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WO1999022001A1 - Procede de regulation de la differenciation/ proliferation des cellules souches hematopoietiques - Google Patents

Procede de regulation de la differenciation/ proliferation des cellules souches hematopoietiques Download PDF

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Publication number
WO1999022001A1
WO1999022001A1 PCT/JP1998/004884 JP9804884W WO9922001A1 WO 1999022001 A1 WO1999022001 A1 WO 1999022001A1 JP 9804884 W JP9804884 W JP 9804884W WO 9922001 A1 WO9922001 A1 WO 9922001A1
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gene
cells
differentiation
hematopoietic stem
stem cells
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PCT/JP1998/004884
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English (en)
Japanese (ja)
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Masatake Osawa
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Kirin Beer Kabushiki Kaisha
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Priority to AU96498/98A priority Critical patent/AU9649898A/en
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a method for regulating the differentiation and proliferation of hematopoietic stem cells and / or hematopoietic progenitor cells, and to vectors and cells used in the method.
  • Hematopoietic stem cells and hematopoietic progenitor cells can be used for blood cell transplantation instead of bone marrow transplantation and cord blood transplantation, or for gene therapy.
  • BACKGROUND ART Mature blood cells flowing in the living body have a short life span (about 120 days for red blood cells and about 7 days for platelets in humans), and mature blood cells are differentiated and proliferated daily from hematopoietic progenitor cells. Maintains homeostasis of peripheral blood mature blood cells.
  • the number of mature blood cells supplied to peripheral blood is 200 billion cells / day for red blood cells and 700 billion cells / day for neutrophils in humans.
  • the progenitor cells of each of these differentiation lineages proliferate while differentiating from undifferentiated hematopoietic stem cells to form a system in which peripheral blood cells do not constantly die (Tsuda Suda, Blood Stem Cells Destiny, Sheepsha, 199 2).
  • hematopoietic stem cells pluripotency
  • hematopoietic stem cells pluripotency
  • a transplantation experiment system using irradiated mice, and an in vitro (in vitro) colony formation method (Bradley, TR, J. Exp. Med., 44: 287-299). , 1966), and knowledge on the differentiation of hematopoietic stem cells and hematopoietic progenitor cells has been accumulated.
  • the transplantation experiment system using irradiated mice is the most direct way to analyze the properties of hematopoietic stem cells.
  • Transplantation of bone marrow cells isolated from another mouse (Donna 1) into a mouse (recipient) that has damaged the hematopoietic system by irradiation may reconstitute the donor-derived hematopoietic system in the recipient mouse. it can.
  • Various differentiation antigens expressed on hematopoietic cells (Spangrude, GJ, Proc. Natl. Acad. Sci.
  • Bone marrow cells were fractionated using substances (Rhl23, Hoechst 33342) (Wolf, NS, Exp. Hematol., 21:61 4-622, 1993) and the transplantation experiments described above were performed. Attempts have been made to identify hematopoietic stem cells.
  • hematopoietic stem cells can differentiate into lymphoid cells and myeloid cells by transplanting a single cell, and hematopoietic stem cells can be transplanted for a long time It has also been demonstrated that systems can be constructed (Osawa, M., Science, 273: 242-245, 1996). Hematopoietic stem cells can survive over a long period of time in the recipient individual and supply differentiated blood cells. On the other hand, in transplantation experiments using hematopoietic progenitor cells, the cells transplanted from the recipient individual disappear in a short period of time and cannot undergo hematopoiesis for a long period of time (Osawa, M., Science, 278: 242- 245, 1996).
  • hematopoietic progenitor cells can only differentiate into mature blood cells of a limited lineage even in the presence of many cytokins, hematopoietic stem cells that can build a long-term hematopoietic system in transplanted mice It is possible to differentiate into many cell lineages. From these results, the differentiation from hematopoietic stem cells to mature blood cells flowing in peripheral blood is interpreted as follows.
  • Hematopoietic stem cells can be differentiated into various lineages It is capable of self-replication while maintaining this pluripotency property (pluripotency). Hematopoietic stem cells self-renew and partially differentiate, are affected by various site forces, gradually narrow the lineage of cells that can be differentiated, differentiate and proliferate into hematopoietic progenitor cells that can only differentiate into a limited number of cell types, It eventually becomes a mature blood cell (Hematopoietic Stem Cell, Levitt, D., Marcel Dekker, Inc., 1995). Bone marrow transplantation and umbilical cord blood transplantation are therapies to transplant hematopoietic cells to patients. It depends on what you can do.
