cell Signaling Technology). Immunoblots were revealed by chemiluminescence using a ChemiDoc MP System (Bio-Rad). Protein levels were quantified using Image Lab software.

[0052] Holotomography

[0053] To measure the refractive index within cells (i.e., label-free visualization of cell organelles), cell samples were seeded in 35 mm glass-bottom dishes (Ibidi-Dishes™ #81156; Ibidi, GmbH) and placed in the observation field of a 3D Cell Explorer microscope (Nanolive SA). Cell images were processed using STEVE software (Nanolive).

[0054] In vitro culture of human umbilical cord blood CD34+ cells

[0055] Cord blood CD34+ HSPCs were cultured overnight (as previously described) 5 ), and then transduced with the appropriate lentiviral supernatant at an infection multiplicity of 80. After 2 days of culture, GFP+ cells were measured by flow cytometry prior to in vitro culture. The ability of CD34+ cells to differentiate along the granulocytic or T cell lineages was measured as described elsewhere. 6,29 .

[0056] Flow cytometry, Mitotracker DIIC1(5), CellRox staining

[0057] Monoclonal antibodies against CD7 (MT-701), CD11b (D12), CD34 (8G12), and mouse IgG1k, IgG2a, and IgG2b isotype controls, as well as the reagents Annexin V and 7-aminoactinomycin D (7AAD) were obtained from BD Biosciences (San Jose, CA). CD15 (80H5) and mouse IgM control antibodies were purchased from Beckman Coulter (San Diego). Mitochondrial membrane potential was measured using the MitoProbe™ DiIC1(5) Assay Kit (M34151), and ROS levels were measured using the CellRox® Assay Kit (C10492) according to the manufacturer's instructions (Life Technologies). After staining, cells were analyzed on a Gallios flow cytometer, and data were processed using Kaluza software (all from Beckman Coulter).

[0058] Homology modeling

[0059] A three-dimensional homology model was constructed for the G12R mutant of human RAC2 (Uniprot P15153) using MODELLER software v20 (Webb and Sali, 2016, 2017). The crystal structure of WT human RAC2 (PDB code: 1DS6) was used as a template.

[0060] statistical analysis

[0061] In all analyses, three or more independent experiments were performed. Data are reported as the mean ± standard error of the mean (SEM). Unpaired two-tailed t-tests were performed using Prism 4 software (GraphPad). The threshold for statistical significance was set at p<0.05.

[0062] result

[0063] A missense mutation (G12R) in the RAC2 GTPase protein leads to a SCID phenotype.

[0064] In our cohort of patients with SCID phenotype, several individuals do not yet have a molecular diagnosis. Three patients from two unrelated families (P1 from one family, and P2 and P3 as mother and daughter, respectively, from another family) presented all clinical features of RD except hearing loss (Figure 1A). As observed in patients with RD, the complete absence of neutrophils was responsible for the onset of severe infections earlier than usually observed in other forms of SCID (Table 1). picard 2 Analysis of other blood cell lineages highlighted the absence of T and B lymphocytes as well as circulating monocytes. Conversely, platelet counts were normal, whereas hemoglobin levels were slightly lower than normal in only one patient (Table 1). Overall, the peripheral cell count profile corresponded to the severe hypoplastic morphology of the patient's bone marrow (Table 1 and data not shown). Hematopoietic stem cell transplantation (HSCT) performed soon after birth was consistent with the diagnosis of P1 (P4 12 (previously described as ) and P2, demonstrating that the genetic defect is intrinsic and not exogenous (i.e., not microenvironmental) (Table 1).

[0065] By performing whole genome sequencing (WES) of the patient's fibroblasts, we identified a heterozygous missense mutation (c.34G>A, p.G12R) in the RAC2 gene as the only possible genetic cause. This mutation was confirmed by Sanger sequencing in three patients, but was absent in P3's father (the only relative from whom we could obtain a DNA sample) (Figure 1B). The same phenotype and the presence of the same RAC2 G12R missense mutation in P2 and his daughter P3 underscored the inheritance pattern of AD.

