WO2001025405A1 - C-myc antisense-treated hematopoietic stem cell composition and method - Google Patents

C-myc antisense-treated hematopoietic stem cell composition and method Download PDF

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WO2001025405A1
WO2001025405A1 PCT/US2000/027311 US0027311W WO0125405A1 WO 2001025405 A1 WO2001025405 A1 WO 2001025405A1 US 0027311 W US0027311 W US 0027311W WO 0125405 A1 WO0125405 A1 WO 0125405A1
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hsc
hematopoietic stem
antisense oligomer
cells
population
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French (fr)
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Stephen H. Bartelmez
Patrick L. Iversen
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Avi Biopharma, Inc.
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Priority to CA002386192A priority Critical patent/CA2386192A1/en
Priority to JP2001528559A priority patent/JP2003527096A/en
Priority to EP00968646A priority patent/EP1224261A1/en
Priority to AU78529/00A priority patent/AU785056B2/en
Publication of WO2001025405A1 publication Critical patent/WO2001025405A1/en

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1135—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against oncogenes or tumor suppressor genes
    • 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
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0647—Haematopoietic stem cells; Uncommitted or multipotent progenitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development

Definitions

  • the present invention relates to methods for increasing the number of hematopoietic stem cells (HSC) in vitro and in vivo by exposing the cells to a c-myc antisense oligonucleotide and to methods for treatment of such HSC in a manner effective to increase the population of lineage committed progenitor cells and their progeny.
  • the invention further relates compositions of HSC produced by such methods.
  • the hematopoietic stem cell is a pluripotent progenitor cell that has been characterized as a cell that is transplantable, can self-replicate and has multilineage potential. Differentiation of HSCs results in a loss of such multilineage potential, and corresponding lineage commitment. It has been demonstrated that self-renewal of HSC occurs in vivo, as indicated by transplantation studies wherein a single HSC repopulated the marrow of an immunodeficient mouse (Smith, et al., 1991 ; Osawa, et al, 1996). It has also been demonstrated that hematopoietic stem cells can be infected with recombinant retroviruses, and can serve as cellular targets for gene therapy (Keller and Snodgrass, 1990).
  • patients suffering from various cell-based diseases including, but not limited to, myeloproliferative diseases, blood cell prohferative diseases and autoimmune diseases often have an imbalance in the number of cells of particular lineages.
  • patients undergoing chemotherapy or irradiation often have defective hematopoiesis.
  • High-dose chemotherapy and/or radiation therapy together with bone marrow transplantation or transplantation of a cell population enriched for hematopoietic stem cells are standard treatment regimens for some malignancies, including acute lymphocytic leukemia, chronic myelogenous leukemia, neuroblastoma, lymphoma, breast cancer, colon cancer, lung cancer and myelodysplastic syndrome, as well as for other non-malignant hematopoietic diseases, e.g. thrombocytopenia.
  • Clinical trials are underway using such regimens for the treatment of various cancers, including ovarian cancer, thymomas, germ cell tumors, multiple myeloma, melanoma, testicular cancer, lung cancer, and brain tumors.
  • the percentage of cells in a cell population enriched for hematopoietic stem cells that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need for techniques for purification and expansion of hematopoietic stem cells.
  • Complications of transplantation with a cell population enriched for HSC have been observed due to graft versus host disease (GVHD) in allogeneic transplants, and residual tumor cells in autologous and allogeneic transplants that may result in recurrence of disease.
  • Current treatment regimens are directed to removal of cells that pose a risk to the transplant recipient, including T-cells and residual tumor cells.
  • antisense oligonucleotides and antibodies can specifically interfere with synthesis of a target protein of interest. Due to their hydrophobicity, antisense oligonucleotides interact well with phospholipid membranes (Akhtar, S., et al, 1991), and it has been suggested that following the interaction with the cellular plasma membrane, oligonucleotides are actively transported into living cells (Loke, S.L., et al, 1989; Yakubov, L.A., et al, 1989; Anderson, CM., et al, 1999).
  • oligonucleotides Although inhibition of genes associated with cellular development has been achieved using antisense technology, naturally occurring oligonucleotides have a nuclease-sensitive phosphodiester backbone. However, naturally occurring oligonucleotides have been rendered resistant to degradation by nucleases, e.g., by utilizing a methylphosphonate, phosphorothioate or phosphorodiamidate linkage instead of a phosphodiester one (Spitzer and Eckstein, 1988; Baker, et al. 1990; Hudziak, 1996). Phosphorodiamidate morpholino oligonucleotides (PMOs) have been demonstrated to exhibit high binding affinity for RNA targets with favorable uptake into cells and minimal nonspecific binding interactions. (See, e.g., Summerton, et al., 1997a).
  • a synthetic phosphorothioate antisense oligonucleotide specific to the actylcholinesterase gene has been described as a tool for inhibiting (1) abnormal hematopoiesis, (2) abnormal platelet proliferation, (3) increasing macrophage and hematopoietic stem cell counts, (4) reducing the immune response to allogeneic organ transplants, and (5) for treating malignant tumors (U.S. Pat. No. 5,891,725).
  • c-myc is a proto-oncogene which regulates cell growth, differentiation, and apoptosis, and its aberrant expression has been associated with a number of human cancers including lung cancer, colorectal cancer, breast cancer, bladder cancer, leukemia, lung cancer, etc.
  • Transplantation of hematopoietic stem cells derived from peripheral blood and/or bone marrow is increasingly used in combination with chemotherapy and/or radiation therapy for the treatment of a variety of disorders including numerous forms of cancer.
  • the percentage of cells in such transplants that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need to develop techniques for purification and expansion of hematopoietic stem cells.
  • the invention provides a method of producing a hematopoietic stem cell (HSC) composition prepared by carrying out the steps of obtaining an HSC-containing cell population from a subject, treating the cell population in manner effective to enrich for HSC; and exposing the enriched HSC population, ex vivo to a c-myc antisense oligomer, at a concentration and for a period of time sufficient to increase the number of hematopoietic stem cells at least two-fold, preferably at least four-fold and more preferably at least 8-fold or more.
  • the HSC are characterized as lacking the expression of lineage markers (lin-), positive for cell surface expression of c-kit (CD 1 17) and positive for cell surface expression of Sea 1.
  • the HSC-containing cell population for practicing the method is obtained from human bone marrow or mobilized human peripheral blood.
  • the subject is a human cancer patient and the patient has lymphoma or leukemia.
  • the c-myc antisense oligomers for use in practicing the invention are typically characterized by one or more of: (1 ) a length of about 12 to 25 bases; (2) a backbone which is substantially uncharged; (3) the ability to hybridize with the complementary sequence of a target RNA with high affinity at a Tm greater than 50°C, (4) nuclease resistance, (5) the capability for active or facilitated transport into cells, and (6) a structure selected from the group consisting of the structures presented in Figures 2 A-A through 2 E-E
  • the antisense oligomer has the sequence identified as SEQ ID NO 1
  • the invention also provides a hematopoietic stem cell (HSC) composition, prepared by the method set forth above for use as a medicament in treating cancer in a subject
  • an expanded HSC population prepared by the method set forth above is further cultured under conditions effective to result in an expanded population of lineage committed progenitor cells and their progeny
  • a method for delivering one or more of (1) a c-myc antisense oligomer- treated hematopoietic stem cell (HSC) composition, (2) a c-myc antisense oligomer, and (3) an expanded population of lineage committed progenitor cells and their progeny to a cancer patient, is ftirther provided by the invention.
  • HSC hematopoietic stem cell
  • Such an ex vivo expanded cell population enriched for hematopoietic stem cells and lineage committed progenitor cells and their progeny may be re-infused into a patient as part of a therapeutic regiment for the treatment of va ⁇ ous malignancies, non-malignant hematopoietic abnormalities, autoimmune disease and to augment vaccines
  • Figure 1A-E show several preferred morphohno subunits having 5-atom (A), six-atom (B) and seven-atom (C-E) linking groups suitable for forming polymers
  • Figures 2A-A through 2E-E show the repeating subunit segment of exemplary morphohno oligonucleotides, constructed using subunits A-E, respectively, of Figure 1
  • Figure 3 depicts the day 0 and day 3 results for high prohferative potential colony forming cell (HPP-CFC) colony formation in an vitro assay wherein single c-k ⁇ t+, Sea 1+ cells were treated with no antisense oligomer, a scrambled antisense oligomer, or 1, 5, 25, or 125 mM c- myc antisense oligomer and cultured for 3 days in the presence of IL-6 and SCF The results are indicated as (A) the total number of HPP-CFC, (B) the frequency of HPP-CFC and (C) the cell number/HPP-CFC frequency
  • Figures 4A-D depict the results of analysis of the pe ⁇ pheral blood of mice treated intraperitoneally on a daily basis for 7 days with various doses (30, 100, 300 or 1000 ⁇ g/day) of a PMO antisense to c-myc, with samples collected at 7, 21 and 28 days
  • the figures represent the number of erythrocytes (A), granulocytes (B), lymphocytes (C), and white blood cells (D) present at each time point
  • a cell population enriched for hematopoietic stem cells refers to the cell population obtained using the positive and negative selection techniques described herein, wherein the hematopoietic stem cells may be LTR- or STR-HSCs.
  • long term repopulating hematopoietic stem cells and "LTR- HSC”, refer to hematopoietic stem cells that are transplantable, and upon differentiation contribute to all lineages of hematopoietic cells for an undefined period of time when transplanted into totally immunosuppressed recipients. LTR-HSC do not undergo clonal extinction.
  • STR-HSC short term repopulating hematopoietic stem cells
  • STR-HSC murine hematopoietic stem cells that are transplantable, and contribute to all lineages of hematopoietic cells for a period of from about one week to 6 months, then undergo clonal extinction when transplanted into immunosuppressed recipients.
  • clonal extinction refers to the terminal differentiation of a single hematopoietic stem cell and all the progeny produced by clonal expansion of that cell, such that no more daughter cells are produced from the initial clone.
  • pluripotent hematopoietic stem cells refers to hematopoietic stem cells, capable of differentiating into all the possible cell lineages.
  • high proliferative potential colony forming cells or "HPP-CFCs”, as used herein relative to hematopoietic stem cells refers to murine cells that proliferate in response to rat rSCF, mouse rIL-3 and human rIL-6.
  • the cells proliferate in semi-solid media, such as agar or methyl cellulose or as single cells in liquid culture, and form macroclones which have a diameter greater than 1 mm, generally having greater than 100,000 cells per clone with dense multicentric centers.
  • This population includes all murine HSCs, however, not all HPP-CFC are HSCs, and the HPP-CFC assay is not a specific assay for LTR-HSC.
  • low proliferative potential (LPP) clones contain from 2 to 100,000 cells per clone.
  • lineage-committed hematopoietic stem cells are hematopoietic stem cells that have differentiated sufficiently to be committed to one or more particular cell lineages, but not all cell lineages.
  • T-cells As used herein, the term 'Tin -" or “lineage-depleted”, refers to a cell population which lacks expression of cell surface antigens specific to T-cells, B-cells, neutrophils, monocytes and erythroid cells, and does not express antigens recognized by the "YW 25.12.7” antibody. (See, e.g., Bertoncello I et al., 1991.)
  • the terms “develop”, “differentiate” and “mature” are used interchangeably and refer to the progression of a cell from a stage of having the potential to differentiate into multiple cellular lineages to becoming a more specialized cell committed to one or more defined lineages.
  • purified relative to hematopoietic stem cells refers to HSCs that have been enriched (isolated or purified) relative to some or all of the other types of cells with which they are normally found in a particular tissue in nature, e.g., bone marrow or peripheral blood.
  • a "purified" population of HSCs has been subjected to density gradient fractionation, lineage depletion and positive selection for c-kit and Sca-1 expression in addition to low level staining with both Hoechst 33342 and Rhodamine 123.
  • antisense oligonucleotide and “antisense oligomer” are used interchangeably and refer to a sequence of nucleotide bases and a subunit-to-subunit backbone that allows the antisense oligomer to hybridize to a target sequence in an RNA by Watson- Crick base pairing, to form an RNA:oligomer heteroduplex within the target sequence.
  • the oligomer may have exact sequence complementarity to the target sequence or near complementarity.
  • antisense oligomers may block or inhibit translation of the mRNA containing the target sequence, or inhibit gene transcription, may bind to double-stranded or single stranded sequences, and may be said to be "directed to" a sequence with which it hybridizes.
  • Exemplary structures for antisense oligonucleotides for use in the invention include the ⁇ - morpholino subunit types shown in Fig 1 A-E. It will be appreciated that a polymer may contain more than one linkage type.
  • Subunit A in Figure 1 contains a 1-atom phosphorous-containing linkage which forms the five atom repeating-unit backbone shown at A-A in Figure 2, where the morphohno rings are linked by a 1-atom phosphonamide linkage.
  • Subunit B in Figure 1 is designed for 6-atom repeating-unit backbones, as shown at B-B, in Figure 2.
  • the atom Y linking the 5' morphohno carbon to the phosphorous group may be sulfur, nitrogen, carbon or, preferably, oxygen.
  • the X moiety pendant from the phosphorous may be any of the following: fluorine; an alkyl or substituted alkyl; an alkoxy or substituted alkoxy; a thioalkoxy or substituted thioalkoxy; or, an unsubstituted, monosubstituted, or disubstituted nitrogen, including cyclic structures.
  • Subunits C-E in Figure 1 are designed for 7-atom unit-length backbones as shown for C-C through E-E in Figure 2.
  • the X moiety is as in Structure B and the moiety Y may be a methylene, sulfur, or preferably oxygen.
  • the X and Y moieties are as in Structure B.
  • X is as in Structure B and Y is O, S, or NR.
  • Z is O or S. and P, or P, is adenine, cytosine, guanine or uracil.
  • a "morphohno oligomer” refers to a polymeric molecule having a backbone which supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose sugar backbone moiety, and more specifically a ribose backbone linked by phosphodiester bonds which is typical of nucleotides and nucleosides, but instead contains a ring nitrogen with coupling through the ring nitrogen.
  • a preferred "morphohno" oligonucleotide is composed of morphohno subunit structures of the form shown in Fig.
  • Fig. 2B shows two such subunits joined by a phosphorodiamidate linkage.
  • Morphohno oligonucleotides are detailed, for example, in co-owned U.S. Patent Nos. 5,698.685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521 ,063, and 5,506,337.
  • nuclease-resistant oligomeric molecule is one whose backbone is not susceptible to nuclease cleavage of a phosphodiester bond.
  • exemplary nuclease resistant antisense oligomers are oligonucleotide analogs, such as phosphorothioate and phosphate-amine DNA (pnDNA), both of which have a charged backbone, and methyl- phosphonate, morphohno. and peptide nucleic acid (PNA) oligonucleotides, all of which may have uncharged backbones.
  • an oligonucleotide or antisense oligomer "specifically hybridizes" to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions, with a Tm substantially greater than 37°C, preferably at least 50° C, and typically 60°C-80°C or higher.
  • Such hybridization preferably corresponds to stringent hybridization conditions, selected to be about 10° C, and preferably about 5° C lower than the thermal melting point ( rrnl) f° r t ne specific sequence at a defined ionic strength and pH.
  • the Tt m ⁇ is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide.
  • Polynucleotides are described as "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides.
  • a double- stranded polynucleotide can be “complementary” to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second.
  • Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules.
  • analog with reference to an oligomer means a substance possessing both structural and chemical properties similar to those of a reference oligomer.
  • a first sequence is an "antisense sequence" with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically binds to, or specifically hybridizes with, the second polynucleotide sequence under physiological conditions.
  • a “base-specific intracellular binding event involving a target RNA” refers to the sequence specific binding of an oligomer to a target RNA sequence inside a cell.
  • a single-stranded polynucleotide can specifically bind to a single-stranded polynucleotide that is complementary in sequence.
  • nuclease-resistant heteroduplex refers to a heteroduplex formed by the binding of an antisense oligomer to its complementary target, which is resistant to in vivo degradation by ubiquitous intracellular and extracellular nucleases.
  • c-myc refers to an oncogene or gene that gives directs cells toward the development and growth of cancer or a tumor
  • c- yc has been associated with gene amplification in various types of cancer, as further detailed below.
  • c-myc antisense oligomer refers to a nuclease-resistant antisense oligomer having high affinity (ie, which "specifically hybridizes") to a complementary or near- complementary c-myc nucleic acid sequence.
  • the term "modulating expression" relative to an oligonucleotide refers to the ability of an antisense oligonucleotide (oligomer) to either enhance or reduce the expression of a given protein by interfering with the expression, or translation of RNA.
  • the antisense oligomer may block expression of a suppressor gene, e.g., a tumor suppressor gene.
  • the antisense oligomer may directly block expression of a given gene, or contribute to the accelerated breakdown of the RNA transcribed from that gene.
  • tumor and cancer refer to a cell that exhibits a loss of growth control and forms unusually large clones of cells. Tumor or cancer cells generally have lost contact inhibition and may be invasive and/or have the ability to metastasize.
  • an antisense oligomer refers to the amount of antisense oligomer administered to a mammalian subject, either as a single dose or as part of a series of doses and which is effective to inhibit expression of a selected target nucleic acid sequence.
  • treatment of an individual or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of e.g., a pharmaceutical composition, and may be performed either prophylactically, or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
  • the term "improved therapeutic outcome" relative to a cancer patient refers to a slowing or diminution of the growth of cancer cells or a solid tumor, or a reduction in the total number of cancer cells or total tumor burden.