  • hematopoietic stem cells are considered to be an optimal target because they can survive for a long time in the transplant recipient as described above.
  • gene therapy that complements the genetic deficiency of hematopoietic stem cells is considered to be a fundamental treatment for the disease.
  • hematopoietic stem cells have been clinically recognized, and it has been an issue how to grow hematopoietic stem cells without differentiation.
  • Attempts have been made to expand hematopoietic stem cells using cytokin-blood cell stimulating factors, but have not succeeded in efficiently expanding hematopoietic stem cells (Trevisan, M., Blood, 88: 4149, 1996).
  • hematopoietic stem cells are differentiated and proliferation of hematopoietic progenitor cells is dominant. Therefore, if the differentiation of hematopoietic stem cells could be suppressed, hematopoietic stem cells could be expanded in the presence of cytokines and hematopoietic cell stimulating factors.
  • Notch / Delta has been shown by genetic analysis to be involved in the formation of various organs such as nerves and wings during embryo development in Drosophila (Artavanis-T sakonas, S., Science, 268: 225, 1995).
  • the ligand, Delta protein binds to the receptor, Notch protein, and transmits signals through Notch to suppress differentiation.
  • homologous genes to the Notch / Delta gene family are known (Blaumueller, CM, Perspectives on Developmental Neurobiology, 4: 325, 1997).
  • the regulation of differentiation by the Notch signaling system in neural cell differentiation is considered as follows (Artavanis-Tsakonas, S., Science, 268: 225, 1995; Simpson, P., Perspectives on Developmental Neurobiology, 4). : 297, 1997).
  • the dorsal-ventral axis and anterior-posterior axis are determined during embryonic development, neuroblasts appear in the ectoderm and proneural clusters that can differentiate into epidermal cells appear.
  • the Notch / Delta signaling system plays an important role in selecting cells capable of differentiating into neurons from this cell cluster.
  • the cells present in this cell cluster express Notch equally, but some cells begin to express Notch ligand, which suppresses neighboring cells from differentiating into neural cells ( Lateral suppression). Cells that express Notch ligand differentiate into neuroblasts, and undergo terminal differentiation under various stimuli to achieve terminal differentiation into functional neurons. On the other hand, cells in which the Notch / Delta signaling system has been activated by binding to Notch ligand cannot differentiate into neurons, but differentiate into epidermal cells.
  • Notch is activated by binding to Delta, Interacts with the nuclear localization protein RBP-J / c (CBF-1, also called KBF-2) through the region (Honjo, T., Genes to Cells, 1: 1, 1996).
  • RBP-J / translocates into the nucleus following Notch activation and induces expression of the HLH (helix-loop-helix) transcription factor HES-1 (Ryuichiro Kageyama, Biochemistry, 67: 1093, 1995) I do.
  • An HLH-type transcription factor is a transcription factor having a three-domain structure, that is, 1) a helix-one-loop part that forms a three-dimensional structure of one helix, which binds to itself or interacts with other HLH-type transcription factors. It comprises a binding portion for forming a homodimer or a heterodimer by binding, 2) a portion capable of binding to basic DNA, and 3) a portion having a transcription promoting activity.
  • a characteristic of HLH-type transcription factors is that their DNA binding ability is regulated by the partner that forms the heterodimer.
  • Mash-1 and MATH are known as HLH-type transcription factors that positively regulate the differentiation of nerve cells. Both Mash-1 and MATH are HLH-type transcription factors that form heterodimers with the ubiquitous HLH-type transcription factor E12 / E47, and recognize specific nucleotide sequences to promote neuronal cell differentiation. Activating genes (Johnson, JE, Proc. Natl. Acc. Sci. USA, 89: 3596, 1992).
  • HES-1 binds to Mash-1 and MATH in a competitive manner with E12 / E47 and suppresses its DM binding activity, thereby inhibiting the expression of genes that promote differentiation into nerve cells, and It is thought to suppress the differentiation of cells (Sasai, Y., Genes Dev., 6: 2620, 1992).