[0066] The p.G12R missense mutation is responsible for the G1 box (a highly conserved guanine nucleotide-binding domain). 13) and was not annotated in our in-house database (n=14154 in the cohort) or in the Human Genome Aggregation Database (gnomAD). This mutation was predicted to be deleterious by four different in silico prediction software (including Combined Annotation Depletion). It is noteworthy that this genetic mutation is distinct from loss-of-function (LOF) or gain-of-function (GOF) mutations previously reported to be responsible for mild neutrophil deficiency and / or lymphopenia. 14-18 It is noteworthy that the G12R missense mutation was associated with normal levels of RAC2 protein expression in patient fibroblasts (Figure 1C).

[0067] The G12R mutation located in the GDP / GTP-binding domain disrupts cellular homeostasis

[0068] To understand the functional impact of this G12R mutation, we determined the X-ray structure of wild-type (WT) RAC2. 19 was used as a template to generate a three-dimensional homology model (data not shown). The bulky, flexible arginine is predicted to block the entrance to the GDP / GTP binding pocket in the G1 box (data not shown). Thus, it is possible that the charged guanidinium group of arginine disrupts the positively charged pocket and thus may affect the rate of GTP hydrolysis (as previously demonstrated for other small GTPases). 20 ).

[0069] To test this model biochemically, we quantified the active RAC2 GTP-bound form in extracts of naturally RAC2-negative HEK293T cells, which were transfected with either an empty lentiviral backbone (WPI) with green fluorescent protein (GFP) as a tracker, or with either the WT form of RAC2 cDNA, the mutant form described herein (G12R), or the constitutively active RAC2 GTP form (G12V) as a positive control. 21The cells were transduced with a vector containing the following: High levels of the activated GTP-bound RAC2 form were observed with both G12V and G12R (FIG. 1D), demonstrating that the substitution with arginine at position 12 leads to sustained activation of the RAC2 signaling pathway. The G12R mutation is therefore a GOF mutation.

[0070] To determine how expression of a constitutively active form of RAC2 affects cell division and survival, we performed holotomography analysis of primary fibroblasts from P3. Consistent with the low observed cell proliferation rate in vitro (data not shown), the patient's fibroblasts were characterized by very slow cell kinetics and fragmented nuclei suggesting cytokinesis failure (data not shown). Furthermore, in contrast to control fibroblasts, cell shape and mitochondrial network were disrupted in P3 fibroblasts (data not shown). These two observations highlight the link between the G12R mutation, defective mitochondrial activity, and the dramatic changes in cell mitosis that reflect defects in the RAC signaling pathway that drives cytokinesis. 22 .

[0071] The G12R mutation disrupts mitochondrial activity and blocks HSPC differentiation

[0072] To understand the impact of constitutive RAC2 activation on hematopoiesis, CD34 positive human cord blood HSPCs (assuming the absence of patient bone marrow samples) were transduced with WPI, WT, G12R, or G12V RAC2 cDNA (using GFP as a tracker) and then cultured with cytokines for 7 days (Figure 2B). Expression of constitutively active RAC2 forms (G12R and G12V) led to the disappearance of GFP+ transduced cells within 4 days, at which time point reactive oxygen species (ROS) production (quantified as the percentage of "ROS low" cells) was significantly blocked, and mitochondrial membrane depolarization (percentage of "DilC1(5) low" cells) and apoptosis (percentage of Annexin V positive cells) were significantly increased (Figure 2A). It is noteworthy that these changes were not observed in the GFP negative subset. Overall, these findings demonstrated a specific correlation between mutations at position 12 in the RAC2 protein and mitochondrial dysfunction. Our results show that (i) mitochondrial activity is required for HSPC function; 23,24 and (ii) disruption of the RAC2 signaling pathway impairs mitochondrial dynamics. 25 and reactive oxygen production 26-28 This is consistent with previous reports showing that it affects

