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WO2021084540A1 - Inhibiteurs de la voie mmej pour la prévention et le traitement de malignités myéloïdes et pré-myéloïdes - Google Patents

Inhibiteurs de la voie mmej pour la prévention et le traitement de malignités myéloïdes et pré-myéloïdes Download PDF

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WO2021084540A1
WO2021084540A1 PCT/IL2020/051130 IL2020051130W WO2021084540A1 WO 2021084540 A1 WO2021084540 A1 WO 2021084540A1 IL 2020051130 W IL2020051130 W IL 2020051130W WO 2021084540 A1 WO2021084540 A1 WO 2021084540A1
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myeloid
mmej
inhibitor
dna
deletions
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Liran Shlush
Tzah FELDMAN
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Yeda Research And Development Co. Ltd.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/55—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having seven-membered rings, e.g. azelastine, pentylenetetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

Definitions

  • the present invention in some embodiments thereof, relates to methods of preventing and treating myeloid malignancies or pre-malignancies.
  • HSPCs Human aged Heamatopoietic Stem and Progenitor Cells
  • HSPCs Human aged Heamatopoietic Stem and Progenitor Cells
  • ADH age related clonal hematopoiesis
  • Somatic pre-leukemic mutations pLMs
  • pLMs do not usually spread randomly across the possible physical positions of a gene, but rather occur at apparent mutational hotspots. Whether mutational hotspots are the result of a specific selective advantage, or are due to increased mutation rate in specific positions remains unclear for most pLMs.
  • SNVs single nucleotide variants
  • CML chronic myeloid leukemia
  • an inhibitor of a component of the Microhomology Mediated End Joining (MMEJ) pathway for use in treating or prevention of a pre-myeloid or myeloid malignancy in a subject in need thereof, wherein the pre-myeloid or myeloid malignancy is not chronic myeloid leukemia (CML).
  • MMEJ Microhomology Mediated End Joining
  • the pre-myeloid or myeloid malignancy is characterized by an MMEJ deletion.
  • the MMEJ deletion is comprised in a gene selected from the group consisting of CALR, ASXL1 and SRSF2.
  • the MMEJ deletion is comprised in a sequence selected from the group consisting of SEQ ID NO: 70-75.
  • the pre-myeloid or myeloid malignancy is pre-leukemia or myeloid leukemia.
  • the myeloid leukemia is selected from the group consisting of myelodisplastic syndrome, polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML).
  • the myeloid leukemia is selected from the group consisting of myelodisplastic syndrome, polycythemia vera, essential thrombocythemia and primary myelofibrosis.
  • the component is selected from the group consisting of CtlP MRN, PARP1, EXOl, BLM, DNA2, XPF, ERCC1, Ligl, Lig3, XRCC1 and Pol Q.
  • the component is selected from the group consisting of Mrell, Rad50, Nbsl, ATM and FEN1.
  • the inhibitor is a small molecule.
  • the method does not comprise treating with a CDK inhibitor.
  • the malignancy is not CML.
  • FIGs. 1A-C show that recurrent deletions in myeloid malignancies share a similar signature of MMEI mediated DSB repair, successfully recapitulated in primary human CD34+ HSPCs.
  • Figure 1A Absolute number of samples carrying somatic deletion (represented by gene and mutation CDS (coding DNA sequence) names) in myeloid malignancies identified in 10 or more samples in COSMIC dataset. Deletion signatures are Microhomology Mediated End loining (MMEI) (orange), Microsatellites (MS) (purple) and deletions with no clear signatures (blue).
  • Figure IB MMEI deletion signature in ASXL1, CALR and SRSF2 genes.
  • FIGs. 2A-C show deletion distribution in ASXL1 and SRSF2 genes following sequential CRISPR Cas9 DSBs in K-562.
  • Canonical Microhomologies (MHs) (orange and yellow backgrounds) are marked along the sequences.
  • FIGs. 3A-B show that recurrent MMEJ deletions occur predominantly in myeloid malignancies.
  • Figure 3A Absolute number of samples carrying deletions with MMEJ signature (represented by gene and mutation CDS (coding DNA sequence) names) identified in 10 or more samples in COSMIC dataset in all cancers.
  • Figure 3B The proportion of deletions with MMEJ signatures (orange) and non-MMEJ signature (blue) out of the total number of deletions reported for each gene presented in Figure 3A. Significant proportion differences (chi square analysis) are defined as p ⁇ 0.0005 and are indicated by asterisks (***).
  • FIGs. 4A-D show that MMEJ canonical deletions are detected in multipotent HSCs.
  • Figure4A - Variant allele frequency (VAFs) (%) of the canonical CALR MMEJ deletion as detected by droplet digital PCR (ddPCR) in various cell populations sorted directly from peripheral blood of two myelofibrosis (MF) patients (samples 140122 and 140681), or in myeloid (CD33+) and B-cells (BC, CD19+) isolated from xenografts generated in NGS mice 16 weeks post intrafemoral transplantation of sample 140122.
  • VAFs Variant allele frequency
  • HSC/MPP haematopoietic stem cell/multipotent progenitor
  • MLP multi-lymphoid progenitor
  • CMP/MEP common myeloid progenitor/megakaryocyte erythroid progenitor
  • GMP granulocyte monocyte progenitor
  • TC T cell
  • BC B cell
  • NK Natural killer cell. Black squares indicate populations without sufficient DNA amounts for variant detection; dark grey bars indicate VAF.
  • BasP basophil progenitor
  • BcP B-cell progenitor
  • CLP common lymphoid progenitor
  • CMP common myeloid progenitor
  • DcP dendritic cell progenitor
  • Early-Gran early granulocyte
  • Early-Neut early neutrophil
  • EP erythroid progenitor
  • LMPP lymphoid-primed multipotential progenitors
  • MegP megakaryocyte progenitor
  • MEP megakaryocyte erythrocyte progenitor
  • MonP monocyte progenitor
  • TcP T- cell progenitor Figure 4C - Log fold change (lfp) in UMI content for DSBs repair genes in the various HSPCs metacells.
  • FIG. 5 is a schematic illustration of the MMEJ pathway as adapted from Seol J.H. et ah, Mutat Res. 2018 (www(dot)ncbi(dot)nlm(dot)nih(dot)gov/pubmed/28754468).
  • the present invention in some embodiments thereof, relates to methods and agents for use for the treatment or prevention of myeloid malignancies and pre-malignancies.
  • the present inventors analyzed the genomic regions around recurrent deletions in myeloid malignancies, and identified microhomology-mediated end-joining (MMEJ) signatures in recurrent deletions in CALR, ASXL1 and SRSF2 loci. Since MMEJ deletions are the result of DNA double-strand breaks (DSBs), the present inventors introduced CRISPR Cas9 DSBs into exon 12 of ASXL1, successfully generating recurrent ASXL1 deletion in human hematopoietic stem and progenitor cells (HSPCs). A systematic search of COSMIC dataset for MMEJ deletions in all cancers revealed that recurrent deletions enrich myeloid malignancies.
  • MMEJ microhomology-mediated end-joining
  • MMEJ deletions occur in multipotent HSCs.
  • An analysis of DNA repair pathway gene expression in single human adult bone marrow HSPCs could not identify a subpopulation of multipotent HSPCs with increased MMEJ expression, however exposed differences between myeloid and lymphoid biased progenitors.
  • An association is indicated between MMEJ-repaired DSBs and recurrent MMEJ deletions in human HSCs and in myeloid leukemia.
  • MMEJ Microhomology Mediated End Joining
  • an inhibitor of a component of the Microhomology Mediated End Joining (MMEJ) pathway for use in treating or prevention of a pre-myeloid or myeloid malignancy in a subject in need thereof, wherein the pre- myeloid or myeloid malignancy is not chronic myeloid leukemia (CML).
  • treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition (also referred to as a disease or disorder).
  • condition refers to a myeloid malignancy or pre-myeloid malignancy, as is explained and exemplified hereinbelow.
  • the term “preventing” refers to keeping a condition from occurring in a subject who may be at risk for the condition, but has not yet been diagnosed as having the disease or disorder, i.e., myeloid malignancy or pre-myeloid malignancy.
  • the term “subject” or “subject in need thereof’ refers to mammals, preferably human beings, male or female, who are diagnosed with, or are at risk of developing a myeloid malignancy or pre-myeloid malignancy.
  • the subject is diagnosed with cancer but has not been subject to anti cancer therapy (e.g., chemotherapy, radiation, radiotherapy or immunotherapy).
  • anti cancer therapy e.g., chemotherapy, radiation, radiotherapy or immunotherapy.