  • HES-1 plays an important role in regulating the differentiation of the Notch / Delta system by inhibiting the activity of transcription factors that positively regulate differentiation at the final stage. Similar regulatory mechanisms have been inferred for the HES-1 similar genes HES-3 and HES-5. (Ryuichiro Kageyama, Biochemistry, 67: 1093, 19995)
  • the differentiation of the skeletal muscle system is regulated by the Notch / Delta system, but even if the HES-1 gene is directly introduced into and expressed in skeletal myoblasts, differentiation into skeletal muscle cells is suppressed.
  • a different regulation mechanism from the signal transduction system from RBP-Jc to the HES family gene has been speculated (Shawber, C., Development, 122: 3765, 1996). In other words, differentiation control is not performed through HES-1 in all cell types.
  • Notch-1 gene also known as TAN-1
  • T cell leukemia Ellison, LW, Cell, 66: 649, 1991; Reynolds, TC, Cell, 50: 107, 1987.
  • transgenic mice transfected with an activated Notch gene are involved in the control of T cell proliferation because leukemia with immature T cell properties is produced (Pear, WS, J. Exp. Med., 183: 2283, 1996).
  • the Notch gene has been reported to be expressed in hematopoietic stem cells (Milner, LA, Blood, 83: 2057, 1994), but during the process of differentiating hematopoietic stem cells into various differentiated cells.
  • the Notch / Delta feature has not been reported.
  • Dll-1 Choitnis, A., Nature, 375: 761, 1995
  • Dll-3 Dunwoodie, SL, Development, 124: 3065, 1997)
  • Jagge d-1 Lidsell , CE, Cell, 80: 909, 1995
  • Jagged-2 Jagged-2
  • DLK also called SCP-1 or Pref-1
  • DLK When DLK is expressed in stromal cells that support hematopoietic cells and cocultured with hematopoietic stem cell fractions, activity to support the proliferation of hematopoietic progenitor cells has been confirmed. However, no activity has been observed when DLK, a membrane protein, is solubilized and DLK acts directly on hematopoietic stem cells. Therefore, it is not clear whether DLK exerts its activity of inhibiting differentiation by directly acting on hematopoietic progenitor cells. Furthermore, because DLK has low homology to Delta, it is unclear at this time whether DLK signals through Notch.
  • the present invention has been made in view of the above, and provides a method for suppressing the differentiation of hematopoietic stem cells and / or hematopoietic progenitor cells, and a method for allowing the cells to survive and preferably further proliferate in a state where the differentiation is suppressed. And to provide means used for these methods.
  • Notch / Delta-based molecules could be used to control the differentiation of hematopoietic stem cells.
  • Notch-l, Notch-2, Notch-3, Notch-4 and Notch-4 were actually used in hematopoietic cells including hematopoietic stem cells.
  • HES-1, HES-3 and HES-5 were examined in detail.
  • Notch and HES genes were widely expressed in hematopoietic cells at various stages of differentiation. From this expression status, it was speculated that the signaling system of the Notch / Delta system plays an important role also in cell differentiation from hematopoietic stem cells.
  • the present invention provides a method for enhancing the expression of a differentiation-suppressing gene in mammalian hematopoietic stem cells and causing a blood cell stimulating factor to act thereon, whereby the hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells are activated. It is a method of regulating the differentiation and proliferation of the plant.
  • the present invention also provides a gene transfer vector in which a differentiation-suppressing gene has been incorporated into a viral vector.
  • the present invention further provides mammalian hematopoietic stem cells in which the expression of the differentiation inhibitory gene is enhanced.
  • the present invention provides a hematopoietic stem cell characterized by culturing a mammalian hematopoietic stem cell in which the expression of a differentiation inhibitory gene is enhanced or a hematopoietic progenitor cell differentiated from the hematopoietic stem cell while allowing a blood cell stimulating factor to act. And / or provide a method for producing hematopoietic progenitor cells.
  • a hematopoietic stem cell is a cell having a pluripotency capable of differentiating into all differentiation lineages of blood cells, and a cell capable of self-renewal while maintaining the pluripotency.
  • CFU-Emix colony containing cell types of multiple differentiating lineages including erythrocytes in an in vitro Atsushi system. These cells are thought to contain cells that self-renew in vivo and survive for long periods of time.
  • Hematopoietic progenitor cells are blood cells that have been slightly differentiated from hematopoietic stem cells, and have the ability to differentiate into single or two types of lineages.