[0073] Since RAC2 is highly expressed in human hematopoietic myeloid subsets and during human thymus formation, we transduced HSPCs with WPI, WT, G12R, or G12V RAC2 cDNA and stimulated them to differentiate along granulocytic, monocytic, and T-lymphoid lineages (all absent in three patients). After 7 days of culture with granulocyte colony-stimulating factor, the number of GFP+CD15+CD11b+ neutrophils was significantly lower in G12R and G12V conditions than in WPI or WT conditions (Figure 2B). Mitochondrial membrane potential and ROS production were also found to be disrupted and associated with elevated levels of apoptosis (Figure 2B). Differentiation towards the monocytic lineage and in colony-forming unit assays gave similar results (data not shown). The Notch ligand delta-like 4 (DL4) culture system (allowing differentiation of HSPCs into CD7+ T-cell progenitors in 7 days) was used to culture HSPCs in the presence of RAC2 cDNA. 29 Upon exposure to RAC2, the number of GFP+ subsets in G12R and G12V conditions was very low (compared to WT and WPI conditions) and GFP+CD7+ T cell precursors were almost completely absent (Figure 2C). These results are consistent with previous reports that expression of the G12V constitutively active form of RAC2 prevents mouse thymocyte differentiation and drives them into apoptosis. 30 Overall, our results indicate a critical function for the GDP / GTP-bound RAC2 balance in the survival and differentiation of lymphoid-myeloid compartments and highlight the involvement of the RAC2 signaling pathway in HSPC function. We next examined the effect of G12R on the proliferation of human cord blood HSPCs by comparing the effects of previously described LOF and GOF missense mutations in RAC2 (p.D57N and p.E62K, respectively). 14、15、18 During 8 days of culture, the number of GFP+ transduced HSPCs was lower in the E62K and G12R groups, but was significantly higher in the E62K condition than in the G12R condition. Unlike the G12R mutation, the D57N and E62K mutations had no effect on mitochondrial activity.

[0074] Overall, our observations suggest that the G12R missense mutation in RAC2 correlates with the levels of the active GTP-bound form of RAC2 and with HSPC survival and function. These findings are compatible with the clinical and immunological phenotype of SCID observed in our three patients, as well as with the less severe phenotype exhibited by patients with E62K or D57 mutations.

[0075] Three types of LAM were tested (Molm13, MV4-11, and HL60).

[0076] Molm-13 cells from acute myeloid leukemia (AML, translocation MLL-AF9) were transduced in the presence of lentiviral constructs containing an empty vector (WPI), a vector containing a wild form of RAC2 (WT), or a vector containing a mutated form of RAC2 (G12R). All transduced cells express the reporter gene GFP (green fluorescent protein), allowing to follow the percentage of GFP+ cells and the number of transduced GFP+ cells by flow cytometry. Expression of the constitutively activated RAC2 form (G12R) led to the disappearance of GFP+ transduced cells within 7 days (Figure 3A). This observation correlates with an increased percentage of GFP+ apoptotic cells (% Annexin V+) with defective mitochondrial membrane polarization and defective reactive oxygen species (ROS) production (Figure 3B).

[0077] MV4-11 cells from AML (translocation MLL-AF4) were also transduced in the presence of lentiviral constructs containing an empty vector (WPI), a vector containing a wild-type form of RAC2 (WT), or a vector containing a mutated form of RAC2 (G12R). Transduced cells expressing a GFP reporter gene were followed by flow cytometry, and we observed a decreased percentage and number of GFP+ cells in the G12R condition (Figure 4). Cells were also stained with May-Grunwald Giemsa, and nuclear fragmentation was observed only in cells transduced with the G12R construct (data not shown).