  • Antisense oligonucleotides of 15-20 bases are usually long enough to have one complementary sequence in the mammalian genome.
  • antisense compounds having a length of at least 17 nucleotides in length have been demonstrated to hybridize well with their target mRNA (Cohen, et al. , 1991 ).
  • a heteroduplex formed between the oligonucleotide and mRNA is a substrate for RNase H, leading to cleavage of the mRNA.
  • Oligonucleotides belonging, or proposed to belong, to this class include phosphorothioates, phosphotriesters, and phosphodiesters (unmodified "natural" oligonucleotides). Such compounds generally show high activity, and phosphorothioates are currently the most widely employed oligonucleotides in antisense applications.
  • a second class of oligonucleotide analogs termed “steric blockers” or, alternatively, “RNase H inactive” or “RNase H resistant”, have not been observed to act as a substrate for RNase H, and are believed to act by sterically blocking target RNA formation, nucleocytoplasmic transport, or translation.
  • This class includes methylphosphonates (Toulme, et al, 1996), morphohno oligonucleotides, peptide nucleic acids (PNA's), 2'-0-allyl or 2'-0-alkyl modified oligonucleotides (Bonham, 1995), and N3' P5' phosphoramidates (Gee, 1998).
  • Naturally occurring oligonucleotides have a phosphodiester backbone which is sensitive to degradation by nucleases, however, certain modifications of the backbone increase the resistance of native oligonucleotides to such degradation. (See, e.g. , Spitzer and Eckstein 1988).
  • Candidate antisense oligomers are evaluated, according to well known methods, for acute and chronic cellular toxicity, such as the effect on protein and DNA synthesis as measured via incorporation of - ⁇ H-leucine and ⁇ H-thymidine, respectively.
  • control oligonucleotides e.g., control oligonucleotides such as sense, nonsense or scrambled antisense sequences, or sequences containing mismatched bases, in order to confirm the specificity of binding of candidate antisense oligomers.
  • control oligonucleotides such as sense, nonsense or scrambled antisense sequences, or sequences containing mismatched bases.
  • sequences may be modified as needed to limit non-specific binding of antisense oligomers to non-target sequences.
  • the effectiveness of a given antisense oligomer molecule in forming a heteroduplex with the target RNA may be determined by screening methods known in the art. For example, the oligomer is incubated a cell culture expressing c-myc, and the effect on the target RNA is evaluated by monitoring the presence or absence of (1) heteroduplex formation with the target sequence and non-target sequences using procedures known to those of skill in the art, (2) the amount of c-myc mRNA, as determined by standard techniques such as RT-PCR or Northern blot, or (3) the amount of c-myc protein, as determined by standard techniques such as ELISA or Western blotting. (See, for example, Pari, et al, 1995; Anderson, et al, 1996).
  • Exemplary antisense oligomers for use in the methods of the invention include morphohno oligomers (Figs. 2A-D), peptide nucleic acids and methyl phosphonate oligomers.
  • Preferred antisense oligonucleotides are nuclease-resistant oligomers with substantially uncharged backbones, and particularly morpholino oligomers having, in addition to a base sequence complementary to a region of a selected mRNA target sequence, an oligomer backbone, defined by the nucleotide subunits of the oligomer and the linkages between them such that the oligomer can bind the target RNA sequence by Watson-Crick base pairing between complementary bases in the target RNA and the oligomer.
  • the preferred oligomers are composed of morpholino subunits of the form shown in the above cited patents, where (i) the morpholino groups are linked together by substantially uncharged linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) the base attached to the morpholino group is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine.
  • PNAs Peptide Nucleic Acids
  • PNAs are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone. It consists of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm et al, 1993).
  • the backbone of PNAs are formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes which exhibit greater than normal thermal stability.
  • PNAs are not recognized by nucleases or proteases.
  • PNA antisense agents has been observed to display slow membrane penetration in cell cultures, possibly due to poor uptake (transport) into cells. (See, e.g., Ardhammar M et al., 1999).
  • Methylphosphonate oligonucleotides are uncharged and therefore predicted to exhibit enhanced cellular uptake relative to charged oligonucleotides (Blake, et al, 1985). However, methylphosphonate antisense oligomers have been shown to be incapable of inhibiting N-ras expression in vitro (Tidd, et al, 1988).
  • Preferred Antisense Oligomers mRNA transcribed from the relevant region of a gene of interest is generally targeted by antisense oligonucleotides, however, in some cases double-stranded DNA may be targeted using a non-ionic probe designed for sequence-specific binding to major-groove sites in duplex DNA.
  • probe types are described in U.S. Patent No. 5,166,315 (Summerton and Weller, 1992), and are generally referred to herein as antisense oligomers, referring to their ability to block expression of target genes.
  • the antisense oligomer is designed to hybridize to a region of the c-myc nucleic acid sequence, under physiological conditions with a Tm substantially greater than 37°C, e.g. , at least 50°C and preferably 60°C-80°C.
  • the oligomer is designed to have high-binding affinity to the nucleic acid and may be 100% complementary to the c-myc target sequence, or may include mismatches, e.g.. to accommodate allelic variants, as long as the heteroduplex formed between the oligomer and c-myc target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation during its transit from cell to body fluid.
  • Mismatches are less destabilizing toward the end regions of the hybrid duplex than in the middle.
  • the number of mismatches allowed will depend on the length of the oligomer, the percentage of G:C base pair in the duplex and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability.
  • an antisense oligomer is not necessarily 100% complementary to the c-myc target sequence, it is effective to stably and specifically bind to the target sequence such that expression of c-myc is modulated.
  • the appropriate length of the oligomer to allow stable, effective binding combined with good specificity is about 8-40 nucleotide base units, and preferably about 12-25 nucleotides.
  • the target for modulation of gene expression using the antisense methods of the present invention comprises a sequence spanning the mRNA translational start codon for c- myc.
  • other regions of the c-myc mRNA may be targeted, including one or more of, an initiator or promoter site, an intron or exon junction site, a 3 '-untranslated region, and a 5'-untranslated region.
  • both spliced and unspliced RNA may serve as the template for design of antisense oligomers for use in the methods of the invention.
  • Preferred antisense oligomers for use in the methods of the invention preferably, have one or more properties including: ( 1) a backbone which is substantially uncharged (e.g. , Uhlmann, et al, 1990), (2) the ability to hybridize with the complementary sequence of a target RNA with high affinity, that is, Tm substantially greater than 37°C, preferably at least 50° C, and typically 60°C-80°C or higher, (3) a subunit length of at least 8 bases, generally about 8-40 bases, preferably 12-25 bases, (4) nuclease resistance (Hudziak, et al , 1996) and (5) capable of active or facilitated transport as evidenced by (i) competitive binding with a phosphorothioate antisense oligomer, and/or (ii) the ability to transport a detectable reporter into the cells.
  • the antisense oligomer comprises the sequence presented as SEQ ID NO: 1.
  • c-myc c-myc is a proto-oncogene which is the cellular homologue of the transforming gene of the chicken retrovirus MC29. c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. Aberrant, constitutive or overexpression of c-myc has been associated with a number of human cancers including lung cancer, colorectal cancer, breast cancer, bladder cancer, leukemia, lung cancer, etc.
  • Proto-oncogenes are activated to oncogenes by a variety of mechanisms which include: (1 ) promoter insertion, (2) enhancer insertion, (3) chromosomal translocation, (4) gene amplification and (5) point mutation.
  • activation relative to a proto-oncogene means transcription of the gene is increased, e.g.. from no expression to low level expression.
  • Mechanisms (l)-(4) result in an increase in the expression level of an oncogene. while (5) results in expression of an altered gene product.
  • Evidence suggests that some form of oncogene expression together with inactivation of tumor suppressor genes is required for the development of cancer.
  • the myc proto-oncogenes have been described as transcription factors that directly regulate the expression of other genes, examples of which include ECA39, p53, ornithine decarboxylase (ODC), alpha-prothymosin and Cdc25A. (Ben-Yosef, et al, 1998).
  • a provirus In chickens, following infection of chicken B-cells with certain avian leukemia viruses, a provirus becomes integrated near the myc gene, which is activated by a viral long terminal repeat (LTR) that acts either as a promoter or an enhancer, resulting in expression of myc and formation of a B-cell tumor (Murray, et al, 1996). Similarly, in Burkitt's lymphoma, an enhancer sequence is translocated resulting in expression of myc. c-myc is expressed in normal hematopoietic stem cells and has been shown to promote the differentiation of human epidermal stem cells (Gandarillas A and Watt FM, Genes Dev 1 1(21):2869-2882, 1997).
  • LTR viral long terminal repeat
  • c-myc has been shown to be overexpressed or aberrently expressed in numerous cancers. (See, e.g., Bieche I et al., Cancer Res 59(12):2759-65, 1999.) It has been observed that when quiescent cells re-enter the cell cycle c-myc expression is upregulated, and that ectopic expression of c-myc prevents cell cycle arrest in response to growth- inhibitory signals, differentiation stimuli, or mitogen withdrawal.
  • a 20- to 100-fold decrease in c-myc mRNA was detected in the colon cancer cell line and the leukemic cell line, respectively, using competitive reverse transcription-polymerase chain reaction (RT-PCR), in addition to inhibition of cellular proliferation.
  • RT-PCR competitive reverse transcription-polymerase chain reaction
  • a phosphordiamidate oligomer antisense to c-myc has been demonstrated to act at the RNA level to inhibit normal pre-mRNA splicing and to produce an aberrantly spliced mRNA in a sequence specific manner (Hudziak RM et al., 2000).
  • hematopoietic stem cells when an antisense oligomer to c-myc was used to treat an enriched population of hematopoietic stem cells, the inventors discovered that the development of the hematopoietic stem cell population was modulated.
  • a population of pluripotent hematopoietic stem cells may be maintained and expanded by exposue to an antisense oligonucleotide, directed to a region spanning the start codon of an mRNA transcribed from a gene encoding c-myc. It was also discovered that such an expanded population of hematopoietic stem cells may be further treated under conditions effective to result in an expanded population of lineage committed progenitor cells and their progeny.
  • Hematopoietic stem cells for use in the methods of the invention may be derived from human bone marrow, human newborn cord blood, fetal liver, or adult human peripheral blood after appropriate mobilization.
  • hematopoietic stem cells can be dramatically increased by treatment of a subject with certain compounds including cytokines.
  • Such "mobilized" peripheral blood hematopoietic stem cells have become an important alternative to bone marrow-derived hematopoietic stem cells in transplantation procedures primarily because engraftment is more rapid. (See, e.g., Tanaka, et al, 1999).
  • Such mobilization may be accomplished using for example, one or more of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (TPO), and a chemotherapeutic agent (i.e., cyclophosphamide).
  • G-CSF granulocyte colony-stimulating factor
  • SCF stem cell factor
  • TPO thrombopoietin
  • chemotherapeutic agent i.e., cyclophosphamide
  • hematopoietic stem cell isolation Numerous methods for hematopoietic stem cell isolation are known in the art and generally include obtaining hematopoietic stem cells from bone marrow, newborn cord blood, fetal liver or adult human peripheral blood. Once obtained, a hematopoietic stem cell population is enriched by performing one or more of a density gradient separation, immunoaffinity purification using positive and/or negative selection by techniques such as panning, FACS and magnetic bead separation. Following such enrichment steps, the cell population is further characterized phenotypically and functionally.
  • HPP-CFC high proliferative potential colony-forming cells
  • Hematopoietic stem cells have been historically defined as transplantable cells, capable of self-renewal as well as possessing the ability to generate daughter cells of any hematopoietic lineage.
  • Lineage-committed progenitor cells are defined as more differentiated cells derived from hematopoietic stem cells.
  • hematopoietic stem cell The phenotypic markers which characterize the hematopoietic stem cell have been the subject of extensive debate and numerous publications. As yet, there is no consensus as to which markers are definitive for murine or human hematopoietic stem cells.
  • LTR-HSC have been isolated and characterized in mice herein using fluorescence-activated cell sorter (FACS) selection of density gradient-enriched, lineage-depleted bone marrow cells that are negative for expression of the CD34 antigen, positive for expression of the CD1 17 (c- kit) antigen, and exhibit low-level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and the mitochondrial binding dye, Rhodamine 123 (Rh- 123), (Wolf, et al, 1993).
  • FACS fluorescence-activated cell sorter
  • the STR-HSC population may be selected by FACS sorting and is phenotypically defined herein as light density gradient-enriched bone marrow cells which lack the expression of lineage markers (lin-), are positive for c-kit (CD 1 17), Seal and CD34, exhibit low-level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and high-level binding of the mitochondrial binding dye. Rhodamine 123 (Rh-123).
  • Functional readouts that have been used to detect and characterize hematopoietic stem cells include the ability to form colonies under particular assay conditions in cell culture (in vitro).
  • Exemplary assays include the long term culture initiating cell (LTCIC) assay (Pettengell R et al., 1994), and the high proliferative potential-colony-forming cell (HPP-CFC) assay.
  • LTCIC long term culture initiating cell
  • HPP-CFC high proliferative potential-colony-forming cell
  • Further functional characterization includes in vivo assay for long-term repopulating hematopoietic stem cells (LTR-HSC) and short-term repopulating hematopoietic stem cells (STR-HSC), as described above.
  • Hematopoietic stem cells are often functionally characterized by activity in the high proliferative potential colony-forming cell (HPP-CFC) assay, as defined above.
  • HPP-CFC have been characterized by: ( 1 ) a relative resistance to treatment in vivo with the cytotoxic drug 5-fluorouracil; (2) high correlation with cells capable of repopulating the bone marrow of lethally irradiated mice; (3) their ability to generate cells of the macrophage, granulocyte, megakaryocyte and erythroid lineages under appropriate conditions; and (4) their multifactor responsiveness. (See, e.g., McNiece, 1990).
  • Hematopoietic stem cells for use in the methods of the invention are enriched, as described in Example 1.
  • the cells were also characterized functionally in the HPP-CFC assay (in vitro) and in an assay for LTR-HSCs (in vivo), as further described in Example 2.
  • cytokines for the culture of hematopoietic stem cells include one or more of interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin- 1 1 (IL- 1 1), interleukin- 12 (IL- 12), stem cell factor (SCF).
  • IL-3 interleukin-3
  • IL-6 interleukin-6
  • IL- 1 1 interleukin- 1 1
  • IL- 12 interleukin- 12
  • SCF stem cell factor
  • TPO thrombopoietin
  • LTR-HSCs Long-term reconstitution with murine LTR-HSCs following complete immunosuppression has been shown to require the transplantation of unfractionated bone marrow cells together with less differentiated long term repopulating cells, in order to provide initial, albeit unsustained engraftment, which allows the completely immunosuppressed host to survive until the long term repopulating cells differentiate sufficiently to repopulate the host.
  • LTR-HSCs may take several months to effectively repopulate the hematopoietic system of the host following complete immunosuppression.
  • CD45 locus defined as CD45.1 and recipient hematopoietic stem cells defined as CD45.2, such that monoclonal antibodies may be used to distinguish donor and recipient cells, i.e. by FACS analysis and/or sorting.
  • the recipient is infused with sufficient CD45.2 positive bone marrow cells to keep the mouse alive until differentiation of CD45.1 donor cells occur to an extent sufficient to repopulate the hematopoietic system of the recipient.
  • Such methods may be used to differentiate LTR-HSCs from STR-HSCs, and donor cells from recipient cells.
  • the cells may be used immediately or frozen in liquid nitrogen and stored for long periods of time, under standard conditions, such that they can later be thawed and used, e.g., for administration to a patient.
  • the cells will usually be stored in 10% DMSO, 50% fetal calf serum (FCS), and 40% cell culture medium.
  • the invention provides methods and compositions for maintaining and expanding the population of pluripotent hematopoietic stem cells (HSC) by exposing an enriched population of HSC to an antisense oligonucleotide directed against a region spanning the start codon of an mRNA transcribed from a gene encoding c-myc.
  • HSC pluripotent hematopoietic stem cells
  • the antisense oligonucleotide has the sequence presented as SEQ ID NO: 1.
  • the antisense oligonucleotide is substantially uncharged and is further characterized by stability, high Tm, and capable of active or facilitated transport as evidenced by (i) competitive binding with a phosphorothioate antisense oligomer, and/or (ii) the ability to transport a detectable reporter into the cells.
  • Hematopoietic stem cells may be exposed to the c-myc antisense oligomer ex vivo (in vitro) and/or a c-myc antisense oligomer may be administered to a subject in an amount effective to result in an expanded population of hematopoietic stem cells in vivo.
  • hematopoietic stem cells are exposed to a c-myc antisense oligomer in an amount effective to result in an expanded population of hematopoietic stem cells, and further treated under conditions effective to result in an expanded population of lineage committed progenitor cells and their progeny.
  • the invention is based on the discovery that a stable, substantially uncharged antisense oligonucleotide, characterized by high Tm, capable of active or facilitated transport into cells, and capable of binding with high affinity to a complementary or near-complementary c-myc nucleic acid sequence, can be administered to a hematopoietic stem cell, and inhibit expression of c-myc in the cell, either in vitro, or in vivo in a subject, resulting in modulation of the development of the cell.
  • hematopoietic stem cells derived from peripheral blood and/or bone marrow is increasingly used in combination with chemotherapy and/or radiation therapy for the treatment of a variety of disorders including numerous forms of cancer.
  • the percentage of cells in such transplants that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need to develop techniques for purification and expansion of hematopoietic stem cells.