  • the treatment described herein is the first line treatment.
  • the subject is undergoing a routine well-being check-up.
  • the subject is an infant, a child, an adolescent or an adult as defined by the classification tables of the Food and Dmg Administration (FDA).
  • FDA Food and Dmg Administration
  • the subject is under 70 years old, under 65 years old, under 60 years old, under 55 years old, under 50 years old, under 45 years old, under 40 years old, under 35 years old, under 30 years old, under 25 years old or under 20 years old.
  • the subject is 18-75 years old.
  • the subject is up to 18 years old.
  • the subject is 3-18 years old.
  • the subject is 0-3 years old.
  • the subject is at risk of developing a myeloid malignancy or pre-myeloid malignancy (e.g., a human who is genetically or otherwise, e.g., environmentally predisposed to developing a myeloid malignancy or pre-myeloid malignancy) and who has not been diagnosed with the malignancy or pre-myeloid malignancy.
  • a myeloid malignancy or pre-myeloid malignancy e.g., a human who is genetically or otherwise, e.g., environmentally predisposed to developing a myeloid malignancy or pre-myeloid malignancy
  • the genome of the subject is characterized by at least one Microhomology Mediated End Joining (MMEJ) deletion, inversion and/or translocation.
  • MMEJ Microhomology Mediated End Joining
  • the genome of the subject is characterized by at least one Microhomology Mediated End Joining (MMEJ) deletion.
  • MMEJ Microhomology Mediated End Joining
  • an “MMEJ deletion” refers to a mutation in which one of two identical sequences in the DNA (termed “microhomologies”, see orange color in Figure IB for illustration) together with the gap between these microhomolgies (see grey color in Figure IB for illustration) are deleted, resulting in only one microhomology in the mutated DNA.
  • an MMEJ is in one of the alleles of a diploid genome. In other embodiments, the MMEJ is in the two alleles.
  • the deletion can be a few bases long e.g., about 10 bp to about 100 bp long but can also be much longer at the sub-chromosomal level e.g., as manifested by as copy number variation and chromosomal translocations encompassing thousands of bases.
  • MMEJ is frequently associated with chromosome abnormalities such as deletions, translocations, inversions and other complex rearrangements.
  • the deletion length is typically about 20-100, 20, 80, 20-70 or 20-60 bp long (according to a specific embodiment the deletion is 23, 24 or 52 bp long.
  • the microhomology is typically between about 2-25 bp long.
  • the microhomology is typically between about 2-20 bp long.
  • the microhomology is typically between about 2-10 bp long.
  • the microhomology is typically between about 4-10 bp long.
  • the microhomology is about 5 bp long.
  • a “high risk subject” is a subject who is likely to develop the malignancy or pre-myeloid malignancy due to one or more so-called risk factors, which are measurable parameters that correlate with development of with the malignancy or pre-myeloid malignancy.
  • a subject having one or more of these risk factors has a higher probability of developing the malignancy or pre-myeloid malignancy as compared to an individual without these risk factor(s).
  • Additional risk factors may include, for example, age, gender, race, diet, weight, history of a previous disease, presence of a precursor disease (e.g. pre-leukemia), genetic (e.g., hereditary) considerations, and environmental exposure (e.g. radiation or chemical exposure).
  • a subject at high risk of developing a pre-myeloid or myeloid malignancy include, for example, a subject whose relatives have experienced this disease, and whose risk is determined by analysis of genetic or biochemical marker/s. Such subjects may be identified by the presence of certain genetic aberrations, as discussed in detail below.
  • a subject at high risk of developing a pre-myeloid or myeloid malignancy if the subject exhibits any of, but not limited to, larger cell clones (measured by peripheral blood variant allele fraction (PB-VAF)), more than one ARCH defining event, increased red cell distribution width (RDW), reduced monocyte cell counts, reduced platelet cell counts, reduced red blood cell counts, reduced white blood cell counts, reduced hemoglobin levels, reduced cholesterol levels, prolonged fever, enlarged lymph nodes and/or spleen, or any combination thereof, as compared to subject not at high risk of developing a pre-myeloid or myeloid malignancy, i.e., a healthy subject.
  • PB-VAF peripheral blood variant allele fraction
  • a myeloid malignancy refers to any one of heterogeneous disorders characterized by uncontrolled proliferation and/or blockage of differentiation of abnormal myeloid progenitor cells.
  • Myeloid malignant diseases comprise chronic (including, but not limited to, myelodysplastic syndromes, myeloproliferative neoplasms and chronic myelomonocytic leukemia) or acute (such as acute myeloid leukemia) stages. They are clonal diseases arising in hematopoietic stem or progenitor cells. Mutations responsible for these diseases occur in several genes whose encoded proteins belong principally to five classes: signaling pathways proteins (e.g. CBL, FLT3, JAK2, RAS), transcription factors (e.g. CEBPA, ETV6, RUNX1), epigenetic regulators (e.g.
  • signaling pathways proteins e.g. CBL, FLT3, JAK2, RAS
  • transcription factors e.g. CEBPA, ETV6, RUNX1
  • epigenetic regulators e.g.
  • tumor suppressors e.g. TP53
  • components of the spliceosome e.g. SF3B1, SRSF2.
  • a pre-myeloid malignancy refers to medical conditions in which asymptomatic subjects for a myeloid malignant disease, as times also referred to as healthy subjects, display (also referred to as “positive for”) somatic mutations carrying high risk for development of myeloid malignancies in a DNA of the peripheral blood (e.g., peripheral blood cells).
  • pre-leukemic hematopoietic stem and progenitor cells pre-leukemic hematopoietic stem and progenitor cells (preLHSPCs) carrying pre-leukemic mutations (pLMs) are the cells of origin in AML and MDS.
  • preLHSPCs acquire leukemia-related mutations years before diagnosis and maintain almost normal function for years before transformation to overt disease.
  • pLMs can be found among individuals destined to develop AML and MDS, they are also present in 20-30 % of healthy individuals.
  • Prevention of disease development in those individuals who are at risk for myeloid malignancies is one of the major embodiments of the present invention.
  • the subject is positive for one or more MMEJ deletion in a gene selected from the group consisting of CALR, ASXL1 and SRSF2.
  • CALR refers to the protein coding gene (gene symbol “CALR”) Calreticulin, a multifunctional protein that acts as a major Ca(2+)-binding (storage) protein in the lumen of the endoplasmic reticulum.
  • the MMEJ deletion in CALR is as set forth in SEQ ID NO: 1
  • ASXL1 refers to the protein coding gene (gene symbol “ASXL1”). This gene is similar to the Drosophila additional sex combs gene, which encodes a chromatin-binding protein required for normal determination of segment identity in the developing embryo.
  • the protein is a member of the Polycomb group of proteins, which are necessary for the maintenance of stable repression of homeotic and other loci.
  • the MMEJ deletion in ASXL1 is as set forth in SEQ
  • SRSF2 refers to serine/arginine-rich splicing factor 2, a protein-coding gene.
  • the protein encoded by this gene is a member of the serine/arginine (SR)-rich family of pre- mRNA splicing factors, which constitute part of the spliceosome.
  • the MMEJ deletion in SRSF2 is as set forth in SEQ ID NO: 1
  • the term “positive” refers to a genome of the subject that displays said MMEJ deletion or any mutation associated with the myeloid malignancy (or pre-myeloid).
  • the subject’s genome is characterized in MMEJ deletions in ASXL1 and CALR, as exemplified below.
  • the subject’s genome is characterized in MMEJ deletions in ASXL1, SRSF2 and CALR.
  • the pre-myeloid or myeloid malignancy is pre leukemia or myeloid leukemia.
  • myeloid malignancies include, but are not limited to, myelodisplastic syndrome (MDS), Polycythemia vera, Essential Throbocythemia, Primary Myelofibrosis, Chronic myelomonocytic leukemia and Acute Myeloid Leukemia (AML).
  • MDS myelodisplastic syndrome
  • Polycythemia vera Essential Throbocythemia
  • Primary Myelofibrosis Primary Myelofibrosis
  • Chronic myelomonocytic leukemia Chronic myelomonocytic leukemia
  • AML Acute Myeloid Leukemia
  • the myeloid leukemia is selected from the group consisting of myelodisplastic syndrome (MDS), polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic myelomonocytic leukemia (CMML) and acute myeloid leukemia (AML).
  • MDS myelodisplastic syndrome
  • CMML chronic myelomonocytic leukemia
  • AML acute myeloid leukemia
  • the myeloid leukemia is selected from the group consisting of myelodisplastic syndrome (MDS), polycythemia vera, essential thrombocythemia and primary myelofibrosis.