  • the differentiation inhibitory gene refers to a gene that encodes a transcription factor having an activity of suppressing the differentiation of a cell when expressed in a cell capable of differentiating.
  • Blood cell stimulating factors are biological factors such as so-called site force in, inuichi leukin, growth stimulating factor, interferon, chemokine, etc.
  • a stimulator that causes a change in activity is so-called site force in, inuichi leukin, growth stimulating factor, interferon, chemokine, etc.
  • the source of hematopoietic stem cells used in the present invention may be, for example, umbilical cord blood, fetal liver, bone marrow, fetal bone marrow, peripheral blood, peripheral blood, cytokine and / or peripheral mobilized stem cells by administration of an anticancer agent in mammals such as humans and mice.
  • Examples include a cell group derived from blood and peripheral blood, and any tissue may be used as long as it contains hematopoietic stem cells.
  • Hematopoietic stem cells can be obtained from these tissues according to Seaberg, L.A., “Weir's Handbook of Experimental Imlunology, 5th edition, Blackwell Science Inc. 1997. That is, an anti-CD34 antibody It can be immunologically stained using an anti-CD33 antibody, an anti-CD38 antibody, or the like, and can be separated by the staining property of these antibodies using a cell sorter.
  • the differentiation inhibitory gene used in the present invention examples include the HES-1, HES-3, and HES-5 genes. As shown in the Examples below, by enhancing the expression of the HES-1 gene in hematopoietic stem cells, the differentiation and proliferation of the hematopoietic stem cells and hematopoietic progenitor cells differentiated from the hematopoietic stem cells can be regulated. .
  • the effect of HES-1 gene expression on hematopoietic stem cells is also similar to that of HES-1 similar proteins, HES-3 (Ryuichiro Kageyama, Biochemistry, 67: 1093, 1995), HES-5 (Ryuichiro Kageyama, Chemistry, 6 7: 1093, 1995).
  • HES-1 gene human-derived one is also called HRY
  • HES-3 gene and HES-5 gene are all known genes, and are described in Sasai et al. (Genes Dev., 6: 2620, 1992; mouse-derived HES- 1 and HES-3), disclosed by Akazawa et al. (J. Biol. Chem., 267: 21879, 1992; HES-5 derived from mouse) and Feder et al. (Genomics, 20: 56, 1994; HES-1 derived from human) It can be obtained by amplifying a DNA fragment containing each gene by PCR (polymerase chain, reaction) using oligo nucleotides prepared based on the given sequence.
  • PCR polymerase chain, reaction
  • to enhance the expression of a differentiation inhibitory gene in a hematopoietic stem cell means to operate the hematopoietic stem cell so that the expression level of the differentiation inhibitory gene in the hematopoietic stem cell is higher than the normal expression level for at least a certain period of time. That means.
  • the expression level of the differentiation-suppressing gene does not need to be always high, but may be high as long as the present invention can increase the level of differentiation and proliferation of hematopoietic stem cells and hematopoietic progenitor cells.
  • a differentiation inhibitory gene is incorporated in a form that can be expressed in a vector for mammalian cells, and the obtained recombinant vector is introduced into hematopoietic stem cells.
  • the expression suppressor gene in an expressible form can be obtained by ligating an expression control factor such as a promoter upstream of the coding sequence of the differentiation suppressor gene.
  • the expression of the differentiation inhibitory gene is regulatable.
  • the differentiation inhibitory gene is expressed only during culture, and it is not preferable to express the cells even after transplanting the cells into a living body. Therefore, it is preferable that the expression can be regulated as needed.
  • expression control include an expression control system using tetracycline (Gossen, M., Proc. Natl. Acad. Sci. USA, 89: 5547, 1992) and an expression control system using the insect hormone ecdysone (No. Natl. Acad. Sci.
  • IPTG isoprovir 1 /?-D-thiogalactobilanoside
  • the above vectors include retrovirus vector, adenovirus vector (Neering, SJ, Blood, 88: 1147, 1996), herpesvirus vector (Dilloo, D., Blood, 89: 119, 1997), and HIV vector.
  • adenovirus vector Neering, SJ, Blood, 88: 1147, 1996)
  • herpesvirus vector Dilloo, D., Blood, 89: 119, 1997)
  • HIV vector HIV vector.