  • Complications of transplantation therapy with a cell population enriched for hematopoietic stem cells include removal of cells in the transplant that pose a risk to the transplant recipient, including T-cells that are responsible for graft versus host disease (GVHD) in allogeneic grafts, and tumor cells in autologous transplants that may cause recurrence of disease.
  • GVHD graft versus host disease
  • Platelets are necessary for effective blood clotting at a site of injury. Frequently, following chemotherapy, radiation therapy, transplantation of a cell population enriched for hematopoietic stem cells or bone marrow transplantation, a patient will suffer from an insufficient platelet count for a time period of from about 4 to 6 weeks, or a longer time period resulting in the patient being easily bruised and excessive bleeding.
  • the invention includes in vitro (ex vivo) exposure of a nuclease-resistant antisense oligomer capable of binding with high affinity to a complementary or near-complementary c-myc sequence to a hematopoietic stem cell, and subsequent modulation of the development of the hematopoietic stem cell in vitro.
  • Such treatment can effectively influence the composition of a culture of hematopoietic stem cells.
  • Hematopoietic stem cells may be treated in vitro (ex vivo) with c-myc antisense oligonucleotides, followed by administration to a subject.
  • the subject may be the same individual from whom the hematopoietic stem cells were obtained (autologous transplantation) or a different individual (allogeneic transplantation).
  • allogeneic transplantation the donor and recipient are matched based on similarity of HLA antigens in order to minimize the immune response of both donor and recipient cells against the other.
  • the invention is directed to methods of modifying the development of hematopoietic stem cells, by obtaining a population of HSCs, exposing them ex vivo to a nuclease-resistant antisense oligomer having high affinity to a complementary or near- complementary c-myc sequence, and subsequently re-infusing the expanded HSC population into a subject.
  • Hematopoietic stem cell populations for use in the methods of the invention are extracted from a subject, purified and cultured ex vivo in the presence of one or more cytokines.
  • Preferred cytokines include IL-3, IL-6, SCF and TPO.
  • a hematopoietic stem cell population for use in the methods of the invention is preferably both human and allogeneic, or autologous.
  • Hematopoietic stem cells may be obtained from a patient in need of hematopoietic stem cell transplantation or from an allogeneic donor.
  • An enriched population of hematopoietic stem cells is exposed to oligomers antisense to c-myc under conditions effective to result in an increase the number of viable hematopoietic stem cells in vitro, in culture.
  • the method of the invention results in an increase in the hematopoietic stem cell population which is at least 2-fold greater than the number of hematopoietic stem cells present prior to exposure to a c-myc antisense oligomer.
  • the treatment of hematopoietic stem cells with a c- yc antisense oligomer using the method described herein results in a hematopoietic stem cell population that is increased at least 4-fold to 8-fold relative to the number of hematopoietic stem cells present prior to exposure to the c- yc antisense oligomer.
  • the treatment of hematopoietic stem cells with a c-myc antisense oligomer using the method described herein results in a hematopoietic stem cell population that is increased more than 8-fold relative to the number of hematopoietic stem cells present prior to exposure to the c-myc antisense oligomer.
  • hematopoietic stem cells are cultured in medium containing a c-myc antisense oligonucleotide together with cytokines or growth factors effective to promote hematopoietic stem cell replication, for a time sufficient to result in an increase in the population of hematopoietic stem cells, without differentiation thereof.
  • the culture time required for a at least a 2-fold, 4-fold, 8-fold, or greater than 8-fold increase in the number of HSC following exposure to medium containing a c-myc antisense oligonucleotide together with cytokines or growth factors effective to promote hematopoietic stem cell replication will vary dependent upon the cell source (including the tissue source and the health of the subject from whom the cells were taken), culture conditions, the percentage of HSC in the enriched population, etc.
  • the present inventions represents two related aspects: (1) a cell population having an increased number of HSC; and (2) a cell population wherein the increased HSC population is differentiated into committed progenitor cells of particular lineages. Longer culture times may result in a cell population with increased numbers of committed progenitor cells of a particular lineage due to differentiation of the HSC originally produced by c-myc antisense treatment of the cell population enriched for HSC.
  • the c-myc antisense oligonucleotides of the invention find utility in methods to increase the number of differentiated and committed progenitor cells of various lineages obtained by differentiation of the increased population of hematopoietic stem cells in vitro. Such an increased population of differentiated and committed progenitor cells may then be re-infused into a patient.
  • hematopoietic stem cells are obtained from a patient in need of transplantation of a particular type of cell, e.g., neutrophils, platelets, lymphocytes, erthryrocytes or monocytes.
  • a particular type of cell e.g., neutrophils, platelets, lymphocytes, erthryrocytes or monocytes.
  • Re-infused ex vivo c-myc antisense oligomer-treated hematopoietic stem cells provide a means to rapidly increase the number of both neutrophils and platelets in the circulation of a patient following chemotherapy or radiation therapy.
  • an enriched population of HSC are obtained, then cultured in medium containing a c-myc antisense oligonucleotide together with one or more growth factors effective to promote hematopoietic stem cell replication without differentiation.
  • the cells are cultured for a time sufficient to result in an increase in the population of hematopoietic stem cells, followed by removal of the antisense oligomer from the culture media and subsequent culture under conditions effective to result in differentiation of the hematopoietic stem cells.
  • an increase in the various cell lineages produced by differentiation of hematopoietic stem cells is obtained from the increased population of hematopoietic stem cells, following removal of the antisense oligomer from the culture media.
  • large numbers of neutrophils and platelets maybe obtained using this procedure.
  • the invention is directed to a method for modifying the development of hematopoietic stem cells, by exposing them in vivo to an antisense oligomer having high affinity to a complementary or near-complementary c-myc sequence.
  • a subject is in need of an increased number of hematopoietic stem cells, e.g., following chemotherapy or radiation therapy.
  • administration of a c-myc antisense oligomer to a subject results in an increase in the number of hematopoietic stem cells in the subject, e.g., following chemotherapy or radiation therapy.
  • a c-myc antisense oligomer is administered to the subject in a manner effective to result in an increase in the number of hematopoietic stem cells in the subject.
  • a subject is in need of an increased number of committed progenitor cells produced by differentiation of hematopoietic stem cells such as neutrophils and/or platelets, e.g., following chemotherapy or radiation therapy.
  • a c-myc antisense oligomer is administered to the subject in a manner effective to result in the differentiation of HSC and a corresponding increase in the number of neutrophils and platelets in the subject.
  • a c-myc antisense oligomer to a subject using the methods described herein can result in an increase in the population of hematopoietic stem cells, and/or the population of lineage committed progenitor cells and their progeny, dependent upon a number of factors including ( 1) the duration, dose and frequency of c-myc antisense administration, and (2) the general condition of the subject.
  • Effective delivery of an antisense oligomer to the target c- yc nucleic acid sequence is an important aspect of the methods of the invention.
  • effective delivery of an oligomer antisense to c-myc may include, but is not limited to, various systemic routes, including oral and parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular; as well as inhalation and transdermal delivery.
  • Transdermal delivery of antisense oligomers may be accomplished by use of a pharmaceutically acceptable carrier adapted for e.g., topical administration.
  • a pharmaceutically acceptable carrier adapted for e.g., topical administration.
  • morpholino oligomer delivery is described in PCT patent application WO 97/40854.
  • the oligomer is a phosphorodiamidate morpholino oligomer (PMO), contained in a pharmaceutically acceptable carrier, and delivered orally.
  • PMO phosphorodiamidate morpholino oligomer
  • a morpholino c-myc antisense oligonucleotide is administered at regular intervals for a short time period, e.g., daily for two weeks or less. However, in some cases the antisense oligomer is administered intermittently over a longer period of time.
  • one or more doses of antisense oligomer are administered, generally at regular intervals for a period of about one to two weeks.
  • Preferred doses for oral administration are from about 1 mg oligomer/patient to about 25 mg oligomer/patient (based on an adult weight of 70 kg). In some cases, doses of greater than 25 mg oligomer/patient may be necessary.
  • the preferred doses are from about 0.5 mg oligomer/patient to about 10 mg oligomer/patient (based on an adult weight of 70 kg).
  • the antisense compound is generally administered in an amount sufficient to result in a peak blood concentration of at least 200-400 nM c-myc antisense oligomer.
  • the method comprises administering to a subject, in a suitable pharmaceutical carrier, an amount of the antisense agent effective to inhibit expression of the c-myc nucleic acid target sequence.
  • a suitable pharmaceutical carrier may include any of a variety of standard physiologically acceptable carriers employed by those of ordinary skill in the art.
  • Such pharmaceutical carriers include, but are not limited to, saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions such as oil/water emulsions, triglyceride emulsions, wetting agents, tablets and capsules. It will be understood that the choice of suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.
  • liposomes may be employed to facilitate uptake of the antisense oligonucleotide into cells.
  • Hydrogels may also be used as vehicles for antisense oligomer administration, for example, as described in WO 93/01286.
  • the oligonucleotides may be administered in microspheres or microparticles. (See, e.g., Wu and Wu, (1987).
  • Sustained release compositions are also contemplated within the scope of this application. These may include semipermeable polymeric matrices in the form of shaped articles such as films or microcapsules.
  • the subject is a human subject.
  • the subject may also be a cancer patient, in particular a patient diagnosed as having a form of leukemia, lymphoma, neuroblastoma breast cancer, colon cancer, lung cancer, or any type of cancer where the patient is being treated or has been treated with chemotherapy or radiation therapy.
  • the method is also applicable to treatment of non-cancerous hematopoietic disorders, including, but not limited to, aplastic anemia, severe combined immunodeficiency, sickle cell anemia, thalassemia and myelodysplastic syndromes.
  • the effective in vivo dose of a c-myc antisense oligonucleotide for use in the methods of the invention will vary according to the frequency and route of administration as well as the condition of the subject under treatment. Accordingly, such in vivo therapy will generally require monitoring by tests appropriate to the condition being treated and a corresponding adjustment in the dose or treatment regimen in order to achieve an optimal therapeutic outcome.
  • the invention provides a method to produce an increased population of more differentiated cells derived from an increased population of hematopoietic stem cells, such as monocytes, granulocytes, platelets, lymphocytes and red blood cells, by exposing a hematopoietic stem cell to the antisense oligomers described herein, under conditions effective to increase the population of hematopoietic stem cells, then further treating the increased population of hematopoietic stem cells in a manner effective to promote differentiation of the hematopoietic stem cells to cell having a more mature phenotype.
  • hematopoietic stem cells such as monocytes, granulocytes, platelets, lymphocytes and red blood cells
  • a therapeutic regimen may include administration of c-myc antisense treated HSC and/or direct administration of a c-myc antisense oligomer to the subject. Such administration may be concurrent or sequential.
  • the patient may be further treated by one or more of chemotherapy, radiation therapy and other agents typically used by those of skill in the art to treat individuals diagnosed with the same or a similar condition.
  • Such further treatment may be concurrent with, sequential or alternating relative to administration of c-myc antisense treated HSC and/or direct administration of a c-myc antisense oligomer to the subject.
  • the efficacy of a given therapeutic regimen involving the methods described herein may be monitored, e.g., by conventional FACS assays for the phenotype of cells in the circulation of the subject under treatment in order to monitor changes in the numbers of cells of various lineages in response to antisense treatment.
  • Phenotypic analysis is generally carried out using monoclonal antibodies specific to the cell type being analyzed.
  • the use of monoclonal antibodies in such phenotypic analyses is routinely employed by those of skill in the art for cellular analyses.
  • Monoclonal antibodies specific to particular cell types are commercially available.
  • Hematopoietic stem cells are characterized phenotypically as detailed above. Such phenotypic analyses are generally carried out in conjunction with biological assays for each particular cell type of interest, for example (1) hematopoietic stem cells (LTCIC, cobblestone forming assays, and assays for HPP-CFCs), (2) granulocytes or neutrophils (clonal agar or methyl cellulose assays wherein the medium contains G-CSF or GM-CSF), (3) megakaryocytes
  • erythroid cells clonal agar or methyl cellulose assays wherein the medium contains EPO and SCF or EPO, SCF and IL-3).
  • the status of the cancer is also monitored using diagnostic techniques appropriate to the type of cancer under treatment.
  • the antisense oligomer treatment regimen may be adjusted (dose, frequency, route, etc.), as indicated, based on the results of the phenotypic and biological assays described above.
  • hematopoietic stem cells treated with a c-myc antisense oligonucleotide find utility in a variety of applications.
  • treatment of HSC with a c-myc antisense oligonucleotide yields an in vitro expanded hematopoietic stem cells, which may be used for a variety of purposes.
  • such a population of in vitro expanded hematopoietic stem cells serve as a source for production of committed progenitor cells and their progeny, e.g., neutrophils or platelets.
  • Ex vivo expanded cell HSC and committed progenitor cell populations find utility in both autologous and allogeneic hematopoietic engraftment when administered to a patient, where the cells are freed of neoplastic cells and graft-versus-host disease can be avoided.
  • hematopoietic stem cells may be cultured ex vivo in the presence of one or more cytokines and a c-myc antisense oligomer.
  • an antisense oligomer-treated hematopoietic stem cell culture finds utility in a variety of applications, including, but not limited to: (1) expanding or multiplying the population of hematopoietic stem cells ex vivo for subsequent in vivo administration to a subject for purposes of (a) hematopoietic stem cell replacement therapy, (b) treatment of autoimmune disease, (c) reducing the immune response to allogeneic transplants, or (d) treatment of HIV-infection in a subject; and (2) inhibiting or arresting growth of cancer cells wherein the cancer is associated with c-myc expression.
  • hematopoietic stem cells may be cultured ex vivo in the presence of one or more cytokines and a c-myc antisense oligomer for a time sufficient to increase the population of particular lineage-committed progenitor cells and their progeny.
  • This process may be carried out ex vivo followed by in vivo administration to a subject for (a) cell replacement therapy, and (b) augmenting vaccination.
  • Autologous hematopoietic stem cell transplantation has been used to treat many solid tumors, including, but not limited to, breast cancer and ovarian cancer.
  • a chemotherapy regimen to reduce the amount of tumor present, generally followed by: (1) the collection of the patient's hematopoietic stem cells from either bone marrow or peripheral blood; (2) culture of hematopoietic stem cells in the presence of cytokines or cryopreservation in liquid nitrogen; (3) high-dose chemotherapy administration intravenously (in most cases);and (4) reinfusion of the patient's hematopoietic stem cells (IV), after the chemotherapy administration is complete; and (5) further treatment of the patient with growth factors to promote the differentiation of the hematopoietic stem cells and repopulation of the hematopoietic system of the patient.
  • the patient is immunocompromised and protective isolation is required.
  • Allogeneic hematopoietic stem cell transplantation has been used to treat patients with leukemia, aplastic anemia, lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), and immune deficiency diseases.
  • An allogeneic hematopoietic stem cell transplantation protocol is similar to that used for autologous transplantation with the exception that in allogeneic transplantation, the donor and recipient must be matched based on the similarity of HLA cell surface antigens in order to minimize the immune response of both donor and recipient cells against the other.
  • hematopoietic stem cells together with committed progenitor cells and their progeny prior to transplantation can shorten the lag period during which the transplanted cells proliferate sufficiently to repopulate the hematopoietic system of the recipient.
  • a c-myc antisense oligomer of the invention may be used for the treatment of a potential bone marrow hematopoietic stem cell donor, prior to the removal of the bone marrow, in order to increase the population of hematopoietic stem cells in the tissue prior to bone marrow removal.
  • the methods of the invention can be used to expose bone marrow or purified hematopoietic stem cell populations to a c-myc antisense oligomer of the invention, prior to their storage in cell banks in order to retain the cells in a form devoid of tissue histocompatibility antigens.
  • the subject is a cancer patient, and the treatment of the patent's hematopoietic stem cells with a c-myc antisense oligomer is used to inhibit or arrest the growth of cancer cells, wherein the cancer is associated with c-myc expression. Once the cells have been treated with c- yc antisense oligomers.
  • the cells may either be (1) directly infused into the patient, and/or (2) be cultured under conditions effective to result in differentiation of the hematopoietic stem cells to produce an expanded population of lineage committed progenitor cells and the progeny thereof, which are then infused into the patient.
  • duration of in vitro culture and the time of exposure to c-myc antisense may be varied to result in different ratios of hematopoietic stem cells and lineage committed progenitor cells and their progeny, prior to re-infusion into the subject.
  • a therapeutic regimen involving such ex vivo c-myc antisense treatment of hematopoietic stem cells for treatment of cancer further includes additional intervention such as radiation therapy and/or chemotherapy.
  • the treatment may occur prior to, during or subsequent to re-infusion of c-myc antisense-treated cells.
  • a transplant recipient's own bone marrow or hematopoietic stem cells e.g., bone marrow extracted from the patient before commencing chemotherapy or radiation therapy
  • a c-myc antisense oligomer may be employed to avoid the need for immune suppression typical of current methodologies, by minimizing the potential for GVHD following transplantation.
  • c-myc antisense oligomers may also be used to treat tissue and organ donors as well as transplant recipients in order to minimize GVHD.
  • GVHD is a frequent complication of allogeneic transplantation. About half of the patients undergoing an allogeneic bone marrow transplant develop some GVHD, which is generally mild, however, the condition can be life threatening. In GVHD, the donor's cells attacks the recipient's organs and tissue. Patients with GVHD have increased susceptibility to infection and the skin, liver, and gastrointestinal tract may be attacked in GVHD.
  • GVHD graft versus host disease
  • T-cells which recognize the patient's cells as foreign, based on differences in human leukocyte antigens (HLA).