  • the myeloid malignancy is MDS .
  • leukemia refers to a disease of the blood forming tissues characterized by an abnormal increase in the number of leukocytes in the tissues of the body with or without a corresponding increase of those in the circulating blood.
  • Leukemia of the present invention includes lymphocytic (lymphoblastic) leukemia and myelogenous (myeloid or nonlymphocytic) leukemia.
  • Exemplary types of leukemia include, but are not limited to, chronic lymphocytic leukemia, (CLL), acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) [also known as acute myelogenous leukemia (AML), acute nonlymphocytic leukemia (ANLL) and acute myeloblastic leukemia (AML)] .
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • AML acute myeloid leukemia
  • AML acute myelogenous leukemia
  • ANLL acute nonlymphocytic leukemia
  • AML acute myeloblastic leukemia
  • acute leukemia means a disease that is characterized by a rapid increase in the numbers of immature blood cells that transform into malignant cells, rapid progression and accumulation of the malignant cells, which spill into the bloodstream and spread to other organs of the body.
  • chronic leukemia means a disease that is characterized by the excessive build up of relatively mature, but abnormal, white blood cells.
  • myelodysplastic syndrome can refer to a heterogeneous group of closely related clonal hematopoietic disorders. All are characterized by a hypercellular or hypocellular marrow with impaired morphology and maturation (dysmyelopoiesis) and peripheral blood cytopenias, resulting from ineffective blood cell production. All three cell lineages in myeloid hematopoiesis can be involved, including erythrocytic, granulocytic, and megakaryocytic cell lines.
  • the diagnosis of myeloid malignancy can be to further diagnose subtypes of disease.
  • the diagnosis utilizes additional tests in combination, such as blood chemistry, cytology, and genetic analysis.
  • additional diagnostic tests may include red cell mass determination (for polycythemia), bone marrow aspirate and trephine biopsy, arterial oxygen saturation and carboxyhaemoglobin level, neutrophil alkaline phosphatase level, vitamin B12 (or B12 binding capacity) and serum urate. Genetic tests have proven to be increasingly important in diagnosis.
  • CML Chronic Myelogenous Leukemia
  • Philadelphia chromosome BCR-ABL translocation which has three breakpoints: u-BCR-ABL (p230): leads to CML with usual neutrophilia and basophilia minor-BCR-ABL (pl90): leads to CML which has a tendency to become acute lymphoblastic leukemia (ALL) usually precursor B ALL and rarely precursor T ALL
  • ALL acute lymphoblastic leukemia
  • major-BCR-ABL p210
  • normal usual breakpoint Essential thrombocythemia (ET) ET is associated with the JAK2V617F mutation in up to 55% of cases and with an MPL (thrombopoietin receptor) mutation in up to 5% of cases: [0360]
  • Cellular phase increased large megakaryocytes with fibrosis and little increase in other bone marrow elements
  • Fibrotic phase collagenous fibrosis with lack of marrow elements Polycythemia Vera (PV)
  • PV is associated most often with the JAK2V617F mutation in greater than 95% of cases, whereas the remainder have a JAK2 exon 12 mutation:
  • Fibrotic phase collagenous fibrosis with lack of marrow elements
  • PMF Primary Myelofibrosis
  • PMF is associated with the JAK2V617F mutation in up to 50% of cases, the JAK2 exon 12 mutations in 1-2% of cases, and the MPL (thrombopoietin receptor) mutation in up to 5% of cases:
  • Cellular phase increased megakaryocytes which cluster, reticulin fibrosis, later trichrome (collagenous) fibrosis, and increased myeloid precursors
  • Fibrotic phase collagenous fibrosis with lack of marrow elements Refractory Anemia with Ring Sideroblasts Associated with Marked Thrombocytosis
  • RARS-T is often considered a myeloid malignancy. Diagnosis of RARS-T may traditionally involve hematology and cytology, analysis of bone marrow, and lack of karyotype abnormalities such as del (5q), t(3;3)(q21;q26) or inv(3)(q21;q26). See Broseus et al., "Clinical features and course of refractory anemia with ring sideroblast associated with marked thrombocytosis" Haematologica 9(7): 1036-1041 (2012).
  • myeloid malignancy While the type of myeloid malignancy guides diagnosis and treatment, individual malignancies may have specific mutations that further determine the prognosis and course of treatment. Genetic markers are particularly useful because they often illuminate the underlying pathogenesis of the disease.
  • treatment and prevention aspects are carried out by the administration of at least one inhibitor to at least one of the components of the MMEJ pathway.
  • MMEJ pathway As mentioned, treatment and prevention aspects are carried out by the administration of at least one inhibitor to at least one of the components of the MMEJ pathway.
  • MMEJ microhomology-mediated end joining
  • MMEJ can occur at any time of the cell cycle and is independent of core NHEJ and HR factors, i.e. Ku70, Ligase IV and Rad52 genes. Instead MMEJ initiation relies on its own set of proteins, the most important ones being the components of the MRN complex (MRX in yeast) comprising Mrell, Rad50 and Nbsl (Xrs2 in yeast), also implicated in the first steps of HR. Apart from the MRN complex many other factors have been proposed to participate in MMEJ, e.g.
  • CTBP- interacting protein CtIP; Yun and Hiom, 2009
  • PARP1 poly (ADP-ribose) polymerase 1
  • ligase III/Xrcc 1 complex ligase I
  • DNA polymerase Q DNA polymerase Q
  • ERCCl/XPF complex ERCCl/XPF complex
  • the DSBs are recognized by PARP1 which then initiates their repair through MMEJ.
  • the repair process similarly to HR, starts with 5' to 3' end resection, which exposes short regions of homology on each side of the break.
  • This processing step is conducted by the MRN complex and regulated by CtIP.
  • the complementary regions present in the 3' ssDNA fragments
  • pair together and the non-complementary segments (flaps) are removed, probably by the ERCCl/XPF complex.
  • Gaps are then filled in by a polymerase (e.g. DNA polymerase Q or d) and breaks joined by the ligase I or ligase III/Xrcc 1 complex.
  • a polymerase e.g. DNA polymerase Q or d
  • Table 2 below lists some of the key genes in the MMEJ pathway, see also Figure 5, with exemplary inhibitors in Table 1 above.
  • Ligase III [protein SEQ ID NO.: 53-57] Ligase III, DNA, ATP-Dependent
  • Xrccl [protein SEQ ID NO.: 58] X-ray repair complementing defective repair in Chinese hamster cells 1
  • DNA-damage checkpoint 1 // Mediator of DNA damage checkpoint protein 1
  • Down regulation in the context of the present invention means that expression or activity of the target gene is reduced, such as by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more in the presence of the inhibitor as compared to the level of expression or activity in the absence of the inhibitor (i.e., control).
  • Complete inhibition means that there is no detectable expression or activity of the target gene such as qualified at the RNA or protein level or appropriate activity assay e.g., DNA repair activity.
  • inhibitor can also be referred to collectively as an “agent”.
  • Non-limiting examples of inhibitors of a components of the MMEJ pathway are described in details hereinbelow.
  • the inhibitor directly downregulates an activity or expression of a component of the MMEJ pathway.
  • the term “directly” means that the inhibitor directly interacts with a component of the MMEJ pathway nucleic acid sequence or protein and not on a co-factor, an upstream activator or downstream effector of the component of the MMEJ pathway. Such an agent typically blocks the MMEJ activity in the cell.
  • the inhibitor refers to a specific inhibitor having a specific activity on a single component of the MMEJ pathway.
  • the inhibitor refers to a non-specific inhibitor having a non-specific activity on a number of components of the MMEJ pathway (e.g., Mirin, see Table 1 above).
  • the agent indirectly downregulates an activity or expression of a component of the MMEJ pathway.
  • directly means that the agent acts upon a co factor, an upstream activator or downstream effector of the component of the MMEJ pathway.
  • the inhibitor can be a small molecule inhibitor such as Rucaparib camsylate and ML216, see Table 1 above.
  • agents capable of downregulating a component of the MMEJ pathway may be any molecule which binds to and/or cleaves the protein (e.g. component of the MMEJ pathway).
  • Such molecules can be small molecules, antagonists, or inhibitory peptides.
  • a non-functional analogue of at least a catalytic or binding portion of the component of the MMEJ pathway can be also used as an agent.
  • Additional agents capable of inhibiting the MMEJ pathway include antibodies, antibody fragments, and ap tamers.
  • the antibody specifically binds at least one epitope of the component of the MMEJ pathway.