  • the gene transfer vector of the present invention can be obtained.
  • the transferred gene exists outside the chromosome and disappears after transient gene expression.
  • the use of such a transient expression vector is advantageous in that the differentiation inhibitory gene can be expressed only during culture.
  • the expression level of the differentiation inhibitory gene on the chromosomal DNA of hematopoietic stem cells may be increased.
  • the expression control sequence such as bromo allyl specific to the differentiation control gene on the chromosomal DNA of hematopoietic stem cells
  • the strong expression control sequence By inserting the gene upstream, the expression level of the gene can be increased.
  • Replacement and insertion of the expression control sequence can be performed by homologous recombination or the like.
  • hematopoietic stem cells and / or hematopoietic progenitor cells differentiated from the hematopoietic stem cells are increased.
  • hematopoietic stem cells and / or hematopoietic progenitor cells can be expanded by culturing hematopoietic stem cells with enhanced expression of differentiation-suppressing genes or hematopoietic progenitor cells differentiated from the hematopoietic stem cells while allowing them to act on blood cell stimulating factors. Can be done.
  • Blood cell stimulating factors are added to the culture medium to promote the growth of hematopoietic stem cells, and include so-called cytokines, interleukins, growth stimulating factors, interferon, chemokines, development-related gene products, etc. (See The Cytokine Factsbook, Callard, RE, Academic Press, 1994 for site power-in). Specific examples of blood cell stimulating factors include SCF (stem cell factor), IL-3 (interleukin-13), IL-6 (interleukin-16), and GM-CSF (requested).
  • SCF stem cell factor
  • IL-3 interleukin-13
  • IL-6 interleukin-16
  • GM-CSF GM-CSF
  • Granulocyte macrophage colony stimulating factor Granulocyte macrophage colony stimulating factor
  • TP0 thrombopoetin
  • EP0 erythropoietin
  • Wnt Thimoth, AW, Blood, 89: 3624-3635, 1997)
  • culture conditions favorable for the growth of hematopoietic stem cells can be improved to be more effective.
  • culture may be performed by adding a culture supernatant of stromal cells capable of maintaining hematopoietic stem cells.
  • the culture medium used for the culture is not particularly limited as long as the growth and survival of hematopoietic stem cells or hematopoietic progenitor cells are not impaired.
  • MEM-G medium GEBCO BRL
  • SF-02 medium Sanko Pure Chemical
  • Opti-MEM medium GIBCO BRL
  • IMDM medium GIBCO BRL
  • PRMI 1640 medium PRMI 1640 medium
  • Materials added to the culture medium include fetal calf serum, human serum, poma serum, insulin, transferrin, lactoferrin, ethanolamine, sodium selenite, sodium monothioglycerol, Melka but-ethanol, ⁇ shea serum albumin, pyruvic Sanna door Li Umm, polyethylene grayed recall, various vitamins, various amino acids, C0 2 is, usually, is a 4 ⁇ , arbitrariness preferred 5%.
  • the hematopoietic stem cells or hematopoietic progenitor cells produced as described above can be used as a transplant for blood cell transplantation instead of conventional bone marrow transplantation and umbilical cord blood transplantation.
  • Construction Blood stem cell transplantation can improve conventional blood cell transplantation therapy because the transplant is semi-permanently engrafted.
  • Hematopoietic stem cell transplantation can be used for various diseases in addition to these treatments when performing systemic X-ray therapy or advanced chemotherapy for leukemia.
  • a treatment that causes bone marrow suppression as a side effect such as chemotherapy or radiation therapy
  • bone marrow is collected before the operation, and hematopoietic stem cells and hematopoietic progenitor cells are expanded in vitro.
  • hematopoietic disorders due to side effects can be recovered early, and stronger chemotherapy can be performed, and the therapeutic effect of chemotherapy can be improved.
  • hematopoietic insufficiency caused by bone marrow hypoplasia exhibiting anemia such as aplastic anemia can be improved.
  • Other diseases for which hematopoietic stem cell transplantation by the method of the present invention is effective include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, acquired immunodeficiency syndrome (AIDS), and the like.
  • Immunodeficiency syndrome group thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage disease such as Gaucher disease and mucopolysaccharidosis, adrenal white matter degeneration, various cancers or tumors, etc.