  • HLA human leukocyte antigens
  • graft versus host disease GVHD
  • treatment to minimize the GVH response is part of the therapeutic regimen for most transplants.
  • hematopoietic stem cell transplantation such treatment generally includes T- cell depletion alone (i.e., by elutriation which removes T-cells based on density gradient centrifugation), or in combination with hematopoietic stem cell enrichment, and drug therapy for prevention of GVHD.
  • Exemplary drug therapy for GVHD is administration of cyclosporine (an immunosuppressive drug), alone or together with mehtotrexate.
  • the invention provides a population of c-myc antisense treated hematopoietic stem cells, such that transplantation of the hematopoietic stem cells into an allogeneic host is unlikely to result in GVHD.
  • the invention provides a population of lineage-committed progenitor cells and their progeny, derived from a population of c-myc antisense treated hematopoietic stem cells such that transplantation of the lineage-committed progenitor cells and their progeny into an allogeneic host is unlikely to result in GVHD. While the mechanism is not part of the invention, such a population of c-myc antisense treated hematopoietic stem cells is expected to lack immunological memory of self and non-self antigens, thereby minimizing the likelihood of GVHD. Such c-myc antisense oligomer treated cells may be used for allogeneic transplantation in order to minimize or eliminate graft versus host disease (GVHD).
  • GVHD graft versus host disease
  • the methods of the invention may be used to generate increased numbers of hematopoietic stem cells alone or together with increased numbers of lineage-committed progenitor cells and their progeny, based on the duration of culture and the duration of exposure of the hematopoietic stem cells to c-myc antisense oligomers.
  • a c- yc antisense oligomer may also be administered to the recipient to further minimize the immune response against the transplanted hematopoietic stem cells, lineage committed progenitor cells and their progeny, tissues, and organs.
  • the present method may be used to expand a population of the recipient's own hematopoietic stem cells in vitro (ex vivo) preserving the multilineage potential of such cells without lineage commitment, e.g., for use in autologous hematopoietic stem cell transplantation.
  • hematopoietic stem cells differentiate they are exposed to the various antigens present on the cells and tissue of the host and immunological tolerance is established during T cell development within the thymus. In general, T cells that would be reactive with host proteins do not survive. However, in some cases, the immune system may recognize self antigens as foreign resulting in an immune reaction against one or more endogenous antigens, leading to an autoimmune condition or disease.
  • Exemplary autoimmune conditions include organ specific forms wherein the immune response is directed against, e.g., the cells of the adrenal glands, causing Addison's disease, against the thyroid causing auto-immune thyroiditis (Hashimoto's disease) or against the beta cells of the islets of Langerhans in the pancreas, resulting in insulin-dependent diabetes mellitus; and non-specific forms wherein the immune response is directed against an antigen that is ubiquitous, e.g., an immune reaction against DNA, resulting in the disease systemic lupus erythematosus.
  • Further examples include, Sjogren's syndrome, caused by the production of auto-antibodies against salivary ducts, rheumatoid arthritis.
  • Auto-immunity may be the result of attack by antibodies, T-cells or both.
  • hematopoietic stem cells may be obtained from the patient, followed by treatment of the patient with chemotherapy, radiation therapy or other means to deplete the patient of residual T-cells.
  • the patients' hematopoietic stem cells or hematopoietic stem cells from an allogeneic donor are then exposed ex vivo to a c-myc antisense oligomer under conditions effective to result in an increase in the number of viable hematopoietic stem cells in vitro, which are then infused into the patient.
  • the expanded population of hematopoietic stem cells develops in the presence of the antigenic repertoire of the host, the newly developed T-cells should not recognize host antigens as foreign and an autoimmune response should not occur.
  • c-myc antisense-treated hematopoietic stem cells lack immunological memory of self antigens, such that transplantation of c-myc-treated hematopoietic stem cells or lineage-committed progenitor and progeny cells derived therefrom may be used in transplantation regimens for treatment of a patient with an autoimmune disease, in order to minimize or eliminate the autoimmune condition.
  • the methods of the invention may be used to generate increased numbers of hematopoietic stem cells alone or together with increased numbers of lineage-committed progenitor cells and their progeny, based on the duration of culture and the duration of exposure of the hematopoietic stem cells to c-myc antisense oligomers.
  • a patient with an autoimmune disease may be treated by in vivo administration of a c-myc antisense oligonucleotide.
  • Such in vivo administration may be carried out alone or in conjunction with administration of ex vivo c-myc antisense oligomer treated HSC and/or lineage-committed progenitor cells and their progeny.
  • additional treatment components include compositions and procedures known in the art for the treatment of autoimmune disease.
  • a c-myc antisense oligomer-treated HSC composition of the invention may be used to modulate the response of a recipient to an organ or tissue transplant.
  • a c-myc antisense oligomer of the invention is administered to an organ and/or tissue transplant recipient, prior to the donation procedure, to decrease the immune response and resulting tissue rejection following transplantation.
  • This may be accomplished by treating the tissue or organ to be transplanted in a manner effective to deplete T-cells in vitro under conditions appropriate for transplantation procedures, as generally employed by those of skill ion the art.
  • Such in vitro treatment may be carried out in conjunction with the administration of a c-myc antisense oligomer to the patient, prior to, during and for a limited time following such transplantation, in a manner effective to minimize or eliminate GVHD.
  • the invention provides methods and compositions for the augmentation of the response to a vaccine.
  • a c-myc antisense oligomer is administered to a subject, prior to or concurrent with, administration of a vaccine, thereby resulting in a greater immune response to the immunogens present in the vaccine.
  • Murine hematopoietic stem cells were obtained from bone marrow starting with unfractionated bone marrow, performing a density separation using 1.080g/ml Nycodenz separation medium (Nycomed Pharma AS OSLO, Norway), followed by isolation of the lin- cell population using Dynal Bead depletion employing lineage-specific monoclonal antibodies, followed by staining with: (1) antibodies to c-kit and Sea 1 ; (2) propidium iodide; and (3) the dyes, Hoescht 33342, Rhodamine 123 and propidium iodide (PI ), then sorting cells using FACS by selecting for either (A) Sea 1+, c-kit+, lin-, Hoescht 33342 low (Ho lmv ), Rhodamine 123 low (Rh low ) and PI negative cells (LTR-HSC), or (B) Sea 1+, c-kit+, lin- cells, (STR-HSC).
  • A Sea
  • A. Culture of HSC in medium containing IL-6 and SCF +/- antisense oligomers 25 cells enriched for STR-HSC, characterized as Sea 1 +, c-kit+, and lin- were directly sorted into medium and cultured for 3 days in the presence of IL-6 and SCF together with either (1 ) no antisense oligomer, (2) 25 ⁇ M of scrambled c- yc antisense oligomer having the sequence presented as SEQ ID NO:2, or (3) 1, 5, 25, or 125 ⁇ M of a morpholino phosphoroamidate c- myc antisense oligomer having the sequence presented as SEQ ID NO: l . After 3 days, the cells were counted, plated in agar, then evaluated for HPP at day 0 and day 3 in a standard HPP-CFC assay. (See Table 1 and Fig. 3)
  • Antisense c-myc mediates self-replication of LTR-HSC daughter cells during the 2 nd - 4 th cell division ex vivo
  • LTR-HSC characterized as lin-, c-kit+, Hoescht low (Ho low ) and Rhodamine low (Rh low )
  • Ho low Hoescht low
  • Rh low Rhodamine low
  • SCF stem cell factor
  • Various amounts of morpholino phosphoroamidate c-myc antisense oligomer having the sequence presented as SEQ ID NO: 1 were injected intraperitoneally (IP) into mice, daily for 7 days. Samples of peripheral blood was taken from the mice and analyzed at 7, 21 and 28 days.
  • Figures 4A-D indicate the number of erythrocytes (A), granulocytes (B), lymphocytes (C), and white blood cells (D) detected in the peripheral blood of the mice.
  • mice for 21 -28 days effected an apparent decrease in granulocytes (B) and white blood cells (D), in the peripheral blood at day 7, followed by an increase at days 21 and 28, wherein the magnitude of the effect greatest at 100 to 300 ⁇ g morpholino phosphoroamidate c-myc antisense oligomer.
  • mice Treated 2 Or 9 Days With A c-mvc Antisense Oligomer 100 ⁇ g morpholino phosphoroamidate c-myc antisense oligomer having the sequence presented as SEQ ID NO: 1 was injected intraperitoneally (IP) into mice, daily for 2 or 1 1 days.
  • IP intraperitoneally
  • the bone marrow compartment of the mice was evaluated at day 1 1 , and the results indicate that the ckit+ (CD1 17 or hematopoietic stem cell) component of the bone marrow was increased in animals treated with 100 ⁇ g c-myc antisense oligomer and that treatment for 2 days was equally effective to treatment for 1 1 days.
  • ckit+ CD1 17 or hematopoietic stem cell

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Abstract

A composition comprising increased numbers of hematopoietic stem cells (HSC) prepared by treating an enriched population of HSC with a c-myc antisense oligonucleotide is described. The invention further relates to methods for producing the same and the use of c-myc antisense oligonucleotide-treated HSC as a medicament.

Description

C-MYC ANTISENSE-TREATED HEMATOPOIETIC STEM CELL
COMPOSITION AND METHOD
Field Of The Invention
The present invention relates to methods for increasing the number of hematopoietic stem cells (HSC) in vitro and in vivo by exposing the cells to a c-myc antisense oligonucleotide and to methods for treatment of such HSC in a manner effective to increase the population of lineage committed progenitor cells and their progeny. The invention further relates compositions of HSC produced by such methods.
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Background Of The Invention
The hematopoietic stem cell (HSC) is a pluripotent progenitor cell that has been characterized as a cell that is transplantable, can self-replicate and has multilineage potential. Differentiation of HSCs results in a loss of such multilineage potential, and corresponding lineage commitment. It has been demonstrated that self-renewal of HSC occurs in vivo, as indicated by transplantation studies wherein a single HSC repopulated the marrow of an immunodeficient mouse (Smith, et al., 1991 ; Osawa, et al, 1996). It has also been demonstrated that hematopoietic stem cells can be infected with recombinant retroviruses, and can serve as cellular targets for gene therapy (Keller and Snodgrass, 1990).
Patients suffering from various cell-based diseases including, but not limited to, myeloproliferative diseases, blood cell prohferative diseases and autoimmune diseases often have an imbalance in the number of cells of particular lineages. In addition, patients undergoing chemotherapy or irradiation often have defective hematopoiesis.
High-dose chemotherapy and/or radiation therapy together with bone marrow transplantation or transplantation of a cell population enriched for hematopoietic stem cells are standard treatment regimens for some malignancies, including acute lymphocytic leukemia, chronic myelogenous leukemia, neuroblastoma, lymphoma, breast cancer, colon cancer, lung cancer and myelodysplastic syndrome, as well as for other non-malignant hematopoietic diseases, e.g. thrombocytopenia.
Clinical trials are underway using such regimens for the treatment of various cancers, including ovarian cancer, thymomas, germ cell tumors, multiple myeloma, melanoma, testicular cancer, lung cancer, and brain tumors.
It follows that modulation of hematopoietic stem cell development and/or replication in patients suffering from any of the above conditions has clinical utility and that such cells are potential targets for genetic engineering-based therapies (Wilson, et al, 1991). Even though HSCs appear to be optimal cellular targets for gene therapy and autologous transplantation, problems have been encountered in (a) identifying the antigenic markers unique to hematopoietic stem cells, (b) obtaining substantial numbers of hematopoietic stem cells, and (c) maintaining and expanding a population of hematopoietic stem cells.
In addition, the percentage of cells in a cell population enriched for hematopoietic stem cells that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need for techniques for purification and expansion of hematopoietic stem cells. Complications of transplantation with a cell population enriched for HSC have been observed due to graft versus host disease (GVHD) in allogeneic transplants, and residual tumor cells in autologous and allogeneic transplants that may result in recurrence of disease. Current treatment regimens are directed to removal of cells that pose a risk to the transplant recipient, including T-cells and residual tumor cells.
In therapeutic regimens used to date which involve transplantation of cell populations enriched for HSC, relapse of the underlying disease due to contamination of the transplanted cells with malignant cells and/or the persistence of malignant cells within the host following myeloablative chemotherapy frequently result in failure of such treatments.
It has been demonstrated that antisense oligonucleotides and antibodies can specifically interfere with synthesis of a target protein of interest. Due to their hydrophobicity, antisense oligonucleotides interact well with phospholipid membranes (Akhtar, S., et al, 1991), and it has been suggested that following the interaction with the cellular plasma membrane, oligonucleotides are actively transported into living cells (Loke, S.L., et al, 1989; Yakubov, L.A., et al, 1989; Anderson, CM., et al, 1999).
Although inhibition of genes associated with cellular development has been achieved using antisense technology, naturally occurring oligonucleotides have a nuclease-sensitive phosphodiester backbone. However, naturally occurring oligonucleotides have been rendered resistant to degradation by nucleases, e.g., by utilizing a methylphosphonate, phosphorothioate or phosphorodiamidate linkage instead of a phosphodiester one (Spitzer and Eckstein, 1988; Baker, et al. 1990; Hudziak, 1996). Phosphorodiamidate morpholino oligonucleotides (PMOs) have been demonstrated to exhibit high binding affinity for RNA targets with favorable uptake into cells and minimal nonspecific binding interactions. (See, e.g., Summerton, et al., 1997a).
A synthetic phosphorothioate antisense oligonucleotide specific to the actylcholinesterase gene has been described as a tool for inhibiting (1) abnormal hematopoiesis, (2) abnormal platelet proliferation, (3) increasing macrophage and hematopoietic stem cell counts, (4) reducing the immune response to allogeneic organ transplants, and (5) for treating malignant tumors (U.S. Pat. No. 5,891,725). c-myc is a proto-oncogene which regulates cell growth, differentiation, and apoptosis, and its aberrant expression has been associated with a number of human cancers including lung cancer, colorectal cancer, breast cancer, bladder cancer, leukemia, lung cancer, etc.
In vitro translation of several oncogene mRNAs has been successfully blocked by phosphodiester and/or phosphorothioate antisense oligonucleotides, as exemplified by antisense to c-myc (McManaway, et al, 1990; Watson, et al, 1991 ; Li, et al, 1995) and antisense to bcl- 2 (Reed, et al, 1990).
Transplantation of hematopoietic stem cells derived from peripheral blood and/or bone marrow is increasingly used in combination with chemotherapy and/or radiation therapy for the treatment of a variety of disorders including numerous forms of cancer. The percentage of cells in such transplants that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need to develop techniques for purification and expansion of hematopoietic stem cells.
Accordingly, there remains a need to develop methods effective to obtain increased numbers of hematopoietic stem cells that are pluripotent, capable of development into a wide variety of cellular lineages and capable of transplantation without the complications of GVHD and residual disease. The present invention addresses this need.
Summary Of The Invention
The invention provides a method of producing a hematopoietic stem cell (HSC) composition prepared by carrying out the steps of obtaining an HSC-containing cell population from a subject, treating the cell population in manner effective to enrich for HSC; and exposing the enriched HSC population, ex vivo to a c-myc antisense oligomer, at a concentration and for a period of time sufficient to increase the number of hematopoietic stem cells at least two-fold, preferably at least four-fold and more preferably at least 8-fold or more. The HSC are characterized as lacking the expression of lineage markers (lin-), positive for cell surface expression of c-kit (CD 1 17) and positive for cell surface expression of Sea 1.
In one embodiment, the HSC-containing cell population for practicing the method is obtained from human bone marrow or mobilized human peripheral blood.
In another embodiment, the subject is a human cancer patient and the patient has lymphoma or leukemia. The c-myc antisense oligomers for use in practicing the invention are typically characterized by one or more of: (1 ) a length of about 12 to 25 bases; (2) a backbone which is substantially uncharged; (3) the ability to hybridize with the complementary sequence of a target RNA with high affinity at a Tm greater than 50°C, (4) nuclease resistance, (5) the capability for active or facilitated transport into cells, and (6) a structure selected from the group consisting of the structures presented in Figures 2 A-A through 2 E-E
In one preferred embodiment, the antisense oligomer has the sequence identified as SEQ ID NO 1
The invention also provides a hematopoietic stem cell (HSC) composition, prepared by the method set forth above for use as a medicament in treating cancer in a subject
In a related application of the method, an expanded HSC population prepared by the method set forth above is further cultured under conditions effective to result in an expanded population of lineage committed progenitor cells and their progeny
A method for delivering one or more of (1) a c-myc antisense oligomer- treated hematopoietic stem cell (HSC) composition, (2) a c-myc antisense oligomer, and (3) an expanded population of lineage committed progenitor cells and their progeny to a cancer patient, is ftirther provided by the invention Such an ex vivo expanded cell population enriched for hematopoietic stem cells and lineage committed progenitor cells and their progeny may be re-infused into a patient as part of a therapeutic regiment for the treatment of vaπous malignancies, non-malignant hematopoietic abnormalities, autoimmune disease and to augment vaccines
These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples
Brief Description Of The Figures
Figure 1A-E show several preferred morphohno subunits having 5-atom (A), six-atom (B) and seven-atom (C-E) linking groups suitable for forming polymers
Figures 2A-A through 2E-E show the repeating subunit segment of exemplary morphohno oligonucleotides, constructed using subunits A-E, respectively, of Figure 1
Figure 3 depicts the day 0 and day 3 results for high prohferative potential colony forming cell (HPP-CFC) colony formation in an vitro assay wherein single c-kιt+, Sea 1+ cells were treated with no antisense oligomer, a scrambled antisense oligomer, or 1, 5, 25, or 125 mM c- myc antisense oligomer and cultured for 3 days in the presence of IL-6 and SCF The results are indicated as (A) the total number of HPP-CFC, (B) the frequency of HPP-CFC and (C) the cell number/HPP-CFC frequency
Figures 4A-D depict the results of analysis of the peπpheral blood of mice treated intraperitoneally on a daily basis for 7 days with various doses (30, 100, 300 or 1000 μg/day) of a PMO antisense to c-myc, with samples collected at 7, 21 and 28 days The figures represent the number of erythrocytes (A), granulocytes (B), lymphocytes (C), and white blood cells (D) present at each time point
Detailed Description Of The Invention
I Definitions
The terms below, as used herein, have the following meanings unless indicated otherwise As used herein, the term "a cell population enriched for hematopoietic stem cells" refers to the cell population obtained using the positive and negative selection techniques described herein, wherein the hematopoietic stem cells may be LTR- or STR-HSCs.