  • epitope refers to any antigenic determinant on an antigen to which the paratope of an antibody binds.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three dimensional structural characteristics, as well as specific charge characteristics.
  • an antibody or antibody fragment capable of specifically binding the component of the MMEJ pathway is typically an intracellular antibody or is modified to cross the cell membrane (e.g., with a cell penetrating moiety such as a cell penetrating peptide (CPP) which is relevant to any agent which is incapable of crossing the cell membrane.
  • a cell penetrating moiety such as a cell penetrating peptide (CPP) which is relevant to any agent which is incapable of crossing the cell membrane.
  • CPP cell penetrating peptide
  • aptamer refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein.
  • specific molecular target such as a protein.
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • SELEX Systematic Evolution of Ligands by Exponential Enrichment
  • Down-regulation at the nucleic acid level is typically effected using a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same.
  • the nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se.
  • RNA silencing refers to a group of regulatory mechanisms [e.g. RNA interference (RNAi), transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS), quelling, co-suppression, and translational repression] mediated by RNA molecules which result in the inhibition or "silencing" of the expression of a corresponding protein coding gene.
  • RNA silencing has been observed in many types of organisms, including plants, animals, and fungi.
  • RNA silencing agent refers to an RNA which is capable of specifically inhibiting or “silencing" the expression of a target gene.
  • the RNA silencing agent is capable of preventing complete processing (e.g, the full translation and/or expression) of an mRNA molecule through a post-transcriptional silencing mechanism.
  • RNA silencing agents include non-coding RNA molecules, for example RNA duplexes comprising paired strands, as well as precursor RNAs from which such small non-coding RNAs can be generated.
  • Exemplary RNA silencing agents include dsRNAs such as siRNAs, miRNAs and shRNAs.
  • the RNA silencing agent is capable of inducing RNA interference.
  • the RNA silencing agent is capable of mediating translational repression.
  • the RNA silencing agent is specific to the target RNA (i.e., component of the MMEJ pathway) and does not cross inhibit or silence other targets or a splice variant which exhibits 99% or less global homology to the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global homology to the target gene; as determined by PCR, Western blot, Immunohistochemistry and/or flow cytometry.
  • RNA interference refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs).
  • RNA silencing agents that can be used according to specific embodiments of the present invention.
  • DsRNA, siRNA and shRNA - The presence of long dsRNAs in cells stimulates the activity of a ribonuclease III enzyme referred to as dicer.
  • Dicer is involved in the processing of the dsRNA into short pieces of dsRNA known as short interfering RNAs (siRNAs).
  • Short interfering RNAs derived from dicer activity are typically about 21 to about 23 nucleotides in length and comprise about 19 base pair duplexes.
  • the RNAi response also features an endonuclease complex, commonly referred to as an RNA-induced silencing complex (RISC), which mediates cleavage of single- stranded RNA having sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA takes place in the middle of the region complementary to the antisense strand of the siRNA duplex.
  • RISC RNA-induced silencing complex
  • some embodiments of the invention contemplate use of dsRNA to downregulate protein expression from mRNA.
  • dsRNA longer than 30 bp are used.
  • dsRNA is provided in cells where the interferon pathway is not activated, see for example Billy et al., PNAS 2001, Vol 98, pages 14428- 14433. and Diallo et al, Oligonucleotides, October 1, 2003, 13(5): 381-392. doi: 10.1089/154545703322617069.
  • the long dsRNA are specifically designed not to induce the interferon and PKR pathways for down-regulating gene expression.
  • Shinagwa and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have developed a vector, named pDECAP, to express long double-strand RNA from an RNA polymerase II (Pol II) promoter. Because the transcripts from pDECAP lack both the 5'-cap structure and the 3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm, long ds-RNA from pDECAP does not induce the interferon response.
  • siRNAs small inhibitory RNAs
  • siRNA refers to small inhibitory RNA duplexes (generally between 18-30 base pairs) that induce the RNA interference (RNAi) pathway.
  • RNAi RNA interference
  • siRNAs are chemically synthesized as 21mers with a central 19 bp duplex region and symmetric 2-base 3'-overhangs on the termini, although it has been recently described that chemically synthesized RNA duplexes of 25-30 base length can have as much as a 100-fold increase in potency compared with 21mers at the same location.
  • RNA silencing agent of some embodiments of the invention may also be a short hairpin RNA (shRNA).
  • RNA agent refers to an RNA agent having a stem-loop structure, comprising a first and second region of complementary sequence, the degree of complementarity and orientation of the regions being sufficient such that base pairing occurs between the regions, the first and second regions being joined by a loop region, the loop resulting from a lack of base pairing between nucleotides (or nucleotide analogs) within the loop region.
  • the number of nucleotides in the loop is a number between and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to 11. Some of the nucleotides in the loop can be involved in base-pair interactions with other nucleotides in the loop.
  • RNA silencing agents suitable for use with some embodiments of the invention can be effected as follows. First, the component of the MMEJ pathway mRNA sequence is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3’ adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex [Tuschl ChemBiochem. 2:239-245].
  • UTRs untranslated regions
  • siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5’ UTR mediated about 90 % decrease in cellular GAPDH mRNA and completely abolished protein level ( w w w . ambion . com/techlib/tn/91/912.html) .
  • potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www(dot)ncbi(dot)nlm(dot)nih.gov/BLAST/). Putative target sites which exhibit significant homology to other coding sequences are filtered out.
  • Qualifying target sequences are selected as template for siRNA synthesis.
  • Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %.
  • Several target sites are preferably selected along the length of the target gene for evaluation.
  • a negative control is preferably used in conjunction.
  • Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome.
  • a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.
  • siRNAs directed against a component of the MMEJ pathway can be commercially obtained from Santa Cruz Biotechnology, Inc.
  • RNA silencing agent of some embodiments of the invention need not be limited to those molecules containing only RNA, but further encompasses chemically-modified nucleotides and non-nucleotides. miRNA and miRNA mimics - According to another embodiment the RNA silencing agent may be a miRNA.
  • miRNA refers to a collection of non-coding single- stranded RNA molecules of about 19-28 nucleotides in length, which regulate gene expression. miRNAs are found in a wide range of organisms (viruses. fwdarw.humans) and have been shown to play a role in development, homeostasis, and disease etiology.
  • the pri-miRNA is typically part of a polycistronic RNA comprising multiple pri-miRNAs.
  • the pri-miRNA may form a hairpin with a stem and loop.
  • the stem may comprise mismatched bases.
  • the hairpin structure of the pri-miRNA is recognized by Drosha, which is an RNase III endonuclease. Drosha typically recognizes terminal loops in the pri-miRNA and cleaves approximately two helical turns into the stem to produce a 60-70 nucleotide precursor known as the pre-miRNA. Drosha cleaves the pri-miRNA with a staggered cut typical of RNase III endonucleases yielding a pre-miRNA stem loop with a 5' phosphate and ⁇ 2 nucleotide 3' overhang. It is estimated that approximately one helical turn of stem ( ⁇ 10 nucleotides) extending beyond the Drosha cleavage site is essential for efficient processing. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by Ran-GTP and the export receptor Ex-portin-5.
  • the double- stranded stem of the pre-miRNA is then recognized by Dicer, which is also an RNase III endonuclease. Dicer may also recognize the 5' phosphate and 3' overhang at the base of the stem loop. Dicer then cleaves off the terminal loop two helical turns away from the base of the stem loop leaving an additional 5' phosphate and ⁇ 2 nucleotide 3' overhang.
  • the resulting siRNA- like duplex which may comprise mismatches, comprises the mature miRNA and a similar-sized fragment known as the miRNA*.
  • the miRNA and miRNA* may be derived from opposing arms of the pri-miRNA and pre-miRNA. miRNA* sequences may be found in libraries of cloned miRNAs but typically at lower frequency than the miRNAs.
  • RISC RNA-induced silencing complex
  • the miRNA strand of the miRNA:miRNA* duplex When the miRNA strand of the miRNA:miRNA* duplex is loaded into the RISC, the miRNA* is removed and degraded.
  • the strand of the miRNA:miRNA* duplex that is loaded into the RISC is the strand whose 5' end is less tightly paired. In cases where both ends of the miRNA:miRNA* have roughly equivalent 5' pairing, both miRNA and miRNA* may have gene silencing activity.
  • the RISC identifies target nucleic acids based on high levels of complementarity between the miRNA and the mRNA, especially by nucleotides 2-7 of the miRNA.
  • miRNAs may direct the RISC to downregulate gene expression by either of two mechanisms: mRNA cleavage or translational repression.