  • congenital anemia such as sickle cell disease
  • lysosomal storage disease such as Gaucher disease and mucopolysaccharidosis
  • adrenal white matter degeneration various cancers or tumors, etc.
  • Transplantation of hematopoietic stem cells may be performed in the same manner as conventional bone marrow transplantation or umbilical cord blood transplantation, except for the cells used.
  • hematopoietic stem cells that may be used for hematopoietic stem cell transplantation as described above is not limited to bone marrow, and stem cells can be obtained by administering fetal liver, fetal bone marrow, peripheral blood, cytokines, and / or anticancer drugs as described above. Mobilized peripheral blood, umbilical cord blood, and the like can be used.
  • the transplant may be a composition containing a buffer solution or the like in addition to the hematopoietic stem cells and hematopoietic progenitor cells produced by the method of the present invention.
  • hematopoietic stem cells or hematopoietic progenitor cells produced by the present invention can be used for ex vivo gene therapy.
  • a foreign gene (therapeutic gene) is introduced into hematopoietic stem cells or hematopoietic progenitor cells, and the resulting transfected cells are used. Done.
  • the foreign gene to be introduced is appropriately selected depending on the disease.
  • Diseases targeted by gene therapy targeting blood cells include chronic granulomatosis, double immunodeficiency syndrome, agammaglobulinemia, Wiskott-Aldrich syndrome, and acquired immune deficiency syndrome (AIDS).
  • Examples include immunodeficiency syndrome, thalassemia, hemolytic anemia due to enzyme deficiency, congenital anemia such as sickle cell disease, lysosomal storage diseases such as Gaucher disease and mucopolysaccharidosis, adrenal leukemia, various cancers and tumors, etc. .
  • retrovirus vector such as Moroni murine leukemia virus, adenovirus vector, adeno-associated virus (AAV) Vectors, simple herpes virus vectors, vector vectors for animal cells used in gene therapy for viruses derived from HIV vectors, etc.
  • AAV adeno-associated virus
  • retrovirus vectors see Verma, I.M. , Nature, 389: 239, 1997), calcium phosphate coprecipitation method, DEAE-dextran method, electoral poration method, liposome method, lipofection method, microinjection method, and the like.
  • a retrovirus vector, an adeno-associated virus vector, or an HIV vector is preferable, since the gene can be expected to be permanently expressed by being integrated into the chromosomal DNA of the target cell.
  • an adeno-associated virus (AAV) vector can be constructed as follows. First, 293 cells were transfected with a vector plasmid containing a therapeutic gene inserted between the ITRs (inverted termina 1 repeats) at both ends of the wild-type adeno-associated virus DNA, and helper plasmid for complementing the viral proteins. I do. Subsequently, when infected with an adenovirus of a helper virus, virus particles containing the AAV vector are produced. Alternatively, instead of adenovirus, a plasmid expressing an adenovirus gene responsible for helper function may be transfected.
  • the obtained virus particles are used to infect hematopoietic stem cells or hematopoietic progenitor cells. It is preferable to insert an appropriate promoter and enhancer upstream of the target gene in the vector DNA, and to regulate the expression of the gene by these. Furthermore, when a marker gene such as a drug resistance gene is used in addition to the therapeutic gene, it becomes easy to select cells into which the therapeutic gene has been introduced. Therapeutic genes are sense sense It may be a gene or an antisense gene.
  • the composition for gene therapy may be a composition containing a buffer, a novel active substance, and the like, in addition to the hematopoietic stem cells and hematopoietic progenitor cells produced by the method of the present invention.
  • FIG. 1 is a diagram showing a construction procedure of pFLAG-CMV2-HES-1.1.
  • FIG. 2 is a diagram showing a procedure for constructing pFLAG-CMV2-HES-1.2.
  • FIG. 3 is a diagram showing a procedure for constructing pEGFP-CI-HES-1.
  • FIG. 4 is a diagram showing a procedure for constructing pMSCV-EH. BEST MODE FOR CARRYING OUT THE INVENTION
  • all antibodies used for cell separation were purchased from Pharmingen.
  • All the restriction enzymes used for gene recombination were purchased from Behringer Mannheim. Separation of each blood cell was generally carried out by Sea, L.A., "Weir, s Handbook of Experimental Immunology, 5th edition", Blackwell Science Inc. 1997.