As used herein, the terms "long term repopulating hematopoietic stem cells" and "LTR- HSC", refer to hematopoietic stem cells that are transplantable, and upon differentiation contribute to all lineages of hematopoietic cells for an undefined period of time when transplanted into totally immunosuppressed recipients. LTR-HSC do not undergo clonal extinction.
As used herein, the term "short term repopulating hematopoietic stem cells" or "STR-HSC", refers to murine hematopoietic stem cells that are transplantable, and contribute to all lineages of hematopoietic cells for a period of from about one week to 6 months, then undergo clonal extinction when transplanted into immunosuppressed recipients.
The term "clonal extinction", as used herein refers to the terminal differentiation of a single hematopoietic stem cell and all the progeny produced by clonal expansion of that cell, such that no more daughter cells are produced from the initial clone.
The term "pluripotent hematopoietic stem cells" refers to hematopoietic stem cells, capable of differentiating into all the possible cell lineages.
As used herein, the term "high proliferative potential colony forming cells" or "HPP-CFCs", as used herein relative to hematopoietic stem cells refers to murine cells that proliferate in response to rat rSCF, mouse rIL-3 and human rIL-6. The cells proliferate in semi-solid media, such as agar or methyl cellulose or as single cells in liquid culture, and form macroclones which have a diameter greater than 1 mm, generally having greater than 100,000 cells per clone with dense multicentric centers. This population includes all murine HSCs, however, not all HPP-CFC are HSCs, and the HPP-CFC assay is not a specific assay for LTR-HSC. In contrast, low proliferative potential (LPP) clones contain from 2 to 100,000 cells per clone.
As used herein, "lineage-committed hematopoietic stem cells" are hematopoietic stem cells that have differentiated sufficiently to be committed to one or more particular cell lineages, but not all cell lineages.
As used herein, the term 'Tin -" or "lineage-depleted", refers to a cell population which lacks expression of cell surface antigens specific to T-cells, B-cells, neutrophils, monocytes and erythroid cells, and does not express antigens recognized by the "YW 25.12.7" antibody. (See, e.g., Bertoncello I et al., 1991.)
As used herein, the terms "develop", "differentiate" and "mature" are used interchangeably and refer to the progression of a cell from a stage of having the potential to differentiate into multiple cellular lineages to becoming a more specialized cell committed to one or more defined lineages.
As used herein, the term "purified", relative to hematopoietic stem cells refers to HSCs that have been enriched (isolated or purified) relative to some or all of the other types of cells with which they are normally found in a particular tissue in nature, e.g., bone marrow or peripheral blood. In general, a "purified" population of HSCs has been subjected to density gradient fractionation, lineage depletion and positive selection for c-kit and Sca-1 expression in addition to low level staining with both Hoechst 33342 and Rhodamine 123. As used herein, the terms "antisense oligonucleotide" and "antisense oligomer" are used interchangeably and refer to a sequence of nucleotide bases and a subunit-to-subunit backbone that allows the antisense oligomer to hybridize to a target sequence in an RNA by Watson- Crick base pairing, to form an RNA:oligomer heteroduplex within the target sequence. The oligomer may have exact sequence complementarity to the target sequence or near complementarity. Such antisense oligomers may block or inhibit translation of the mRNA containing the target sequence, or inhibit gene transcription, may bind to double-stranded or single stranded sequences, and may be said to be "directed to" a sequence with which it hybridizes. Exemplary structures for antisense oligonucleotides for use in the invention include the β- morpholino subunit types shown in Fig 1 A-E. It will be appreciated that a polymer may contain more than one linkage type.
Subunit A in Figure 1 contains a 1-atom phosphorous-containing linkage which forms the five atom repeating-unit backbone shown at A-A in Figure 2, where the morphohno rings are linked by a 1-atom phosphonamide linkage.
Subunit B in Figure 1 is designed for 6-atom repeating-unit backbones, as shown at B-B, in Figure 2. In structure B, the atom Y linking the 5' morphohno carbon to the phosphorous group may be sulfur, nitrogen, carbon or, preferably, oxygen. The X moiety pendant from the phosphorous may be any of the following: fluorine; an alkyl or substituted alkyl; an alkoxy or substituted alkoxy; a thioalkoxy or substituted thioalkoxy; or, an unsubstituted, monosubstituted, or disubstituted nitrogen, including cyclic structures.
Subunits C-E in Figure 1 are designed for 7-atom unit-length backbones as shown for C-C through E-E in Figure 2. In Structure C, the X moiety is as in Structure B and the moiety Y may be a methylene, sulfur, or preferably oxygen. In Structure D the X and Y moieties are as in Structure B. In Structure E, X is as in Structure B and Y is O, S, or NR. In all subunits depicted in Figures 1 A-E, Z is O or S. and P, or P, is adenine, cytosine, guanine or uracil.
As used herein, a "morphohno oligomer" refers to a polymeric molecule having a backbone which supports bases capable of hydrogen bonding to typical polynucleotides, wherein the polymer lacks a pentose sugar backbone moiety, and more specifically a ribose backbone linked by phosphodiester bonds which is typical of nucleotides and nucleosides, but instead contains a ring nitrogen with coupling through the ring nitrogen. A preferred "morphohno" oligonucleotide is composed of morphohno subunit structures of the form shown in Fig. 2B, where (i) the structures are linked together by phosphorous-containing linkages, one to three atoms long, joining the morphohno nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) P, and P, are purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
This preferred aspect of the invention is illustrated in Fig. 2B, which shows two such subunits joined by a phosphorodiamidate linkage. Morphohno oligonucleotides (including antisense oligomers) are detailed, for example, in co-owned U.S. Patent Nos. 5,698.685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,185, 444, 5,521 ,063, and 5,506,337.
As used herein, a "nuclease-resistant" oligomeric molecule (oligomer) is one whose backbone is not susceptible to nuclease cleavage of a phosphodiester bond. Exemplary nuclease resistant antisense oligomers are oligonucleotide analogs, such as phosphorothioate and phosphate-amine DNA (pnDNA), both of which have a charged backbone, and methyl- phosphonate, morphohno. and peptide nucleic acid (PNA) oligonucleotides, all of which may have uncharged backbones. As used herein, an oligonucleotide or antisense oligomer "specifically hybridizes" to a target polynucleotide if the oligomer hybridizes to the target under physiological conditions, with a Tm substantially greater than 37°C, preferably at least 50° C, and typically 60°C-80°C or higher. Such hybridization preferably corresponds to stringent hybridization conditions, selected to be about 10° C, and preferably about 5° C lower than the thermal melting point ( rrnl) f°r tne specific sequence at a defined ionic strength and pH. At a given ionic strength and pH , the Ttm~ι is the temperature at which 50% of a target sequence hybridizes to a complementary polynucleotide.
Polynucleotides are described as "complementary" to one another when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double- stranded polynucleotide can be "complementary" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. Complementarity (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonds with each other, according to generally accepted base-pairing rules. As used herein the term "analog" with reference to an oligomer means a substance possessing both structural and chemical properties similar to those of a reference oligomer.
As used herein, a first sequence is an "antisense sequence" with respect to a second sequence if a polynucleotide whose sequence is the first sequence specifically binds to, or specifically hybridizes with, the second polynucleotide sequence under physiological conditions.
As used herein, a "base-specific intracellular binding event involving a target RNA" refers to the sequence specific binding of an oligomer to a target RNA sequence inside a cell. For example, a single-stranded polynucleotide can specifically bind to a single-stranded polynucleotide that is complementary in sequence. As used herein, "nuclease-resistant heteroduplex" refers to a heteroduplex formed by the binding of an antisense oligomer to its complementary target, which is resistant to in vivo degradation by ubiquitous intracellular and extracellular nucleases.
As used herein, "c-myc", refers to an oncogene or gene that gives directs cells toward the development and growth of cancer or a tumor, "c- yc" has been associated with gene amplification in various types of cancer, as further detailed below.
As used herein, the term "c-myc antisense oligomer" refers to a nuclease-resistant antisense oligomer having high affinity (ie, which "specifically hybridizes") to a complementary or near- complementary c-myc nucleic acid sequence.
As used herein, the term "modulating expression" relative to an oligonucleotide refers to the ability of an antisense oligonucleotide (oligomer) to either enhance or reduce the expression of a given protein by interfering with the expression, or translation of RNA. In the case of enhanced protein expression, the antisense oligomer may block expression of a suppressor gene, e.g., a tumor suppressor gene. In the case of reduced protein expression, the antisense oligomer may directly block expression of a given gene, or contribute to the accelerated breakdown of the RNA transcribed from that gene.
As used herein, the terms "tumor" and "cancer" refer to a cell that exhibits a loss of growth control and forms unusually large clones of cells. Tumor or cancer cells generally have lost contact inhibition and may be invasive and/or have the ability to metastasize.
As used herein, "effective amount" relative to an antisense oligomer refers to the amount of antisense oligomer administered to a mammalian subject, either as a single dose or as part of a series of doses and which is effective to inhibit expression of a selected target nucleic acid sequence.
As used herein "treatment" of an individual or a cell is any type of intervention used in an attempt to alter the natural course of the individual or cell. Treatment includes, but is not limited to, administration of e.g., a pharmaceutical composition, and may be performed either prophylactically, or subsequent to the initiation of a pathologic event or contact with an etiologic agent.
As used herein, the term "improved therapeutic outcome" relative to a cancer patient refers to a slowing or diminution of the growth of cancer cells or a solid tumor, or a reduction in the total number of cancer cells or total tumor burden.
II. c- yc Antisense Oligonucleotides
A. Types Of Antisense Oligonucleotides
Antisense oligonucleotides of 15-20 bases are usually long enough to have one complementary sequence in the mammalian genome. In addition, antisense compounds having a length of at least 17 nucleotides in length have been demonstrated to hybridize well with their target mRNA (Cohen, et al. , 1991 ).
Two general mechanisms have been proposed to account for inhibition of expression by antisense oligonucleotides. (See e.g., Agrawal, et al., 1990; Bonham, et al, 1995; and Boudvillain, et al, 1997).
In the first, a heteroduplex formed between the oligonucleotide and mRNA is a substrate for RNase H, leading to cleavage of the mRNA. Oligonucleotides belonging, or proposed to belong, to this class include phosphorothioates, phosphotriesters, and phosphodiesters (unmodified "natural" oligonucleotides). Such compounds generally show high activity, and phosphorothioates are currently the most widely employed oligonucleotides in antisense applications. However, these compounds tend to produce unwanted side effects due to non- specific binding to cellular proteins (Gee, et al, 1998), as well as inappropriate RNase cleavage of non-target RNA heteroduplexes (Giles, et al, 1993).
A second class of oligonucleotide analogs, termed "steric blockers" or, alternatively, "RNase H inactive" or "RNase H resistant", have not been observed to act as a substrate for RNase H, and are believed to act by sterically blocking target RNA formation, nucleocytoplasmic transport, or translation. This class includes methylphosphonates (Toulme, et al, 1996), morphohno oligonucleotides, peptide nucleic acids (PNA's), 2'-0-allyl or 2'-0-alkyl modified oligonucleotides (Bonham, 1995), and N3' P5' phosphoramidates (Gee, 1998). Naturally occurring oligonucleotides have a phosphodiester backbone which is sensitive to degradation by nucleases, however, certain modifications of the backbone increase the resistance of native oligonucleotides to such degradation. (See, e.g. , Spitzer and Eckstein 1988). Candidate antisense oligomers are evaluated, according to well known methods, for acute and chronic cellular toxicity, such as the effect on protein and DNA synthesis as measured via incorporation of -^H-leucine and ^H-thymidine, respectively. In addition, various control oligonucleotides, e.g., control oligonucleotides such as sense, nonsense or scrambled antisense sequences, or sequences containing mismatched bases, in order to confirm the specificity of binding of candidate antisense oligomers. The outcome of such tests are important to discern specific effects of antisense inhibition of gene expression from indiscriminate suppression. (See, e.g. Bennett, et al, 1995). Accordingly, sequences may be modified as needed to limit non-specific binding of antisense oligomers to non-target sequences.
The effectiveness of a given antisense oligomer molecule in forming a heteroduplex with the target RNA may be determined by screening methods known in the art. For example, the oligomer is incubated a cell culture expressing c-myc, and the effect on the target RNA is evaluated by monitoring the presence or absence of (1) heteroduplex formation with the target sequence and non-target sequences using procedures known to those of skill in the art, (2) the amount of c-myc mRNA, as determined by standard techniques such as RT-PCR or Northern blot, or (3) the amount of c-myc protein, as determined by standard techniques such as ELISA or Western blotting. (See, for example, Pari, et al, 1995; Anderson, et al, 1996).
Exemplary antisense oligomers for use in the methods of the invention include morphohno oligomers (Figs. 2A-D), peptide nucleic acids and methyl phosphonate oligomers.
1. Morpholino Antisense Oligonucleotides
Preferred antisense oligonucleotides are nuclease-resistant oligomers with substantially uncharged backbones, and particularly morpholino oligomers having, in addition to a base sequence complementary to a region of a selected mRNA target sequence, an oligomer backbone, defined by the nucleotide subunits of the oligomer and the linkages between them such that the oligomer can bind the target RNA sequence by Watson-Crick base pairing between complementary bases in the target RNA and the oligomer.
The synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Patent Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5, 166,315, 5,521 ,063, and 5,506,337. The preferred oligomers are composed of morpholino subunits of the form shown in the above cited patents, where (i) the morpholino groups are linked together by substantially uncharged linkages, one to three atoms long, joining the morpholino nitrogen of one subunit to the 5' exocyclic carbon of an adjacent subunit, and (ii) the base attached to the morpholino group is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide. The purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine. Preparation of such oligomers is described in detail in U.S. Patent No. 5, 185,444 (Summerton and Weller, 1993). A variety of types of nonionic linkages may be used to construct a morpholino backbone. Morpholino oligomers exhibit little or no non-specific antisense activity, afford good water solubility, are immune to nucleases, and are designed to have low production costs (Summerton and Weller, 1997b).
2. Peptide Nucleic Acids (PNAs)
It has been demonstrated that the phosphodiester backbone found in naturally occurring oligonucleotides is not essential for a potent structural DNA mimic and not even required for a helical duplex structure, and that peptide or protein nucleic acids (PNAs), may function as effective DNA mimics (Nielsen, 1995).
PNAs are analogs of DNA in which the backbone is structurally homomorphous with a deoxyribose backbone. It consists of N-(2-aminoethyl)glycine units to which pyrimidine or purine bases are attached. PNAs containing natural pyrimidine and purine bases hybridize to complementary oligonucleotides obeying Watson-Crick base-pairing rules, and mimic DNA in terms of base pair recognition (Egholm et al, 1993). The backbone of PNAs are formed by peptide bonds rather than phosphodiester bonds, making them well-suited for antisense applications. The backbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes which exhibit greater than normal thermal stability. PNAs are not recognized by nucleases or proteases. However, PNA antisense agents has been observed to display slow membrane penetration in cell cultures, possibly due to poor uptake (transport) into cells. (See, e.g., Ardhammar M et al., 1999).
3. Methylphosphonate Oligonucleotides
Methylphosphonate oligonucleotides are uncharged and therefore predicted to exhibit enhanced cellular uptake relative to charged oligonucleotides (Blake, et al, 1985). However, methylphosphonate antisense oligomers have been shown to be incapable of inhibiting N-ras expression in vitro (Tidd, et al, 1988). In contrast, in vitro translation of several oncogene mRNAs was shown to be successfully blocked by phosphodiester and/or phosphorothioate antisense oligonucleotides (c-myc McManaway, et al, 1990; Watson, et al, 1991 ; bcl-2: Reed, et al, 1990; myb: Calabrett, et al, 1991 ; bcr-ab: Szczylik, et al, 1991). The synthesis of methyl phosphonate oligomers requires multiple steps which may limit the practical utility of the use of such structure in clinical applications.
4. Preferred Antisense Oligomers mRNA transcribed from the relevant region of a gene of interest is generally targeted by antisense oligonucleotides, however, in some cases double-stranded DNA may be targeted using a non-ionic probe designed for sequence-specific binding to major-groove sites in duplex DNA. Such probe types are described in U.S. Patent No. 5,166,315 (Summerton and Weller, 1992), and are generally referred to herein as antisense oligomers, referring to their ability to block expression of target genes.