  • the miRNA may specify cleavage of the mRNA if the mRNA has a certain degree of complementarity to the miRNA. When a miRNA guides cleavage, the cut is typically between the nucleotides pairing to residues 10 and 11 of the miRNA.
  • the miRNA may repress translation if the miRNA does not have the requisite degree of complementarity to the miRNA. Translational repression may be more prevalent in animals since animals may have a lower degree of complementarity between the miRNA and binding site.
  • any pair of miRNA and miRNA* there may be variability in the 5’ and 3’ ends of any pair of miRNA and miRNA*. This variability may be due to variability in the enzymatic processing of Drosha and Dicer with respect to the site of cleavage. Variability at the 5’ and 3’ ends of miRNA and miRNA* may also be due to mismatches in the stem structures of the pri-miRNA and pre-miRNA. The mismatches of the stem strands may lead to a population of different hairpin structures. Variability in the stem structures may also lead to variability in the products of cleavage by Drosha and Dicer.
  • miRNA mimic refers to synthetic non-coding RNAs that are capable of entering the RNAi pathway and regulating gene expression. miRNA mimics imitate the function of endogenous miRNAs and can be designed as mature, double stranded molecules or mimic precursors (e.g., or pre-miRNAs). miRNA mimics can be comprised of modified or unmodified RNA, DNA, RNA-DNA hybrids, or alternative nucleic acid chemistries (e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA)).
  • nucleic acid chemistries e.g., LNAs or 2'-0,4'-C-ethylene-bridged nucleic acids (ENA)
  • the length of the duplex region can vary between 13-33, 18-24 or 21-23 nucleotides.
  • the miRNA may also comprise a total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.
  • the sequence of the miRNA may be the first 13-33 nucleotides of the pre-miRNA.
  • the sequence of the miRNA may also be the last 13-33 nucleotides of the pre-miRNA.
  • Preparation of miRNAs mimics can be effected by any method known in the art such as chemical synthesis or recombinant methods. It will be appreciated from the description provided herein above that contacting cells with a miRNA may be effected by transfecting the cells with e.g. the mature double stranded miRNA, the pre-miRNA or the pri-miRNA.
  • the pre-miRNA sequence may comprise from 45-90, 60-80 or 60-70 nucleotides.
  • the pri-miRNA sequence may comprise from 45-30,000, 50-25,000, 100-20,000, 1,000- 1,500 or 80-100 nucleotides.
  • Antisense - Antisense is a single stranded RNA designed to prevent or inhibit expression of a gene by specifically hybridizing to its mRNA. Downregulation of a component of the MMEJ pathway can be effected using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding component of the MMEJ pathway.
  • the first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide which specifically binds the designated mRNA within cells in a way which inhibits translation thereof .
  • the prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Jaaskelainen et al. Cell Mol Biol Lett. (2002) 7(2):236-7; Gait, Cell Mol Life Sci. (2003) 60(5):844-53; Martino et al. J Biomed Biotechnol. (2009) 2009:410260; Grijalvo et al. Expert Opin Ther Pat. (2014) 24(7):801- 19; Falzarano et al, Nucleic Acid Ther. (2014) 24(1):87-100; Shilakari et al. Biomed Res Int. (2014) 2014: 526391; Prakash et al. Nucleic Acids Res. (2014) 42(13):8796-807 and Asseline et al. J Gene Med. (2014) 16(7-8): 157-65].
  • nucleic acid agents can also operate at the DNA level as summarized infra.
  • Downregulation of component of the MMEJ pathway can also be achieved by inactivating the gene coding for a component of the MMEJ pathway via introducing targeted mutations involving loss-of function alterations (e.g. point mutations, deletions and insertions) in the gene stmcture.
  • loss-of function alterations e.g. point mutations, deletions and insertions
  • loss-of-function alterations refers to any mutation in the DNA sequence of a gene (i.e., coding for a component of the MMEJ pathway) which results in downregulation of the expression level and/or activity of the expressed product, i.e., the mRNA transcript and/or the translated protein.
  • Non-limiting examples of such loss-of-function alterations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the enzymatic activity
  • loss-of-function alteration of a gene may comprise at least one allele of the gene.
  • allele refers to any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
  • loss-of-function alteration of a gene comprises both alleles of the gene.
  • the component of the MMEJ pathway may be in a homozygous form or in a heterozygous form.
  • Genome Editing using engineered endonucleases - this approach refers to a reverse genetics method using artificially engineered nucleases to cut and create specific double-stranded breaks at a desired location(s) in the genome, which are then repaired by cellular endogenous processes such as, homology directed repair (HDS) and non-homologous end-joining (NFfEJ).
  • HDS homology directed repair
  • NFfEJ non-homologous end-joining
  • HDR utilizes a homologous sequence as a template for regenerating the missing DNA sequence at the break point.
  • a DNA repair template containing the desired sequence must be present during HDR.
  • Genome editing cannot be performed using traditional restriction endonucleases since most restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location.
  • restriction enzymes recognize a few base pairs on the DNA as their target and the probability is very high that the recognized base pair combination will be found in many locations across the genome resulting in multiple cuts not limited to a desired location.
  • ZFNs Zinc finger nucleases
  • TALENs transcription-activator like effector nucleases
  • CRISPR/Cas system CRISPR/Cas system.
  • Meganucleases are commonly grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family and the HNH family. These families are characterized by structural motifs, which affect catalytic activity and recognition sequence. For instance, members of the LAGLIDADG family are characterized by having either one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are widely separated from one another with respect to conserved structural elements and, consequently, DNA recognition sequence specificity and catalytic activity. Meganucleases are found commonly in microbial species and have the unique property of having very long recognition sequences (>14bp) thus making them naturally very specific for cutting at a desired location.
  • DNA interacting amino acids of the meganuclease can be altered to design sequence specific meganucleases (see e.g., US Patent 8,021,867).
  • Meganucleases can be designed using the methods described in e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Patent Nos. 8,304,222; 8,021,867; 8, 119,381; 8, 124,369; 8, 129,134; 8,133,697; 8,143,015; 8,143,016; 8, 148,098; or 8, 163,514, the contents of each are incorporated herein by reference in their entirety.
  • meganucleases with site specific cutting characteristics can be obtained using commercially available technologies e.g., Precision Biosciences' Directed Nuclease EditorTM genome editing technology.
  • ZFNs and TALENs Two distinct classes of engineered nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have both proven to be effective at producing targeted double- stranded breaks (Christian et ah, 2010; Kim et ah, 1996; Li et ak, 2011; Mahfouz et ak, 2011; Miller et al., 2010).
  • ZFNs and TALENs restriction endonuclease technology utilizes a non-specific DNA cutting enzyme which is linked to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively).
  • a restriction enzyme whose DNA recognition site and cleaving site are separate from each other is selected. The cleaving portion is separated and then linked to a DNA binding domain, thereby yielding an endonuclease with very high specificity for a desired sequence.
  • An exemplary restriction enzyme with such properties is Fokl. Additionally Fokl has the advantage of requiring dimerization to have nuclease activity and this means the specificity increases dramatically as each nuclease partner recognizes a unique DNA sequence.
  • Fokl nucleases have been engineered that can only function as heterodimers and have increased catalytic activity.
  • the heterodimer functioning nucleases avoid the possibility of unwanted homodimer activity and thus increase specificity of the double-stranded break.
  • ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind adjacent sequences at the targeted site.
  • the nucleases bind to their target sites and the Fokl domains heterodimerize to create a double- stranded break. Repair of these double- stranded breaks through the nonhomologous end-joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair made by NHEJ is unique, the use of a single nuclease pair can produce an allelic series with a range of different deletions at the target site.
  • NHEJ nonhomologous end-joining
  • deletions typically range anywhere from a few base pairs to a few hundred base pairs in length, but larger deletions have successfully been generated in cell culture by using two pairs of nucleases simultaneously (Carlson et ak, 2012; Lee et ak, 2010).
  • the double- stranded break can be repaired via homology directed repair to generate specific modifications (Li et ak, 2011; Miller et a , 2010; Umov et ak, 2005).
  • ZFNs rely on Cys2- His2 zinc fingers and TALENs on TALEs. Both of these DNA recognizing peptide domains have the characteristic that they are naturally found in combinations in their proteins. Cys2-His2 Zinc fingers typically found in repeats that are 3 bp apart and are found in diverse combinations in a variety of nucleic acid interacting proteins. TALEs on the other hand are found in repeats with a one-to-one recognition ratio between the amino acids and the recognized nucleotide pairs.