[0078] HL60 cells from AML were transduced in the presence of lentiviral constructs containing vectors containing the wild-type form of RAC2 (WT) or the mutant form of RAC2 (G12R). After transduction, cells were cultured for 4 days in the presence of retinoic acid to induce their differentiation into granulocytes (CD11b+ CD15+). The number of GFP+ cells was measured on days 2 (J2) and 4 (J4), and the number of granulocytes on day 4. As observed in Figure 5A and Figure 5B, the number of GFP+ and CD11b+ CD15+ cells was dramatically reduced in the G12R condition compared to the WT condition. This observation correlated with a higher mitochondrial membrane depolarization (quantified as the percentage of DILC low cells; Figure 5C) and a higher percentage of cells unable to produce ROS (quantified as % of ROS low; Figure 5D). Of note, the percentage of GFP+ cells entering apoptosis was 2% and 10% for the WT and G12R conditions, respectively. Cells were also stained with May-Grunwald Giemsa and fragmented nuclei were observed only in cells transduced with the G12R construct.

[0079] We also tested the effect of the RAC2 G12R missense mutation on primary cells. Briefly, mononuclear cells from healthy donors were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a mutated form of RAC2 (G12R). Transduced GFP+ were tracked by flow cytometry during 8 days of culture. As observed in Figure 6A, the percentage and number of GFP+ cells dramatically decreased in the G12R condition compared to the control condition. This observation is associated with a higher percentage of apoptotic cells (Figure 6A). B-EBV strains from healthy donors were transduced in the presence of lentiviral constructs containing an empty vector (WPI) or a vector containing a mutated form of RAC2 (G12R). In the time course kinetics depicted in Figure 6B, the percentage of GFP+ cells is significantly decreased in the G12R condition.

[0080] Consideration

[0081] In three patients with SCID, we identified a heterozygous dominant missense mutation (p.G12R) in the highly conserved G1 box domain of the RAC2 protein. This mutation affected the innate and adaptive immune systems. Given the severity of the clinical symptoms, the patients had to undergo hematopoietic stem cell transplantation within the first few weeks of life. The observation of AD inheritance broadens the clinical spectrum of RD. We distinguish here between two forms of RD: a recessive syndromic form associated with hearing loss and AK2 mutations (type I) and a non-syndromic form (without hearing loss) specifically associated with AD G12R mutations in RAC2 (type II). The absence of sensorineural hearing loss in the type II AD-SCID form may be due to the specific expression of RAC2 in the hematopoietic lineage and the fact that RAC1 and RAC3 (but not RAC2) are RAC GTPases involved in the development of the mouse inner ear. 31 .

[0082] The G12R-RAC2 mutation identified herein has a dramatic effect on cell proliferation and survival, especially in HSPC.In addition, this mutation disrupts mitochondrial activity in HSPC, impairing cell differentiation toward lymphoid and myeloid lineages.These features may explain the absence of circulating lymphocytes and neutrophils in our three patients, and highlight the non-redundant regulatory role of RAC2 in different hematopoietic checkpoints.

[0083] Surprisingly, in our experiments, the D57N LOF mutant has no effect on the HSPC pool, and the E62K GOF mutant has only a moderate effect (compared to G12R). Overall, our findings highlight that various RAC2 mutations differ in their effect on HSPCs, possibly explaining the wide spectrum of clinical phenotypes observed in patients with RAC2 deficiency, from neutrophil deficiency and / or leukopenia (without any reported hematopoietic alterations) to the SCID morphology with bone marrow hypoplasia that we describe here. This type of heterogeneity has already been reported for RAG1 mutations, where phenotypes range from autoimmune disease to SCID. 32 .

[0084] The location of the amino acid substitution (within the GDP / GTP-binding pocket (G12R) or in the switch II domain (E62K)) may explain the difference between these two GOF mutations. This hypothesis is consistent with (i) the high level of active (GTP-bound) form of RAC2 observed in the G12R condition (compared to its absence in the E62K condition) and (ii) the low level of RAC2 protein expression observed in the E62K condition, suggesting that the glutamic acid substitution at position 62 may affect the stability of the protein. Consequently, we suggest that the G12R mutation is the only mutation that can activate downstream targets in a constitutive and sustained manner. Our results with the E62K mutant are consistent with the results of Hsu et al. 18 This result is different from that described by .Because they evaluated the GTP binding ability of purified E62K protein; here, we measured the RAC2GTP activity in cell lysate.In light of these results, it seems that the two GOF mutations do not drive the same level of RAC2 GTP activity, and G-LISA assay is a suitable method for measuring the level of RAC2 activation in living cells.