In the methods of the invention, the antisense oligomer is designed to hybridize to a region of the c-myc nucleic acid sequence, under physiological conditions with a Tm substantially greater than 37°C, e.g. , at least 50°C and preferably 60°C-80°C. The oligomer is designed to have high-binding affinity to the nucleic acid and may be 100% complementary to the c-myc target sequence, or may include mismatches, e.g.. to accommodate allelic variants, as long as the heteroduplex formed between the oligomer and c-myc target sequence is sufficiently stable to withstand the action of cellular nucleases and other modes of degradation during its transit from cell to body fluid. Mismatches, if present, are less destabilizing toward the end regions of the hybrid duplex than in the middle. The number of mismatches allowed will depend on the length of the oligomer, the percentage of G:C base pair in the duplex and the position of the mismatch(es) in the duplex, according to well understood principles of duplex stability. Although such an antisense oligomer is not necessarily 100% complementary to the c-myc target sequence, it is effective to stably and specifically bind to the target sequence such that expression of c-myc is modulated. The appropriate length of the oligomer to allow stable, effective binding combined with good specificity is about 8-40 nucleotide base units, and preferably about 12-25 nucleotides. Oligomer bases that allow degenerate base pairing with target bases are also contemplated, assuming base-pair specificity with the target is maintained. In general, the target for modulation of gene expression using the antisense methods of the present invention comprises a sequence spanning the mRNA translational start codon for c- myc. However, in some cases, other regions of the c-myc mRNA may be targeted, including one or more of, an initiator or promoter site, an intron or exon junction site, a 3 '-untranslated region, and a 5'-untranslated region. In addition, both spliced and unspliced RNA may serve as the template for design of antisense oligomers for use in the methods of the invention.
Preferred antisense oligomers for use in the methods of the invention preferably, have one or more properties including: ( 1) a backbone which is substantially uncharged (e.g. , Uhlmann, et al, 1990), (2) the ability to hybridize with the complementary sequence of a target RNA with high affinity, that is, Tm substantially greater than 37°C, preferably at least 50° C, and typically 60°C-80°C or higher, (3) a subunit length of at least 8 bases, generally about 8-40 bases, preferably 12-25 bases, (4) nuclease resistance (Hudziak, et al , 1996) and (5) capable of active or facilitated transport as evidenced by (i) competitive binding with a phosphorothioate antisense oligomer, and/or (ii) the ability to transport a detectable reporter into the cells. In one preferred aspect of this embodiment, the antisense oligomer comprises the sequence presented as SEQ ID NO: 1.
B. c-myc c-myc is a proto-oncogene which is the cellular homologue of the transforming gene of the chicken retrovirus MC29. c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. Aberrant, constitutive or overexpression of c-myc has been associated with a number of human cancers including lung cancer, colorectal cancer, breast cancer, bladder cancer, leukemia, lung cancer, etc.
Proto-oncogenes are activated to oncogenes by a variety of mechanisms which include: (1 ) promoter insertion, (2) enhancer insertion, (3) chromosomal translocation, (4) gene amplification and (5) point mutation. As used herein, "activation" relative to a proto-oncogene means transcription of the gene is increased, e.g.. from no expression to low level expression. Mechanisms (l)-(4) result in an increase in the expression level of an oncogene. while (5) results in expression of an altered gene product. Evidence suggests that some form of oncogene expression together with inactivation of tumor suppressor genes is required for the development of cancer. The myc proto-oncogenes have been described as transcription factors that directly regulate the expression of other genes, examples of which include ECA39, p53, ornithine decarboxylase (ODC), alpha-prothymosin and Cdc25A. (Ben-Yosef, et al, 1998).
In chickens, following infection of chicken B-cells with certain avian leukemia viruses, a provirus becomes integrated near the myc gene, which is activated by a viral long terminal repeat (LTR) that acts either as a promoter or an enhancer, resulting in expression of myc and formation of a B-cell tumor (Murray, et al, 1996). Similarly, in Burkitt's lymphoma, an enhancer sequence is translocated resulting in expression of myc. c-myc is expressed in normal hematopoietic stem cells and has been shown to promote the differentiation of human epidermal stem cells (Gandarillas A and Watt FM, Genes Dev 1 1(21):2869-2882, 1997). In addition, c-myc has been shown to be overexpressed or aberrently expressed in numerous cancers. (See, e.g., Bieche I et al., Cancer Res 59(12):2759-65, 1999.) It has been observed that when quiescent cells re-enter the cell cycle c-myc expression is upregulated, and that ectopic expression of c-myc prevents cell cycle arrest in response to growth- inhibitory signals, differentiation stimuli, or mitogen withdrawal. (See, e.g., Amati B et al., Front Biosci 3:D250-68, 1998.) Further, the expression of an apoptosis inhibitor, bcl-2 has been inversely correlated with expression of c-myc in colorectal cancer cells. (See, e.g., Popescu RA et al., 1998; Peters R et al., 1998.)
When a c-myc antisense phosphorothioate oligomer was used to treat c-myc over-expressing leukemia and colon cancer cell lines, a 20- to 100-fold decrease in c-myc mRNA was detected in the colon cancer cell line and the leukemic cell line, respectively, using competitive reverse transcription-polymerase chain reaction (RT-PCR), in addition to inhibition of cellular proliferation. (Li et al, 1995).
A phosphordiamidate oligomer antisense to c-myc has been demonstrated to act at the RNA level to inhibit normal pre-mRNA splicing and to produce an aberrantly spliced mRNA in a sequence specific manner (Hudziak RM et al., 2000).
Surprisingly, when an antisense oligomer to c-myc was used to treat an enriched population of hematopoietic stem cells, the inventors discovered that the development of the hematopoietic stem cell population was modulated. In summary, a population of pluripotent hematopoietic stem cells may be maintained and expanded by exposue to an antisense oligonucleotide, directed to a region spanning the start codon of an mRNA transcribed from a gene encoding c-myc. It was also discovered that such an expanded population of hematopoietic stem cells may be further treated under conditions effective to result in an expanded population of lineage committed progenitor cells and their progeny. III. Hematopoietic Stem Cell Compositions.
A. Methods Of Obtaining Hematopoietic Stem Cells.
In adults, the large majority of pluripotent hematopoietic stem cells are found in the bone marrow. However, small but significant numbers of such cells can be found in the peripheral circulation, liver and spleen.
Hematopoietic stem cells for use in the methods of the invention may be derived from human bone marrow, human newborn cord blood, fetal liver, or adult human peripheral blood after appropriate mobilization.
The frequency of hematopoietic stem cells can be dramatically increased by treatment of a subject with certain compounds including cytokines. Such "mobilized" peripheral blood hematopoietic stem cells have become an important alternative to bone marrow-derived hematopoietic stem cells in transplantation procedures primarily because engraftment is more rapid. (See, e.g., Tanaka, et al, 1999). Such mobilization may be accomplished using for example, one or more of granulocyte colony-stimulating factor (G-CSF), stem cell factor (SCF), thrombopoietin (TPO), and a chemotherapeutic agent (i.e., cyclophosphamide).
Numerous methods for hematopoietic stem cell isolation are known in the art and generally include obtaining hematopoietic stem cells from bone marrow, newborn cord blood, fetal liver or adult human peripheral blood. Once obtained, a hematopoietic stem cell population is enriched by performing one or more of a density gradient separation, immunoaffinity purification using positive and/or negative selection by techniques such as panning, FACS and magnetic bead separation. Following such enrichment steps, the cell population is further characterized phenotypically and functionally.
Previous studies have demonstrated that primitive hematopoietic cells, characterized as high proliferative potential colony-forming cells (HPP-CFC, in vitro) may be isolated by selecting a fraction of density gradient-enriched, lineage-depleted marrow cells; and further selecting a cell population based on a single step fluorescence-activated cell sorter (FACS) fractionation for cells that bind low levels of the DNA binding dye, Hoechst 33342 and low levels of the mitochondrial binding dye, Rhodamine 123 (Wolf, et al, 1993). Recently, it has been shown that a defined subpopulation of HPP-CFC are transplantable and that a subpopulation of the cells that give rise to HPP-CFC are LTR-HSCs, which can replicate ex vivo. (See, e.g., Yagi, et al, 1999).
B. Characterization Of Hematopoietic Stem Cell Compositions
Hematopoietic stem cells have been historically defined as transplantable cells, capable of self-renewal as well as possessing the ability to generate daughter cells of any hematopoietic lineage. Lineage-committed progenitor cells are defined as more differentiated cells derived from hematopoietic stem cells.
The phenotypic markers which characterize the hematopoietic stem cell have been the subject of extensive debate and numerous publications. As yet, there is no consensus as to which markers are definitive for murine or human hematopoietic stem cells.
LTR-HSC have been isolated and characterized in mice herein using fluorescence-activated cell sorter (FACS) selection of density gradient-enriched, lineage-depleted bone marrow cells that are negative for expression of the CD34 antigen, positive for expression of the CD1 17 (c- kit) antigen, and exhibit low-level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and the mitochondrial binding dye, Rhodamine 123 (Rh- 123), (Wolf, et al, 1993). This isolated cell population has been demonstrated to be transplantable and capable of repopulating lethally irradiated recipients when transplanted together with unfractionated bone marrow cells.
The STR-HSC population may be selected by FACS sorting and is phenotypically defined herein as light density gradient-enriched bone marrow cells which lack the expression of lineage markers (lin-), are positive for c-kit (CD 1 17), Seal and CD34, exhibit low-level binding of the DNA binding dye, Hoechst 33342 (Ho-33342) and high-level binding of the mitochondrial binding dye. Rhodamine 123 (Rh-123).
Functional readouts that have been used to detect and characterize hematopoietic stem cells include the ability to form colonies under particular assay conditions in cell culture (in vitro). Exemplary assays include the long term culture initiating cell (LTCIC) assay (Pettengell R et al., 1994), and the high proliferative potential-colony-forming cell (HPP-CFC) assay. (See, e.g., Yagi, et al , 1999.) Further functional characterization includes in vivo assay for long-term repopulating hematopoietic stem cells (LTR-HSC) and short-term repopulating hematopoietic stem cells (STR-HSC), as described above.
Hematopoietic stem cells are often functionally characterized by activity in the high proliferative potential colony-forming cell (HPP-CFC) assay, as defined above. HPP-CFC have been characterized by: ( 1 ) a relative resistance to treatment in vivo with the cytotoxic drug 5-fluorouracil; (2) high correlation with cells capable of repopulating the bone marrow of lethally irradiated mice; (3) their ability to generate cells of the macrophage, granulocyte, megakaryocyte and erythroid lineages under appropriate conditions; and (4) their multifactor responsiveness. (See, e.g., McNiece, 1990). Hematopoietic stem cells for use in the methods of the invention are enriched, as described in Example 1. The cells were also characterized functionally in the HPP-CFC assay (in vitro) and in an assay for LTR-HSCs (in vivo), as further described in Example 2.
Preferred cytokines for the culture of hematopoietic stem cells include one or more of interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin- 1 1 (IL- 1 1), interleukin- 12 (IL- 12), stem cell factor (SCF). an early acting hematopoietic factor, described, for example in WO
91/05795, and thrombopoietin (TPO).
Long-term reconstitution with murine LTR-HSCs following complete immunosuppression has been shown to require the transplantation of unfractionated bone marrow cells together with less differentiated long term repopulating cells, in order to provide initial, albeit unsustained engraftment, which allows the completely immunosuppressed host to survive until the long term repopulating cells differentiate sufficiently to repopulate the host. (See, e.g., Jones, et al, 1990). LTR-HSCs may take several months to effectively repopulate the hematopoietic system of the host following complete immunosuppression.
Methods have been developed to distinguish the cells of the donor and recipient in murine hematopoietic reconstitution studies, by using donor hematopoietic stem cells, congenic at the
CD45 locus, defined as CD45.1 and recipient hematopoietic stem cells defined as CD45.2, such that monoclonal antibodies may be used to distinguish donor and recipient cells, i.e. by FACS analysis and/or sorting. In such detection methods, the recipient is infused with sufficient CD45.2 positive bone marrow cells to keep the mouse alive until differentiation of CD45.1 donor cells occur to an extent sufficient to repopulate the hematopoietic system of the recipient. Such methods may be used to differentiate LTR-HSCs from STR-HSCs, and donor cells from recipient cells. (Example 2.)
Once a hematopoietic stem cell population is obtained, the cells may be used immediately or frozen in liquid nitrogen and stored for long periods of time, under standard conditions, such that they can later be thawed and used, e.g., for administration to a patient. The cells will usually be stored in 10% DMSO, 50% fetal calf serum (FCS), and 40% cell culture medium.
IV. Methods And Compositions Of The Invention
In one preferred aspect, the invention provides methods and compositions for maintaining and expanding the population of pluripotent hematopoietic stem cells (HSC) by exposing an enriched population of HSC to an antisense oligonucleotide directed against a region spanning the start codon of an mRNA transcribed from a gene encoding c-myc.
In one preferred embodiment the antisense oligonucleotide has the sequence presented as SEQ ID NO: 1. In a further preferred embodiment the antisense oligonucleotide is substantially uncharged and is further characterized by stability, high Tm, and capable of active or facilitated transport as evidenced by (i) competitive binding with a phosphorothioate antisense oligomer, and/or (ii) the ability to transport a detectable reporter into the cells.
Hematopoietic stem cells may be exposed to the c-myc antisense oligomer ex vivo (in vitro) and/or a c-myc antisense oligomer may be administered to a subject in an amount effective to result in an expanded population of hematopoietic stem cells in vivo.
In a related aspect, hematopoietic stem cells are exposed to a c-myc antisense oligomer in an amount effective to result in an expanded population of hematopoietic stem cells, and further treated under conditions effective to result in an expanded population of lineage committed progenitor cells and their progeny.
A. Exposing Cells To Antisense Oligomers The invention is based on the discovery that a stable, substantially uncharged antisense oligonucleotide, characterized by high Tm, capable of active or facilitated transport into cells, and capable of binding with high affinity to a complementary or near-complementary c-myc nucleic acid sequence, can be administered to a hematopoietic stem cell, and inhibit expression of c-myc in the cell, either in vitro, or in vivo in a subject, resulting in modulation of the development of the cell.
Many cancer treatment regimens result in immunosuppression of the patient, leaving the patient unable to defend against infection. Supportive care for immunosuppression may include protective isolation of the patient such that the patient is not exposed to infectious agents; administration of: antibiotics, e.g., antiviral agents and antifungal agents; and/or periodic blood transfusions to treat anemia, thrombocytopenia (low platelet count), or neutropenia (low neutrophil count). Transplantation of hematopoietic stem cells derived from peripheral blood and/or bone marrow is increasingly used in combination with chemotherapy and/or radiation therapy for the treatment of a variety of disorders including numerous forms of cancer. The percentage of cells in such transplants that are capable of long-term hematopoietic reconstitution is very low and therefore there is a need to develop techniques for purification and expansion of hematopoietic stem cells.
Complications of transplantation therapy with a cell population enriched for hematopoietic stem cells include removal of cells in the transplant that pose a risk to the transplant recipient, including T-cells that are responsible for graft versus host disease (GVHD) in allogeneic grafts, and tumor cells in autologous transplants that may cause recurrence of disease.
Current transplantation regimens that employ cell populations enriched for hematopoietic stem cells and/or bone marrow transplantation also suffer from an excessive lag time between transplantation and repopulation of the patient's hematopoietic system, in particular patients often suffer from a deficiency in neutrophils and platelets. Neutrophils are involved in defending the host against infection. Frequently, following a chemotherapy or radiation therapy, a patient will suffer from insufficient neutrophil counts for time period of from about 3 to 4 weeks, or a longer time period resulting in increased susceptibility to infection.
Platelets are necessary for effective blood clotting at a site of injury. Frequently, following chemotherapy, radiation therapy, transplantation of a cell population enriched for hematopoietic stem cells or bone marrow transplantation, a patient will suffer from an insufficient platelet count for a time period of from about 4 to 6 weeks, or a longer time period resulting in the patient being easily bruised and excessive bleeding.
B. Treating Cells Ex Vivo With c-myc Antisense Oligomers
The invention includes in vitro (ex vivo) exposure of a nuclease-resistant antisense oligomer capable of binding with high affinity to a complementary or near-complementary c-myc sequence to a hematopoietic stem cell, and subsequent modulation of the development of the hematopoietic stem cell in vitro. Such treatment can effectively influence the composition of a culture of hematopoietic stem cells.
Hematopoietic stem cells may be treated in vitro (ex vivo) with c-myc antisense oligonucleotides, followed by administration to a subject. The subject may be the same individual from whom the hematopoietic stem cells were obtained (autologous transplantation) or a different individual (allogeneic transplantation). In allogeneic transplantation, the donor and recipient are matched based on similarity of HLA antigens in order to minimize the immune response of both donor and recipient cells against the other.
In one aspect, the invention is directed to methods of modifying the development of hematopoietic stem cells, by obtaining a population of HSCs, exposing them ex vivo to a nuclease-resistant antisense oligomer having high affinity to a complementary or near- complementary c-myc sequence, and subsequently re-infusing the expanded HSC population into a subject. Hematopoietic stem cell populations for use in the methods of the invention are extracted from a subject, purified and cultured ex vivo in the presence of one or more cytokines. Preferred cytokines include IL-3, IL-6, SCF and TPO. A hematopoietic stem cell population for use in the methods of the invention is preferably both human and allogeneic, or autologous. Hematopoietic stem cells may be obtained from a patient in need of hematopoietic stem cell transplantation or from an allogeneic donor. An enriched population of hematopoietic stem cells is exposed to oligomers antisense to c-myc under conditions effective to result in an increase the number of viable hematopoietic stem cells in vitro, in culture.