  • Zinc fingers correlated with a triplet sequence are attached in a row to cover the required sequence
  • OPEN low-stringency selection of peptide domains vs. triplet nucleotides followed by high- stringency selections of peptide combination vs. the final target in bacterial systems
  • ZFNs can also be designed and obtained commercially from e.g., Sangamo BiosciencesTM (Richmond, CA).
  • TALEN Method for designing and obtaining TALENs are described in e.g. Reyon et al. Nature Biotechnology 2012 May;30(5):460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39 (12): e82 and Zhang et al. Nature Biotechnology (2011) 29 (2): 149-53.
  • a recently developed web-based program named Mojo Hand was introduced by Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (can be accessed through www(dot)talendesign(dot)org).
  • TALEN can also be designed and obtained commercially from e.g., Sangamo BiosciencesTM (Richmond, CA).
  • CRISPR-Cas system Many bacteria and archea contain endogenous RNA-based adaptive immune systems that can degrade nucleic acids of invading phages and plasmids. These systems consist of clustered regularly interspaced short palindromic repeat (CRISPR) genes that produce RNA components and CRISPR associated (Cas) genes that encode protein components.
  • CRISPR clustered regularly interspaced short palindromic repeat
  • Cas CRISPR associated genes that encode protein components.
  • the CRISPR RNAs (crRNAs) contain short stretches of homology to specific viruses and plasmids and act as guides to direct Cas nucleases to degrade the complementary nucleic acids of the corresponding pathogen.
  • RNA/protein complex RNA/protein complex and together are sufficient for sequence- specific nuclease activity: the Cas9 nuclease, a crRNA containing 20 base pairs of homology to the target sequence, and a trans-activating crRNA (tracrRNA) (Jinek et al. Science (2012) 337: 816-821.).
  • gRNA chimeric guide RNA
  • transient expression of Cas9 in conjunction with synthetic gRNAs can be used to produce targeted double- stranded brakes in a variety of different species (Cho et al., 2013; Cong et al., 2013; DiCarlo et al., 2013; Hwang et al., 2013a, b; Jinek et al., 2013; Mali et al., 2013).
  • the CRIPSR/Cas system for genome editing contains two distinct components: a gRNA and an endonuclease e.g. Cas9.
  • the gRNA is typically a 20 nucleotide sequence encoding a combination of the target homologous sequence (crRNA) and the endogenous bacterial RNA that links the crRNA to the Cas9 nuclease (tracrRNA) in a single chimeric transcript.
  • the gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement genomic DNA.
  • the genomic target sequence must also contain the correct Protospacer Adjacent Motif (PAM) sequence immediately following the target sequence.
  • PAM Protospacer Adjacent Motif
  • the binding of the gRNA/Cas9 complex localizes the Cas9 to the genomic target sequence so that the Cas9 can cut both strands of the DNA causing a double-strand break.
  • the double- stranded brakes produced by CRISPR/Cas can undergo homologous recombination or NHEJ.
  • the Cas9 nuclease has two functional domains: RuvC and HNH, each cutting a different DNA strand. When both of these domains are active, the Cas9 causes double strand breaks in the genomic DNA.
  • CRISPR/Cas A significant advantage of CRISPR/Cas is that the high efficiency of this system coupled with the ability to easily create synthetic gRNAs enables multiple genes to be targeted simultaneously. In addition, the majority of cells carrying the mutation present biallelic mutations in the targeted genes.
  • nickases Modified versions of the Cas9 enzyme containing a single inactive catalytic domain, either RuvC- or HNH-, are called ‘nickases’. With only one active nuclease domain, the Cas9 nickase cuts only one strand of the target DNA, creating a single-strand break or 'nick'. A single-strand break, or nick, is normally quickly repaired through the HDR pathway, using the intact complementary DNA strand as the template. However, two proximal, opposite strand nicks introduced by a Cas9 nickase are treated as a double-strand break, in what is often referred to as a 'double nick' CRISPR system.
  • a double-nick can be repaired by either NHEJ or HDR depending on the desired effect on the gene target.
  • using the Cas9 nickase to create a double-nick by designing two gRNAs with target sequences in close proximity and on opposite strands of the genomic DNA would decrease off- target effect as either gRNA alone will result in nicks that will not change the genomic DNA.
  • dCas9 Modified versions of the Cas9 enzyme containing two inactive catalytic domains
  • dCas9 can be utilized as a platform for DNA transcriptional regulators to activate or repress gene expression by fusing the inactive enzyme to known regulatory domains.
  • the binding of dCas9 alone to a target sequence in genomic DNA can interfere with gene transcription.
  • both gRNA and Cas9 should be expressed in a target cell.
  • the insertion vector can contain both cassettes on a single plasmid or the cassettes are expressed from two separate plasmids.
  • CRISPR plasmids are commercially available such as the px330 plasmid from Addgene.
  • “Hit and run” or “in-out” - involves a two-step recombination procedure.
  • an insertion-type vector containing a dual positive/negative selectable marker cassette is used to introduce the desired sequence alteration.
  • the insertion vector contains a single continuous region of homology to the targeted locus and is modified to carry the mutation of interest.
  • This targeting construct is linearized with a restriction enzyme at a one site within the region of homology, electroporated into the cells, and positive selection is performed to isolate homologous recombinants. These homologous recombinants contain a local duplication that is separated by intervening vector sequence, including the selection cassette.
  • targeted clones are subjected to negative selection to identify cells that have lost the selection cassette via intrachromosomal recombination between the duplicated sequences.
  • the local recombination event removes the duplication and, depending on the site of recombination, the allele either retains the introduced mutation or reverts to wild type. The end result is the introduction of the desired modification without the retention of any exogenous sequences.
  • the “double-replacement” or “tag and exchange” strategy - involves a two-step selection procedure similar to the hit and run approach, but requires the use of two different targeting constructs.
  • a standard targeting vector with 3' and 5' homology arms is used to insert a dual positive/negative selectable cassette near the location where the mutation is to be introduced.
  • homologously targeted clones are identified.
  • a second targeting vector that contains a region of homology with the desired mutation is electroporated into targeted clones, and negative selection is applied to remove the selection cassette and introduce the mutation.
  • the final allele contains the desired mutation while eliminating unwanted exogenous sequences.
  • Site-Specific Recombinases The Cre recombinase derived from the PI bacteriophage and Flp recombinase derived from the yeast Saccharomyces cerevisiae are site-specific DNA recombinases each recognizing a unique 34 base pair DNA sequence (termed “Lox” and “FRT”, respectively) and sequences that are flanked with either Lox sites or FRT sites can be readily removed via site-specific recombination upon expression of Cre or Flp recombinase, respectively. . Basically, the site specific recombinase system offers means for the removal of selection cassettes after homologous recombination.
  • Transposases refers to an enzyme that binds to the ends of a transposon and catalyzes the movement of the transposon to another part of the genome.
  • transposon refers to a mobile genetic element comprising a nucleotide sequence which can move around to different positions within the genome of a single cell. In the process the transposon can cause mutations and/or change the amount of a DNA in the genome of the cell.
  • transposon systems that are able to also transpose in cells e.g. vertebrates have been isolated or designed, such as Sleeping Beauty [Izsvak and Ivies Molecular Therapy (2004) 9, 147-156] , piggyBac [Wilson et al. Molecular Therapy (2007) 15, 139-145], Tol2 [Kawakami et al. PNAS (2000) 97 (21): 11403-11408] or Frog Prince [Miskey et al. Nucleic Acids Res. Dec 1, (2003) 31(23): 6873-6881].
  • DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner.
  • Genome editing using recombinant adeno-associated virus (rAAV) platform is based on rAAV vectors which enable insertion, deletion or substitution of DNA sequences in the genomes of live mammalian cells.
  • the rAAV genome is a single-stranded deoxyribonucleic acid (ssDNA) molecule, either positive- or negative-sensed, which is about 4.7 kb long.
  • ssDNA deoxyribonucleic acid
  • These single-stranded DNA viral vectors have high transduction rates and have a unique property of stimulating endogenous homologous recombination in the absence of double-strand DNA breaks in the genome.
  • rAAV genome editing has the advantage in that it targets a single allele and does not result in any off- target genomic alterations.
  • rAAV genome editing technology is commercially available, for example, the rAAV GENESISTM system from HorizonTM (Cambridge, EiK).
  • Methods for qualifying efficacy and detecting sequence alteration include, but not limited to, DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis.
  • Sequence alterations in a specific gene can also be determined at the protein level using e.g. chromatography, electrophoretic methods, immunodetection assays such as ELISA and western blot analysis and immunohistochemistry.