[0085] In summary, p.G12R RAC2 mutation has a dramatic impact on the maintenance and differentiation of HSPC compartments, and therefore may explain the severity of the clinical and immunological phenotype of patients. To our knowledge, this study is the first to report an AD form of SCID, and physicians should consider RAC2 gene sequencing for patients with clinical symptoms of SCID and RD. We also observed a dramatic impact of G12R missense mutation in various cell lines (B-EBV, AML) or mature primary cells, suggesting that targeting the GDP / GTP binding domain of RAC2 protein may represent a new therapeutic strategy to induce leukemic cell death. [Table 1] TIFF0007662609000002.tif153165

[0086] References

[0087] 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 this disclosure. [Table 2] TIFF0007662609000004.tif234165 TIFF0007662609000005.tif200165

Claims

1. A pharmaceutical composition for completely ablating hematopoiesis in a patient in need thereof, comprising a therapeutically effective amount of i) a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with an amino acid residue (R), or ii) a polynucleotide encoding a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with an amino acid residue (R).

2. A pharmaceutical composition for inhibiting the proliferation and differentiation of a population of hematopoietic stem cells, comprising an effective amount of i) a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with amino acid residue (R), or ii) a polynucleotide encoding a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with amino acid residue (R).

3. 1. A pharmaceutical composition for inducing cell death in a population of hematopoietic cells, comprising an effective amount of i) a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with amino acid residue (R), or ii) a polynucleotide encoding a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with amino acid residue (R).

4. A pharmaceutical composition for inducing cell death of a population of malignant hematopoietic cells, comprising an effective amount of i) a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with amino acid residue (R), or ii) a polynucleotide encoding a polypeptide comprising an amino acid sequence in which the amino acid residue (G) at position 12 of the amino acid sequence shown in SEQ ID NO:1 is substituted with amino acid residue (R).

5. 1. A pharmaceutical composition for treating a hematopoietic cell malignancy in a patient in need thereof, comprising a therapeutically effective amount of i) a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:1, in which the amino acid residue (G) at position 12 is substituted by amino acid residue (R), or ii) a polynucleotide encoding a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:1, in which the amino acid residue (G) at position 12 is substituted by amino acid residue (R).

6. 4. The pharmaceutical composition of claim 1, 2 or 3, used to prepare a patient for bone marrow transplantation.

7. The pharmaceutical composition according to claim 6, wherein the bone marrow transplant is a hematopoietic stem cell transplant.

8. The pharmaceutical composition of claim 5, wherein the hematopoietic cell malignancy is selected from the group consisting of leukemia, lymphoma, and multiple myeloma.

9. 6. The pharmaceutical composition of claim 5, wherein the hematopoietic cell malignancy is selected from the group consisting of myelodysplastic syndromes (MDS); myeloproliferative disorders; and diseases with features of both myelodysplastic syndromes and myeloproliferative disorders.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the polynucleotide of the present invention is messenger RNA (mRNA).

11. The pharmaceutical composition according to any one of claims 1 to 9, wherein the polynucleotide is inserted into a vector.

12. The pharmaceutical composition according to any one of claims 1 to 10, wherein the polypeptide or polynucleotide is bound to at least one other molecule selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, sugars, vitamins, cofactors, and drugs.

13. The pharmaceutical composition of claim 12, wherein the polypeptide or polynucleotide is bound to an antibody or peptide having binding affinity for a protein expressed on the surface of a hematopoietic stem cell.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the polypeptide or polynucleotide is formulated with a lipidoid.

15. The pharmaceutical composition according to any one of claims 1 to 13, wherein the polypeptide or polynucleotide is formulated using one or more liposomes, lipoplexes or lipid nanoparticles.

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