In one approach, the method of the invention results in an increase in the hematopoietic stem cell population which is at least 2-fold greater than the number of hematopoietic stem cells present prior to exposure to a c-myc antisense oligomer. Preferably, the treatment of hematopoietic stem cells with a c- yc antisense oligomer using the method described herein results in a hematopoietic stem cell population that is increased at least 4-fold to 8-fold relative to the number of hematopoietic stem cells present prior to exposure to the c- yc antisense oligomer. More preferably, the treatment of hematopoietic stem cells with a c-myc antisense oligomer using the method described herein results in a hematopoietic stem cell population that is increased more than 8-fold relative to the number of hematopoietic stem cells present prior to exposure to the c-myc antisense oligomer.
In general, hematopoietic stem cells are cultured in medium containing a c-myc antisense oligonucleotide together with cytokines or growth factors effective to promote hematopoietic stem cell replication, for a time sufficient to result in an increase in the population of hematopoietic stem cells, without differentiation thereof.
The culture time required for a at least a 2-fold, 4-fold, 8-fold, or greater than 8-fold increase in the number of HSC following exposure to medium containing a c-myc antisense oligonucleotide together with cytokines or growth factors effective to promote hematopoietic stem cell replication will vary dependent upon the cell source (including the tissue source and the health of the subject from whom the cells were taken), culture conditions, the percentage of HSC in the enriched population, etc.
It will be understood that the present inventions represents two related aspects: (1) a cell population having an increased number of HSC; and (2) a cell population wherein the increased HSC population is differentiated into committed progenitor cells of particular lineages. Longer culture times may result in a cell population with increased numbers of committed progenitor cells of a particular lineage due to differentiation of the HSC originally produced by c-myc antisense treatment of the cell population enriched for HSC. In this aspect, the c-myc antisense oligonucleotides of the invention find utility in methods to increase the number of differentiated and committed progenitor cells of various lineages obtained by differentiation of the increased population of hematopoietic stem cells in vitro. Such an increased population of differentiated and committed progenitor cells may then be re-infused into a patient.
In such cases, hematopoietic stem cells are obtained from a patient in need of transplantation of a particular type of cell, e.g., neutrophils, platelets, lymphocytes, erthryrocytes or monocytes. Re-infused ex vivo c-myc antisense oligomer-treated hematopoietic stem cells provide a means to rapidly increase the number of both neutrophils and platelets in the circulation of a patient following chemotherapy or radiation therapy.
In this aspect, an enriched population of HSC are obtained, then cultured in medium containing a c-myc antisense oligonucleotide together with one or more growth factors effective to promote hematopoietic stem cell replication without differentiation. The cells are cultured for a time sufficient to result in an increase in the population of hematopoietic stem cells, followed by removal of the antisense oligomer from the culture media and subsequent culture under conditions effective to result in differentiation of the hematopoietic stem cells. As will be appreciated, an increase in the various cell lineages produced by differentiation of hematopoietic stem cells is obtained from the increased population of hematopoietic stem cells, following removal of the antisense oligomer from the culture media. In particular, large numbers of neutrophils and platelets maybe obtained using this procedure.
C. In Vivo Administration Of Antisense Oligomers
In one aspect, the invention is directed to a method for modifying the development of hematopoietic stem cells, by exposing them in vivo to an antisense oligomer having high affinity to a complementary or near-complementary c-myc sequence.
In some cases, a subject is in need of an increased number of hematopoietic stem cells, e.g., following chemotherapy or radiation therapy.
In one exemplary application of the method administration of a c-myc antisense oligomer to a subject results in an increase in the number of hematopoietic stem cells in the subject, e.g., following chemotherapy or radiation therapy.
In this embodiment, a c-myc antisense oligomer is administered to the subject in a manner effective to result in an increase in the number of hematopoietic stem cells in the subject.
In other cases, a subject is in need of an increased number of committed progenitor cells produced by differentiation of hematopoietic stem cells such as neutrophils and/or platelets, e.g., following chemotherapy or radiation therapy.
In this embodiment, a c-myc antisense oligomer is administered to the subject in a manner effective to result in the differentiation of HSC and a corresponding increase in the number of neutrophils and platelets in the subject.
It will be understood that in vivo administration of a c-myc antisense oligomer to a subject using the methods described herein can result in an increase in the population of hematopoietic stem cells, and/or the population of lineage committed progenitor cells and their progeny, dependent upon a number of factors including ( 1) the duration, dose and frequency of c-myc antisense administration, and (2) the general condition of the subject.
1. Treating Patients
Effective delivery of an antisense oligomer to the target c- yc nucleic acid sequence is an important aspect of the methods of the invention. In accordance with the invention, effective delivery of an oligomer antisense to c-myc may include, but is not limited to, various systemic routes, including oral and parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal, and intramuscular; as well as inhalation and transdermal delivery.
It is appreciated that any methods effective to deliver a c-myc antisense oligomer to hematopoietic stem cells or to introduce the drug into the bloodstream of a subject are also contemplated.
Transdermal delivery of antisense oligomers may be accomplished by use of a pharmaceutically acceptable carrier adapted for e.g., topical administration. One example of morpholino oligomer delivery is described in PCT patent application WO 97/40854.
In one preferred embodiment, the oligomer is a phosphorodiamidate morpholino oligomer (PMO), contained in a pharmaceutically acceptable carrier, and delivered orally. In a further aspect of this embodiment, a morpholino c-myc antisense oligonucleotide is administered at regular intervals for a short time period, e.g., daily for two weeks or less. However, in some cases the antisense oligomer is administered intermittently over a longer period of time.
Typically, one or more doses of antisense oligomer are administered, generally at regular intervals for a period of about one to two weeks. Preferred doses for oral administration are from about 1 mg oligomer/patient to about 25 mg oligomer/patient (based on an adult weight of 70 kg). In some cases, doses of greater than 25 mg oligomer/patient may be necessary. For IV administration, the preferred doses are from about 0.5 mg oligomer/patient to about 10 mg oligomer/patient (based on an adult weight of 70 kg). The antisense compound is generally administered in an amount sufficient to result in a peak blood concentration of at least 200-400 nM c-myc antisense oligomer.
In general, the method comprises administering to a subject, in a suitable pharmaceutical carrier, an amount of the antisense agent effective to inhibit expression of the c-myc nucleic acid target sequence. It follows that the antisense oligonucleotide composition may be administered in any convenient vehicle, which is physiologically acceptable. Such an oligonucleotide composition may include any of a variety of standard physiologically acceptable carriers employed by those of ordinary skill in the art. Examples of such pharmaceutical carriers include, but are not limited to, saline, phosphate buffered saline (PBS), water, aqueous ethanol, emulsions such as oil/water emulsions, triglyceride emulsions, wetting agents, tablets and capsules. It will be understood that the choice of suitable physiologically acceptable carrier will vary dependent upon the chosen mode of administration.
In some instances liposomes may be employed to facilitate uptake of the antisense oligonucleotide into cells. (See, e.g., Williams, 1996; Lappalainen, et al, 1994; Uhlmann, et al, 1990; Gregoriadis, 1979.) Hydrogels may also be used as vehicles for antisense oligomer administration, for example, as described in WO 93/01286. Alternatively, the oligonucleotides may be administered in microspheres or microparticles. (See, e.g., Wu and Wu, (1987).
Sustained release compositions are also contemplated within the scope of this application. These may include semipermeable polymeric matrices in the form of shaped articles such as films or microcapsules.
In preferred applications of the method, the subject is a human subject. The subject may also be a cancer patient, in particular a patient diagnosed as having a form of leukemia, lymphoma, neuroblastoma breast cancer, colon cancer, lung cancer, or any type of cancer where the patient is being treated or has been treated with chemotherapy or radiation therapy. The method is also applicable to treatment of non-cancerous hematopoietic disorders, including, but not limited to, aplastic anemia, severe combined immunodeficiency, sickle cell anemia, thalassemia and myelodysplastic syndromes.
It will be understood that the effective in vivo dose of a c-myc antisense oligonucleotide for use in the methods of the invention will vary according to the frequency and route of administration as well as the condition of the subject under treatment. Accordingly, such in vivo therapy will generally require monitoring by tests appropriate to the condition being treated and a corresponding adjustment in the dose or treatment regimen in order to achieve an optimal therapeutic outcome.
In one aspect, the invention provides a method to produce an increased population of more differentiated cells derived from an increased population of hematopoietic stem cells, such as monocytes, granulocytes, platelets, lymphocytes and red blood cells, by exposing a hematopoietic stem cell to the antisense oligomers described herein, under conditions effective to increase the population of hematopoietic stem cells, then further treating the increased population of hematopoietic stem cells in a manner effective to promote differentiation of the hematopoietic stem cells to cell having a more mature phenotype.
A therapeutic regimen may include administration of c-myc antisense treated HSC and/or direct administration of a c-myc antisense oligomer to the subject. Such administration may be concurrent or sequential. In some cases, the patient may be further treated by one or more of chemotherapy, radiation therapy and other agents typically used by those of skill in the art to treat individuals diagnosed with the same or a similar condition. Such further treatment may be concurrent with, sequential or alternating relative to administration of c-myc antisense treated HSC and/or direct administration of a c-myc antisense oligomer to the subject.
2. Monitoring Treatment
The efficacy of a given therapeutic regimen involving the methods described herein, may be monitored, e.g., by conventional FACS assays for the phenotype of cells in the circulation of the subject under treatment in order to monitor changes in the numbers of cells of various lineages in response to antisense treatment.
Phenotypic analysis is generally carried out using monoclonal antibodies specific to the cell type being analyzed. The use of monoclonal antibodies in such phenotypic analyses is routinely employed by those of skill in the art for cellular analyses. Monoclonal antibodies specific to particular cell types are commercially available.
Hematopoietic stem cells are characterized phenotypically as detailed above. Such phenotypic analyses are generally carried out in conjunction with biological assays for each particular cell type of interest, for example (1) hematopoietic stem cells (LTCIC, cobblestone forming assays, and assays for HPP-CFCs), (2) granulocytes or neutrophils (clonal agar or methyl cellulose assays wherein the medium contains G-CSF or GM-CSF), (3) megakaryocytes
(clonal agar or methyl cellulose assays wherein the medium contains TPO, IL-3, IL-6 and IL- 1 1), and (4) erythroid cells (clonal agar or methyl cellulose assays wherein the medium contains EPO and SCF or EPO, SCF and IL-3).
It will be understood that the exact nature of such phenotypic and biological assays will vary dependent upon the condition being treated and whether the treatment is directed to enhancing the population of hematopoietic stem cells or the population of cells of a particular lineage or lineages.
In cases where the subject has been diagnosed as having a particular type of cancer, the status of the cancer is also monitored using diagnostic techniques appropriate to the type of cancer under treatment. The antisense oligomer treatment regimen may be adjusted (dose, frequency, route, etc.), as indicated, based on the results of the phenotypic and biological assays described above.
V. Applications/Utilitv of the Invention
As described herein, hematopoietic stem cells treated with a c-myc antisense oligonucleotide find utility in a variety of applications. For example, treatment of HSC with a c-myc antisense oligonucleotide yields an in vitro expanded hematopoietic stem cells, which may be used for a variety of purposes. In addition, under appropriate conditions, such a population of in vitro expanded hematopoietic stem cells serve as a source for production of committed progenitor cells and their progeny, e.g., neutrophils or platelets. Ex vivo expanded cell HSC and committed progenitor cell populations find utility in both autologous and allogeneic hematopoietic engraftment when administered to a patient, where the cells are freed of neoplastic cells and graft-versus-host disease can be avoided.
In one aspect, once extracted and enriched, hematopoietic stem cells may be cultured ex vivo in the presence of one or more cytokines and a c-myc antisense oligomer. Such an antisense oligomer-treated hematopoietic stem cell culture finds utility in a variety of applications, including, but not limited to: (1) expanding or multiplying the population of hematopoietic stem cells ex vivo for subsequent in vivo administration to a subject for purposes of (a) hematopoietic stem cell replacement therapy, (b) treatment of autoimmune disease, (c) reducing the immune response to allogeneic transplants, or (d) treatment of HIV-infection in a subject; and (2) inhibiting or arresting growth of cancer cells wherein the cancer is associated with c-myc expression.
In another aspect, once extracted and enriched, hematopoietic stem cells may be cultured ex vivo in the presence of one or more cytokines and a c-myc antisense oligomer for a time sufficient to increase the population of particular lineage-committed progenitor cells and their progeny. This process may be carried out ex vivo followed by in vivo administration to a subject for (a) cell replacement therapy, and (b) augmenting vaccination.
Autologous hematopoietic stem cell transplantation has been used to treat many solid tumors, including, but not limited to, breast cancer and ovarian cancer. Prior to hematopoietic stem cell transplantation the patient may or may not receive a chemotherapy regimen to reduce the amount of tumor present, generally followed by: (1) the collection of the patient's hematopoietic stem cells from either bone marrow or peripheral blood; (2) culture of hematopoietic stem cells in the presence of cytokines or cryopreservation in liquid nitrogen; (3) high-dose chemotherapy administration intravenously (in most cases);and (4) reinfusion of the patient's hematopoietic stem cells (IV), after the chemotherapy administration is complete; and (5) further treatment of the patient with growth factors to promote the differentiation of the hematopoietic stem cells and repopulation of the hematopoietic system of the patient. In general, during this time the patient is immunocompromised and protective isolation is required.
Allogeneic hematopoietic stem cell transplantation has been used to treat patients with leukemia, aplastic anemia, lymphomas (Hodgkin's disease and non-Hodgkin's lymphoma), and immune deficiency diseases. An allogeneic hematopoietic stem cell transplantation protocol is similar to that used for autologous transplantation with the exception that in allogeneic transplantation, the donor and recipient must be matched based on the similarity of HLA cell surface antigens in order to minimize the immune response of both donor and recipient cells against the other.
Increasing the population of hematopoietic stem cells together with committed progenitor cells and their progeny prior to transplantation can shorten the lag period during which the transplanted cells proliferate sufficiently to repopulate the hematopoietic system of the recipient.
A c-myc antisense oligomer of the invention may be used for the treatment of a potential bone marrow hematopoietic stem cell donor, prior to the removal of the bone marrow, in order to increase the population of hematopoietic stem cells in the tissue prior to bone marrow removal.
In a related approach, the methods of the invention can be used to expose bone marrow or purified hematopoietic stem cell populations to a c-myc antisense oligomer of the invention, prior to their storage in cell banks in order to retain the cells in a form devoid of tissue histocompatibility antigens. In one exemplary application of the method, the subject is a cancer patient, and the treatment of the patent's hematopoietic stem cells with a c-myc antisense oligomer is used to inhibit or arrest the growth of cancer cells, wherein the cancer is associated with c-myc expression. Once the cells have been treated with c- yc antisense oligomers. the cells may either be (1) directly infused into the patient, and/or (2) be cultured under conditions effective to result in differentiation of the hematopoietic stem cells to produce an expanded population of lineage committed progenitor cells and the progeny thereof, which are then infused into the patient.
It will be understood that the duration of in vitro culture and the time of exposure to c-myc antisense may be varied to result in different ratios of hematopoietic stem cells and lineage committed progenitor cells and their progeny, prior to re-infusion into the subject.
In some cases, a therapeutic regimen involving such ex vivo c-myc antisense treatment of hematopoietic stem cells for treatment of cancer further includes additional intervention such as radiation therapy and/or chemotherapy. The treatment may occur prior to, during or subsequent to re-infusion of c-myc antisense-treated cells. 1. Graft versus Host disease (GVHD)
Treatment of a transplant recipient's own bone marrow or hematopoietic stem cells (e.g., bone marrow extracted from the patient before commencing chemotherapy or radiation therapy), with a c-myc antisense oligomer may be employed to avoid the need for immune suppression typical of current methodologies, by minimizing the potential for GVHD following transplantation. c-myc antisense oligomers may also be used to treat tissue and organ donors as well as transplant recipients in order to minimize GVHD.
GVHD is a frequent complication of allogeneic transplantation. About half of the patients undergoing an allogeneic bone marrow transplant develop some GVHD, which is generally mild, however, the condition can be life threatening. In GVHD, the donor's cells attacks the recipient's organs and tissue. Patients with GVHD have increased susceptibility to infection and the skin, liver, and gastrointestinal tract may be attacked in GVHD.
GVHD is caused by T-cells, which recognize the patient's cells as foreign, based on differences in human leukocyte antigens (HLA). Even when the donor and recipient have similar HLA types, many minor markers differ between them except when the donor and recipient are identical twins. Hence, graft versus host disease (GVHD) is a potential problem and treatment to minimize the GVH response is part of the therapeutic regimen for most transplants. In the case of hematopoietic stem cell transplantation, such treatment generally includes T- cell depletion alone (i.e., by elutriation which removes T-cells based on density gradient centrifugation), or in combination with hematopoietic stem cell enrichment, and drug therapy for prevention of GVHD. Exemplary drug therapy for GVHD is administration of cyclosporine (an immunosuppressive drug), alone or together with mehtotrexate. In one aspect, the invention provides a population of c-myc antisense treated hematopoietic stem cells, such that transplantation of the hematopoietic stem cells into an allogeneic host is unlikely to result in GVHD.
In a related aspect, the invention provides a population of lineage-committed progenitor cells and their progeny, derived from a population of c-myc antisense treated hematopoietic stem cells such that transplantation of the lineage-committed progenitor cells and their progeny into an allogeneic host is unlikely to result in GVHD. While the mechanism is not part of the invention, such a population of c-myc antisense treated hematopoietic stem cells is expected to lack immunological memory of self and non-self antigens, thereby minimizing the likelihood of GVHD. Such c-myc antisense oligomer treated cells may be used for allogeneic transplantation in order to minimize or eliminate graft versus host disease (GVHD).