  • Assays for testing MMEJ activity are well known in the art and include, but are not limited to DNA sequencing, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, plasmid based MMEJ reporter assays.
  • the inhibitors of some embodiments of the invention can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • a "pharmaceutical composition” refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.
  • active ingredient refers to the inhibitor of a component of the MMEJ pathway accountable for the biological effect.
  • physiologically acceptable carrier and “pharmaceutically acceptable carrier” which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound.
  • An adjuvant is included under these phrases.
  • excipient refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient.
  • excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.
  • Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, inrtaperitoneal, intranasal, or intraocular injections.
  • neurosurgical strategies e.g., intracerebral injection or intracerebroventricular infusion
  • molecular manipulation of the agent e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB
  • pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers)
  • the transitory disruption of the integrity of the BBB by hyperosmotic disruption resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide).
  • each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.
  • tissue refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.
  • compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.
  • the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient.
  • Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores.
  • Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • compositions which can be used orally include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers.
  • the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • a suitable propellant e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • compositions described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative.
  • the compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes.
  • Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran.
  • the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.
  • a suitable vehicle e.g., sterile, pyrogen-free water based solution
  • compositions of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.
  • compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients i.e., the inhibitor) effective to prevent, alleviate or ameliorate symptoms of a disorder or prolong the survival of the subject being treated.
  • the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays.
  • a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.
  • Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals.
  • the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human.
  • the dosage may vary depending upon the dosage form employed and the route of administration utilized.
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l).
  • Dosage amount and interval may be adjusted individually to provide pre-leukemic cells (e.g.
  • MEC minimal effective concentration
  • dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.
  • compositions to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.
  • compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may, for example, comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drag Administration for prescription drugs or of an approved product insert.
  • Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.
  • the present invention in order to enhance prevention of the myeloid malignancy, further envisions administering to the subject an additional therapy which may benefit treatment.
  • an additional therapy which may benefit treatment.
  • One of skill in the art is capable of making such a determination.
  • compositions described herein may be administered in conjunction with dietary supplements, hormonal therapy, targeted therapy, immunotherapy, chemotherapy, radiation therapy or surgical procedures.
  • anti-cancer therapies and methods of utilizing same are well known to one of skill in the art.
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range.
  • the phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • any Sequence Identification Number can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.
  • Biological samples from Myelofibrosis (MF) patients were collected with informed consent according to procedures approved by the Research Ethics Board of the University Health Network (REB 01-0573-C).
  • Mobilized peripheral blood autologous transplant products were collected with informed consent according to procedures approved by the University health network ethics committee protocol # 15-9633, and Weizmann institute of science IRB protocol #337-1.
  • Targeted genome editing in K562 cells was performed by using CRISPR Cas9 system according to protocols described elsewhere 28,29 with slight modifications.
  • 20bp sgRNA sequences were designed around the genomic loci of interest using DESKGEN algorithm (www(dot)deskgen(dot)com/landing/#/login) (Extended Data Table 1).
  • Sense and antisense oligonucleotides for each sgRNA with overhangs compatible to Bbsi- digested px330 were designed and ordered from Integrated DNA Technologies (IDT) wwwdotidtdnadotcom.
  • ASXL1 sgRNA sequences CTGCAGGTCCGAGGGGCGAG (SEQ ID NO: 76), CAGTGGTGGCCGCCTCTCTA (SEQ ID NO: 77), AGGTCACCACTGCCATAGAG (SEQ ID NO: 78), TGGCCGCCTCTCTATGGCAG (SEQ ID NO: 79), TCACCACTGCCATAGAGAGG (SEQ ID NO: 80), CCCCCCTCCGATGGCAGTGG (SEQ ID NO: 81), CGGCCACCACTGCCATCGGA (SEQ ID NO: 82).
  • SRSF2 sgRNA sequences TGCGGGGTGGCGGTCCCCGG (SEQ ID NO: 105), GACTCACACCACAGCCGCCG (SEQ ID NO: 83), CGGACTCACACCACAGCCGC (SEQ ID NO: 84),
  • CALR sgRNA sequences ACAAACAGGACGAGGAGCAG (SEQ ID NO: 90), GGACGAGGAGCAGAGGCTTA (SEQ ID NO: 91), CGAGGAGCAGAGGCTTAAGG (SEQ ID NO: 92), AGAAGACAAGAAACGCAAAG (SEQ ID NO: 93),
  • Each oligo pair was further phosphorylated and annealed using T4 PNK (NEB) and T4 Ligation Buffer (NEB). Phosphorylation and annealing reaction was performed at 37°C for 30 min, followed by 95°C for 5 min and ramping down to 25°C at 5°C /min. Annealed oligo pairs were then ligated into a previously Bbsi digested px330 plasmid. Per reaction, 50ng digested px330 was mixed with 1:250 diluted oligo duplex with 2X quick ligation buffer (NEB) and quick ligase (NEB) at 16°C overnight.
  • NEB quick ligation buffer
  • NEB quick ligase
  • BioSuper DH5a competent cells (Biolab) were transformed with Px330. Bacteria was resuspended and plated on LB agar AMP dishes and incubated at 37°C over-night. Colonies were then screened and grown in 2-3 ml LB + Ampicillin at 37°C overnight in a shaker (250rpm). For each colony (guide), plasmid DNA was extracted using the QIAprep Spin Miniprep standard protocol (Qiagen, cat. No. 27104). To validate the presence of the desired inserts, Sanger sequencing reactions were performed for each plasmid using the U6 promoter primer ACTATCATATGCTTACCGTAAC (SEQ ID No: 98). 2.2 Electroporation reactions
  • Electroporation reactions on primary CD34+ enriched and K562 cells were performed by using a synthetic ASXL1 targeted sgRNA ordered from IDT and resuspended in IDTE buffer to a final concentration of lOOuM. K562 sequential sgRNA experiments, were done using purified px330 plasmids at 2ug/reaction. For each unique sample per electroporation, a control sample containing the same cell amount with no RNP/plasmid, underwent electroporation at the same conditions was sequenced.
  • K562 cell line experiments using px330 plasmids were performed according to the manufacture recommendations. Briefly, K562 cells were cultured in ATCC-formulated Iscove's Modified Dulbecco's Medium containing 10%FBS and 1% pen-strep and split to 300,000 cells/ml 48h prior to electroporation. On electroporation day, cells were counted and resuspended in SF nucleofector solution. 200,000 cells /reaction were resuspended in 20ul SF solution and mixed with 2ug px330 plasmids.
  • K562 cell line experiments involving synthetic sgRNA targeted to ASXL1 were done according to IDT (reference above) recommendations.
  • ASXL1 synthetic sgRNA was ordered from IDT according to the following sequence: AGGTCACCACTGCCATAGAG (SEQ ID NO: 78).
  • RNP complexes were generated by mixing 2.1ul PBS, 1.2ul lOOuM sgRNA and 1.7ul synthetic CAS9 (IDT) per reaction, followed by incubation at room temp for 10-20 min. Complexed RNPs were then transformed to 4°C. 5pl of complexed RNP were mixed with 20 pi SF solution containing 200,000 K562 cells Per reaction as was described.
  • CD34+ enrichment and electroporation was done according to IDT protocol “Electroporation of primary human CD34+ hematopoietic stem and progenitor cells”. 48 hours prior to electroporation, CD34+ cells were isolated from mononuclear cells derived from mobilized PBSC autologous transplant products by using CD34 Miltenyi Biotec MicroBead kit. Cell were re-plated in cytokine rich medium in a 24-well plate to reach a density of 250,000cells/ml.
  • CD34+ cells cytokine rich medium was based on SFEMII supplemented with SCF (100 ng/mL), TPO (100 ng/mL), Flt3- Ligand (100 ng/mL), IL-6 (100 ng/mL), streptomycin (20 mg/mL), and penicillin (20 unit/mL).
  • Cells were incubated in a low-oxygen, tissue culture incubator (37°C, 5% C02, 5% 02) according to IDT protocol. 24h prior to electroporation, cells were counted and cell density was maintained below 10 L 6 cells/ml. On electroporation day, cells were washed with PBS and counted.
  • CD34+ cells were resuspended in 20ul P3 nucleofector solution and mixed with 5ul RNP complexes that were generated as described under section 1.3. Program DZ-100 was used. Following electroporation, cells were washed with 80pl pre- warmed cytokine rich medium and transferred into a U-bottom 96-well plate containing lOOul pre- warmed cytokine rich medium. CD34+ cells were cultured for 2 additional days in a low-oxygen incubator followed by a lysate generation that was subsequent to library preparations.