The methods of the invention may be used to generate increased numbers of hematopoietic stem cells alone or together with increased numbers of lineage-committed progenitor cells and their progeny, based on the duration of culture and the duration of exposure of the hematopoietic stem cells to c-myc antisense oligomers. In cases where hematopoietic stem cells, lineage committed progenitor cells and their progeny, tissues, or organs are transplanted, a c- yc antisense oligomer may also be administered to the recipient to further minimize the immune response against the transplanted hematopoietic stem cells, lineage committed progenitor cells and their progeny, tissues, and organs.
The present method may be used to expand a population of the recipient's own hematopoietic stem cells in vitro (ex vivo) preserving the multilineage potential of such cells without lineage commitment, e.g., for use in autologous hematopoietic stem cell transplantation.
2. Autoimmune Disease
As hematopoietic stem cells differentiate they are exposed to the various antigens present on the cells and tissue of the host and immunological tolerance is established during T cell development within the thymus. In general, T cells that would be reactive with host proteins do not survive. However, in some cases, the immune system may recognize self antigens as foreign resulting in an immune reaction against one or more endogenous antigens, leading to an autoimmune condition or disease. Exemplary autoimmune conditions include organ specific forms wherein the immune response is directed against, e.g., the cells of the adrenal glands, causing Addison's disease, against the thyroid causing auto-immune thyroiditis (Hashimoto's disease) or against the beta cells of the islets of Langerhans in the pancreas, resulting in insulin-dependent diabetes mellitus; and non-specific forms wherein the immune response is directed against an antigen that is ubiquitous, e.g., an immune reaction against DNA, resulting in the disease systemic lupus erythematosus. Further examples include, Sjogren's syndrome, caused by the production of auto-antibodies against salivary ducts, rheumatoid arthritis. Auto-immunity may be the result of attack by antibodies, T-cells or both.
The invention provides methods and compositions for the treatment of autoimmune disease. In such methods, hematopoietic stem cells may be obtained from the patient, followed by treatment of the patient with chemotherapy, radiation therapy or other means to deplete the patient of residual T-cells. The patients' hematopoietic stem cells or hematopoietic stem cells from an allogeneic donor are then exposed ex vivo to a c-myc antisense oligomer under conditions effective to result in an increase in the number of viable hematopoietic stem cells in vitro, which are then infused into the patient. As the expanded population of hematopoietic stem cells develops in the presence of the antigenic repertoire of the host, the newly developed T-cells should not recognize host antigens as foreign and an autoimmune response should not occur.
In vitro c-myc antisense-treated hematopoietic stem cells lack immunological memory of self antigens, such that transplantation of c-myc-treated hematopoietic stem cells or lineage-committed progenitor and progeny cells derived therefrom may be used in transplantation regimens for treatment of a patient with an autoimmune disease, in order to minimize or eliminate the autoimmune condition.
The methods of the invention may be used to generate increased numbers of hematopoietic stem cells alone or together with increased numbers of lineage-committed progenitor cells and their progeny, based on the duration of culture and the duration of exposure of the hematopoietic stem cells to c-myc antisense oligomers. In a related aspect of this embodiment, a patient with an autoimmune disease may be treated by in vivo administration of a c-myc antisense oligonucleotide. Such in vivo administration may be carried out alone or in conjunction with administration of ex vivo c-myc antisense oligomer treated HSC and/or lineage-committed progenitor cells and their progeny. It will be understood that such autoimmune disease treatment regimens will further include additional treatment components, as needed to optimize the therapeutic outcome of the patient. Such additional treatment components include compositions and procedures known in the art for the treatment of autoimmune disease.
3. Tissue And Organ Transplantation
In a related approach, a c-myc antisense oligomer-treated HSC composition of the invention may be used to modulate the response of a recipient to an organ or tissue transplant. In this aspect of the invention, a c-myc antisense oligomer of the invention is administered to an organ and/or tissue transplant recipient, prior to the donation procedure, to decrease the immune response and resulting tissue rejection following transplantation.
This may be accomplished by treating the tissue or organ to be transplanted in a manner effective to deplete T-cells in vitro under conditions appropriate for transplantation procedures, as generally employed by those of skill ion the art. Such in vitro treatment may be carried out in conjunction with the administration of a c-myc antisense oligomer to the patient, prior to, during and for a limited time following such transplantation, in a manner effective to minimize or eliminate GVHD.
4. Augmentation Of The Immune Response To A Vaccine
In a further embodiment, the invention provides methods and compositions for the augmentation of the response to a vaccine. In such methods, a c-myc antisense oligomer is administered to a subject, prior to or concurrent with, administration of a vaccine, thereby resulting in a greater immune response to the immunogens present in the vaccine.
The following examples illustrate but are not intended in any way to limit the invention.
EXAMPLE 1
Effect Of c-myc Antisense On LTR-HSC In Vitro
Murine hematopoietic stem cells were obtained from bone marrow starting with unfractionated bone marrow, performing a density separation using 1.080g/ml Nycodenz separation medium (Nycomed Pharma AS OSLO, Norway), followed by isolation of the lin- cell population using Dynal Bead depletion employing lineage-specific monoclonal antibodies, followed by staining with: (1) antibodies to c-kit and Sea 1 ; (2) propidium iodide; and (3) the dyes, Hoescht 33342, Rhodamine 123 and propidium iodide (PI ), then sorting cells using FACS by selecting for either (A) Sea 1+, c-kit+, lin-, Hoescht 33342 low (Holmv), Rhodamine 123 low (Rhlow) and PI negative cells (LTR-HSC), or (B) Sea 1+, c-kit+, lin- cells, (STR-HSC). A. Culture of HSC in medium containing IL-6 and SCF +/- antisense oligomers 25 cells enriched for STR-HSC, characterized as Sea 1 +, c-kit+, and lin- were directly sorted into medium and cultured for 3 days in the presence of IL-6 and SCF together with either (1 ) no antisense oligomer, (2) 25 μM of scrambled c- yc antisense oligomer having the sequence presented as SEQ ID NO:2, or (3) 1, 5, 25, or 125 μM of a morpholino phosphoroamidate c- myc antisense oligomer having the sequence presented as SEQ ID NO: l . After 3 days, the cells were counted, plated in agar, then evaluated for HPP at day 0 and day 3 in a standard HPP-CFC assay. (See Table 1 and Fig. 3)
The results indicate that after 3 days culture in the presence of IL-6 + SCF + 125 μM c-myc antisense, total cell production (cell birth minus death) is arrested, while HPP-CFC are "preserved" or expanded (approximately 33 HPP for cells cultured in the presence of IL-6 + SCF +125 μM c-myc antisense vs. approximately 3 HPP in cells cultured in the presence of IL- 6 + SCF alone).
Table 1. Culture Of HSC In Medium Containing IL-6 And SCF +/- Antisense Oligomers
Figure imgf000028_0001
B. Single HSC escape the proliferation inhibition induced by 25 μM c- vc antisense Single highly purified STR- or LTR-HSC were directly sorted into 96-well plates containing medium with IL-3+ IL-6+ SCF alone or IL-3+ IL-6+ SCF and 25 μM morpholino phosphoroamidate c-myc antisense oligomer having the sequence presented as SEQ ID NO: 1 , or scrambled c-myc antisense oligomer having the sequence presented as SEQ ID NO:2. Table 2. Culture Of Single HSC +/- Antisense Oligomers
Figure imgf000029_0001
The results indicate that when cultured as single cells, both STR- and LTR-HSC continue to increase their clone size.
C. Antisense c-myc mediates self-replication of LTR-HSC daughter cells during the 2nd - 4th cell division ex vivo
In a further experiment, single purified LTR-HSC [characterized as lin-, c-kit+, Hoescht low (Holow) and Rhodamine low (Rhlow)] were directly sorted into 96 well tissue culttire plates containing medium with IL-3, IL-6 and stem cell factor (SCF), with or without 25 μM antisense oligomers, and cultured for 6 days. At day 6 the number of cells per well (clone) was determined and transferred into agar and assayed for HPP-CFC (i.e., the proliferative potential of individual daughter cells could then be determined).
Table 3. Effect of Antisense c-mvc on self-replication of LTR-HSC daughter cells during the 2nd - 4th cell division ex vivo
Figure imgf000029_0002
The results indicate that during the 4 to 8-cell stage, no difference in HPP-CFC were detected amongst treated and untreated cultures. However, during the 4 to approximately 32 cell stage, the presence of 25 μM of c-myc antisense oligomer in the culture medium resulted in a doubling of the number of HPP-CFC per clone compared to either c-myc antisense scramble or growth factor alone-treated cultures, suggesting that reduction in c-myc expression mediates self-replication of HSCs.
EXAMPLE 2 Effect Of In Vivo Administered c-myc Antisense (AS) On Hematopoiesis. A. Mice Treated 7 Days With A c-myc Antisense Oligomer
Various amounts of morpholino phosphoroamidate c-myc antisense oligomer having the sequence presented as SEQ ID NO: 1 were injected intraperitoneally (IP) into mice, daily for 7 days. Samples of peripheral blood was taken from the mice and analyzed at 7, 21 and 28 days.
Figures 4A-D indicate the number of erythrocytes (A), granulocytes (B), lymphocytes (C), and white blood cells (D) detected in the peripheral blood of the mice.
The results indicate that administration of 100 μg c-myc antisense to mice for 21 -28 days effected an apparent decrease in granulocytes (B) and white blood cells (D), in the peripheral blood at day 7, followed by an increase at days 21 and 28, wherein the magnitude of the effect greatest at 100 to 300 μg morpholino phosphoroamidate c-myc antisense oligomer. A minimal effect on the number of erythrocytes (A) or lymphocytes (B) in the peripheral blood, was observed following treatment.
B. Mice Treated 2 Or 9 Days With A c-mvc Antisense Oligomer 100 μg morpholino phosphoroamidate c-myc antisense oligomer having the sequence presented as SEQ ID NO: 1 was injected intraperitoneally (IP) into mice, daily for 2 or 1 1 days. The bone marrow compartment of the mice was evaluated at day 1 1 , and the results indicate that the ckit+ (CD1 17 or hematopoietic stem cell) component of the bone marrow was increased in animals treated with 100 μg c-myc antisense oligomer and that treatment for 2 days was equally effective to treatment for 1 1 days. (Table 4)
Table 4. Effect Of 2 Or 9 Day c-mvc Antisense Treatment On The Bone Marrow Of Mice
Figure imgf000030_0001
Table 5. Sequences Provided In Support Of The Invention
Description SEQ ID NO antisense to c-myc AVI 4126 1
5'-ACG TTG AGG GGC ATC GTC GC-3' c-myc antisense reverse control AVI 4198 2
5'-CGC TGC TAC GGG GAG TTG CA-3' c-myc antisense scramble U2 AVI 4199 _>
5'-GTA TGC GAG CGA CTG CGG CT-3' c-myc antisense scramble control AVI 4144 4
5'- ACT GTG AGG GCG ATC GCT GC-3' c-myc antisense scrambled by triplets control AVI 4166 5
5'-TTG ATC ACG GCG TCA GGG GC-3'

Claims

IT IS CLAIMED:
1. A method of producing a hematopoietic stem cell (HSC) composition, comprising: (a) obtaining an HSC-containing cell population from a subject; (b) treating the cell population in manner effective to enrich the cell population for HSC; and
(c) exposing the enriched HSC population, ex vivo to a c- yc antisense oligomer, at an oligomer concentration, and for a period of time, sufficient to increase the number of hematopoietic stem cells in such population at least two-fold, wherein said HSC are characterized as lacking the expression of lineage markers (lin-), positive for cell surface expression of c-kit (CD 117) and positive for cell surface expression of Seal .
2. The method of claim 1 , wherein said exposing is carried out at a concentration of antisense oligomer, and for a period sufficient to sufficient to increase the number of hematopoietic stem cells in such population at least four-fold relative to the number of HSC present in said cell population prior to treatment with said c-myc antisense oligomer.
3. The method of claim 1, wherein the HSC-containing cell population is obtained from human bone marrow.
4. The method of claim 1, wherein the HSC-containing cell population is obtained from mobilized human peripheral blood.
5. The method of claim 1, wherein the HSC-containing cell population is obtained from a human cancer patient, and said exposing is effective to reduce the number of viable cancer cells in said expanded HSC population, relative to the number of cancer cells present in said enriched HSC population prior to treatment with said c-myc antisense oligomer.
6. The method of claim 5, wherein the HSC-containing cell population is obtained from a patient with lymphoma or leukemia.
7. The method of claim 1, wherein said antisense oligomer has a length of about 12 to 25 bases.
8. The method of claim 1 , wherein said antisense oligomer is characterized by, (a) a backbone which is substantially uncharged;
(b) the ability to hybridize with the complementary sequence of a target RNA with high affinity at a Tm greater than 50°C;
(c) nuclease resistance; and
(d) the capability for active or facilitated transport into cells.
9. The method of claim 1, wherein said antisense oligomer backbone has a structure selected from the group consisting of the structures presented in Figures 2 A-A through 2 E-E.
10. The method of claim 1 , wherein said antisense oligomer has the sequence identified as SEQ ID NO: 1.
1 1. A hematopoietic stem cell (HSC) composition, prepared by the process of:
(a) obtaining an HSC-containing cell population from a subject;
(b) treating the cell population in manner effective to enrich the cell population for HSC; and
(c) exposing the enriched HSC population ex vivo, to a c- yc antisense oligomer, at an oligomer concentration, and for a period of time, sufficient to increase the number of hematopoietic stem cells in such population at least two-fold relative to the number of HSC present prior to treatment with said c-myc antisense oligomer; wherein said HSC are characterized as lacking the expression of lineage markers (lin-), positive for cell surface expression of c-kit (CD 1 17), positive for cell surface expression of Seal .
12. The composition of claim 1 1, wherein said antisense oligomer has a length of about 12 to
25 bases.
13. The composition of claim 1 1, wherein said antisense oligomer is characterized by, (a) a backbone which is substantially uncharged; (b) the ability to hybridize with the complementary sequence of a target RNA with high affinity at a Tm greater than 50°C;
(c) nuclease resistance; and
(d) the capability for active or facilitated transport into cells.
14. The composition of claim 1 1, wherein said antisense oligomer backbone has a structure selected from the group consisting of the structures presented in Figures 2 A-A through 2 E-E.
15. The composition of claim 11, wherein said antisense oligomer has the sequence identified as SEQ ID NO: 1.
16. A composition according to any one of claims 1 1 to 15 for use as a medicament in treating cancer in a subject.
17. A method for delivering a c-myc antisense oligomer-treated hematopoietic stem cell (HSC) composition to a cancer patient, comprising:
(a) obtaining an HSC-containing cell population from said patient;
(b) treating the cell population in manner effective to enrich the cell population for HSC;
(c) exposing the enriched HSC population ex vivo, to a c-myc antisense oligomer, at an oligomer concentration, and for a period of time, sufficient to increase the number of hematopoietic stem cells in such population at least two-fold relative to the number of HSC present prior to treatment with said c-myc antisense oligomer; and (d) introducing said c-myc antisense oligomer-treated hematopoietic stem cell composition into the patient, wherein said HSC are characterized as lacking the expression of lineage markers (lin-), positive for cell surface expression of c-kit (CD 1 17) and positive for cell surface expression of Seal .
18. The method of claim 17, wherein said exposing is carried out at a concentration of antisense oligomer, and for a period sufficient to increase the number of hematopoietic stem cells in such population at least four-fold relative to the number of HSC present in said cell population prior to treatment with said c-myc antisense oligomer.
19. The method of claim 17, wherein said exposing is effective to reduce the number of viable cancer cells in such population relative to the number of viable cancer cells present prior to treatment with said c-myc antisense oligomer.
20. The method of claim 17, wherein the antisense oligomer has a length of about 12 to 25 bases.
21. The method of claim 17, wherein said antisense oligomer is characterized by, (a) a backbone which is substantially uncharged; (b) the ability to hybridize with the complementary sequence of a target RNA with high affinity at a Tm greater than 50°C;
(c) nuclease resistance; and
(d) the capability for active or facilitated transport into cells.
22. The method of claim 17, wherein said antisense oligomer backbone has a structure selected from the group consisting of the structures presented in Figures 2 A-A through 2 E-E.
23. The method of claim 17, wherein said antisense oligomer has the sequence identified as SEQ ID NO: 1.
PCT/US2000/027311 1999-10-07 2000-10-04 C-myc antisense-treated hematopoietic stem cell composition and method WO2001025405A1 (en)

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CN100506978C (en) * 2001-09-21 2009-07-01 中国人民解放军军事医学科学院野战输血研究所 Hemapoietic stem/ancestral cell enriching method and its in vitro directional induction and differentiation
EP2351844A1 (en) 2003-04-29 2011-08-03 AVI BioPharma, Inc. Compositions for enhancing transport and antisense efficacy of nucleic acid analog into cells
US9156912B2 (en) 2008-12-12 2015-10-13 The University Of Tokyo Immunological reconstitution promoter or prophylactic agent for infections each of which maintains graft-versus-tumor effect
WO2015113041A3 (en) * 2014-01-27 2015-11-05 The Children's Hospital Of Philadelphia Compositions and methods for treating autoimmune and inflammatory diseases
US10130650B2 (en) 2014-01-27 2018-11-20 The Children's Hospital Of Philadelphia Compositions and methods for treating autoimmune and inflammatory diseases

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