  • K562 cells were cultured in 96-well plates in 200ul medium/reaction. Cells in four different wells were irradiated with gradient dosages of 0, 5, 10 and 20Gy. Cells were cultured for additional four days. Then, lysate was extracted for targeted sequencing.
  • Dual indexed illumina Libraries were generated using two-step PCR procedure. 1 st PCR primer prefix sequences and 2 nd PCR primer sequences were, as previously described 30 with a modified protocol. In short, target- specific primers were designed by Primer3plus (wwwdotbioinformaticsdotnl/cgi-bin/primer3plus/primer3plusdotcgi) and were ordered with the described 5’ prefixes 30 (IDT) ASXL1 forward primer:
  • CTACACGACGCTCTTCCGATCTaccctcgcagacattaaagc (SEQ ID NO: 99)
  • ASXL1 reverse primer CAGACGTGTGCTCTTCCGATCTgtagatctgacgtacactttcca (SEQ ID NO: 100)
  • SRSF2 forward primer CTACACGACGCTCTTCCGATCTctcagccccgtttacctg (SEQ ID NO: 101)
  • SRSF2 reverse primer CAGACGTGTGCTCTTCCGATCTctgaggacgctatggatg (SEQ ID NO: 102)
  • CALR forward primer CALR forward primer:
  • PCR was applied to target the regions of interest.
  • the reaction mixture was composed of a PCR ready mix (using NEBNext® UltraTM II Q5® Master Mix, NEB), lysate product and a final primer concentration of luM each.
  • PCR protocol was as follows: 98°C for 30 sec, followed by 40 amplification cycles of 98°C for 10 sec, 65°C for 30 sec and a final elongation at 65°C for 5 min.
  • a 2 nd PCR was performed using primers composed of Illumina sequencing primers, indexes and adapters, under the same conditions as the 1 st PCR with the exceptions of final primer concentration of 0.5 mM each and 20 cycles of amplifications. Samples were pooled together at equal volume. Pooled library sizes were selected (2% gel, BluePippin, Sage Science) and sent for 2 x 150-bp deep sequencing (Miseq, Illumina).
  • argetCreator’ and TndelRe aligner’ commands 32 were generated by samtools 1.8 followed by SNVs and small indels detection using varscan2.3.9 ‘pileup2cns’ command to generate VCF files containing consensus variant calls 33 .
  • Short indels at positions around polyG sequences position 20:31022441 in ASXL1 and 17:74732955 in SRSF2
  • Deletion frequencies from bulk cells were obtained by dividing variant allele frequency by the sum of all deletions allele frequencies per sample.
  • a threshold deletion VAF > 20% was set for each single cell derived colony. Frequency of colonies carrying deletions were obtained by dividing colony numbers carrying each variant (at >20% VAF) by the total colonies number.
  • MMEJ deletion signature from all CRISPR experiments were defined as MH>3bp and no mismatches were allowed.
  • Fluorescence-activated cell sorting of human stem/progenitor and mature cell populations was performed on mononuclear cells from peripheral blood of two MF patients according to the sorting strategy described in details elsewhere 34 .
  • Animal experiments were performed in accordance to the IACUC of the Weizmann Institute, its relevant guidelines and regulations (11790319-2).
  • Eight- to 12-week-old female NOD/SCID/IL-2Rgc-null (NSG) mice were sublethally irradiated (225 cGy) 24 hours before transplantation.
  • CD34+ cells were enriched by magnetic beads (Miltneyi Inc.) and 50,000 cells were injected into the right femur as previously described 34 .
  • mice were euthanized 16 weeks following transplantation and human engraftment in the injected right femur and non-injected bone marrow (left femur, tibias) was evaluated by flow cytometry. Subpopulations were sorted as previously described 34 . ddPCR reaction was performed by using probes designed for CALR deletion as described elsewhere 35 . Amplified DNA (2ul from a 1:20 dilution of a 16 h REPLI-g Mini Kit whole- genome amplification, Qiagen) from each sorted population was tested in a 96-well plate in duplicate according to the manufacturer’s protocol.
  • Mutant and wild-type sequences were read using a droplet reader with a two-color fluorescein/HEX fluorescence detector (Bio-Rad).
  • the mutant allele frequency was calculated as the fraction of mutant-positive droplets divided by total droplets containing a target. As previously reported 3 the minimum detection level was 1:1,000 (0.1%). Variants were considered present if there were at least three dots in the mutant fluorescein channel resulting in VAF > 0.1%.
  • COSMIC mutation data containing data from both targeted and genome wide screens was downloaded from www(dot)cancer(dot)sanger(dot)ac(dot)uk/cosmic/download.
  • Deletion table was generated by filtering for rows containing the letters ‘del’ in ’Mutation CDS’ column using ‘awk’ command. Additional filtering for rows containing genomic coordinates in column ‘Mutation genome position’ was done. This resulted in a table of 210,442 reported deletions.
  • MMEJ signature detection was done using an in-house matlab code that analyzed each deletion’s flanking sequences for microhomologies (MHs) according to the ‘MMEJ signatures’ described in Figure IB.
  • MMEJ deletions included the following criteria:
  • FIG. 1A a table of all myeloid deletions was generated by filtering the ‘Primary site’ column of the final table to include ’haematopoietic and lymphoid’ tissue deletions followed by the exclusion of the letters ‘lymph’ from the ‘Primary histology’ column.
  • Signature types were assessed for deletions reported in 10 or more unique samples and a single nucleotide mismatch was allowed in MMEJ signature (mainly in the case of CALR c.l091_1142del52).
  • Published sequencing data was analyzed from healthy individuals where the canonical MMEJ deletion in ASXL1 was identified as occurring in three of 124 pre-AML cases 10.7, 8.8 and 1.7 years prior to AML diagnoses (not shown) and none among the 676 controls 4 .
  • HSCs long-lived HSCs are the cell of origin for canonical MMEJ deletion in ASXL1.
  • VAFs variant allele frequencies of the canonical MMEJ CALR deletion were analyzed in isolated HSPCs and mature cells from two different myelofibrosis (MF) donors.
  • CALR deletion was identified among HSCs, more committed progenitors, and mature myeloid and lymphoid cells (Figure 4A).
  • CD34 positive cells were transplanted from one of the donors into NOD/SCID/IL-2Rgc-null (NSG) mice and after 16 weeks observed a multi-lineage graft.
  • MMEJ repair was shown to be active when the c-NHEJ pathway is knocked out 20,21 , it is possible that HSPCs with low c-NHEJ expression could be primed for MMEJ repair. However, these results could not identify the exact cell population at risk for MMEJ error-prone repair.
  • MMEJ DNA repair pathway leads to pre leukemic deletions it was assumed that enhanced MMEJ pathway is crucial for the survival of pre-leukemic and leukemic hematopoietic stem cells and that inhibiting MMEJ components in these cells will result in pre-leukemic and leukemic cells enhanced apoptosis.
  • the present inventors used xenograft assays in which human CD34+ cells are injected into the right femur of eight- to 12-week-old female NOD/SCID/IL-2Rgc-null (NSG) mice that were sublethally irradiated (225 cGy) 24 hours before transplantation.
  • Mice are euthanized 16 weeks following transplantation and human engraftment (which represents the human pre-leukemic clone) in the injected right femur and non-injected bone marrow (left femur, tibias) is evaluated by flow cytometry.
  • the mutational burden of this human graft is further assessed using Next generation sequencing.
  • mice By treating the mice with different compounds (MMEJ inhibitor) prior to injection it is possible to assess the inhibition of the human pre-leukemic clone by the change in both engraftment capacity and variant allele frequencies of pre-leukemic mutations in the human graft (treated VS non-treated mice).
  • MMEJ inhibitor MMEJ inhibitor

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Abstract

Un inhibiteur d'un composant de la voie de jonction d'extrémité à médiation par la microhomologie (MMEJ) est destinée à être utilisée dans le traitement ou la prévention d'une malignité myéloïde ou myéloïde chez un sujet en ayant besoin, la malignité myéloblastique ou myéloïde n'étant pas la leucémie myéloïde chronique (LMC)
PCT/IL2020/051130 2019-10-30 2020-10-29 Inhibiteurs de la voie mmej pour la prévention et le traitement de malignités myéloïdes et pré-myéloïdes WO2021084540A1 (fr)

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IL270306 2019-10-30
IL270306A IL270306A (en) 2019-10-30 2019-10-30 Prevention and treatment of pre-myeloid and myeloid malignancies

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CN113797338B (zh) * 2021-09-10 2022-07-15 中国医学科学院基础医学研究所 Dbc1调控细胞衰老及其应用

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