  • Bone marrow cells, spleen cells, and thymocytes are separated from 6- to 8-week-old C57B1 / 6 mice (purchased from Nippon Charls River Co., Ltd.), and mononuclear cells and granulocytes are obtained using Celso overnight. , Mature T cells, immature T cells, mature B cells, hematopoietic progenitor cells, and hematopoietic stem cells.
  • Anti-CD32 antibody was added to bone marrow cells removed from mouse femur bone marrow and placed on ice. After standing for 10 minutes, FITC-labeled anti-Mac-1 antibody and PE-labeled anti-Gr-1 antibody were added, and reacted on ice for 30 minutes.
  • the cells were washed twice with a staining buffer (PBS (phosphate buffered saline), 5% FCS (CS fetal serum), 0.05% NaN 3 ), suspended in the staining buffer, and then suspended in a cell sorter (FACS Vantage, Mononuclear cells (Mac-1 positive 'Gr-1 negative cells) and granulocytes (Mac-1 positive and Gr-1 positive cells) were separated using Becton Dickinson.
  • a staining buffer PBS (phosphate buffered saline), 5% FCS (CS fetal serum), 0.05% NaN 3
  • FACS Vantage Mononuclear cells (Mac-1 positive 'Gr-1 negative cells) and granulocytes (Mac-1 positive and Gr-1 positive cells) were separated using Becton Dickinson.
  • the thymocyte suspension is overlaid on a high-density cell separation solution (Nycomed, Lymphoprep), centrifuged at 1500 rpm for 30 minutes at 25 ° C, and cells collected at the interface between the suspension and Lymphoprep are removed. Collected. After the cells were washed twice with a staining buffer, an anti-CD32 antibody, a FITC-labeled anti-CD4 antibody, and a PE-labeled anti-CD8 antibody were added, and reacted on ice for 30 minutes. After the reaction, the cells were washed twice with a staining buffer and suspended in a staining buffer. Then, immature T cells (CD4 negative ⁇ CD8 negative cells and CD4 positive ⁇ CD8 positive cells) and mature T cells (CD4 positive ⁇ CD8-negative cells and CD4-negative and CD8-positive cells).
  • a staining buffer an anti-CD32 antibody, a FITC-labeled anti-CD4 antibody, and
  • the suspension of spleen cells was overlaid on Lymphoprep (Nycomed) and centrifuged at 1500 rpm at 25 ° C for 30 minutes to collect the cells collected at the interface. Wash the cells twice with staining buffer, add anti-CD32 antibody, and leave on ice for 10 minutes, then add FITC-labeled anti-B220 antibody and PE-labeled anti-mouse IgM antibody, and on ice for 30 minutes Reacted. After the reaction, the cells were washed twice with the staining buffer, suspended in the staining buffer, and then mature B cells (B220-positive-IgM-positive cells) were separated using a cell sorter.
  • the bone marrow cell suspension was overlaid on Lymphoprep (Nycomed) and centrifuged at 1500 rpm at 25 C for 30 minutes to collect cells that had collected at the interface. After washing the cells twice with the staining buffer, the cells were washed twice with PBS and suspended in the staining buffer.
  • An antibody against the biotinylated differentiation antigen marker ie, an anti-CD4 antibody, an anti-CD8 antibody, an anti-B220 antibody, an anti-Gr-1 antibody, and an anti-Tell19 antibody, were added to the cell suspension, and the mixture was left on ice for 30 minutes.
  • avidin-coated magnetic beads (Avidinma Gnet beads (Perseptive)) and left on ice for 30 minutes.
  • avidin magnet beads were collected using a magnet, and the cells presenting the differentiation antigen were removed to obtain differentiated antigen-negative cells (Lin-).
  • a FITC-labeled anti-CD34 antibody, a PE-labeled anti-Sea-1 antibody, a Texas red-labeled avidin, and an APC-labeled anti-C-Kit antibody were added to the differentiation antigen-negative cell solution, and left on ice for 30 minutes.
  • hematopoietic progenitor cells (Sea-1 negative, C-kit positive cells and CD34 positive, Sca-1 positive, C-kit positive cells) and hematopoietic stem cells (Selso overnight) CD34 negative to weak positive ⁇ Sea-1 positive ⁇ c-kit positive cells).
  • RNA was obtained according to the instructions for use of the reagent.
  • 5 units of DNase RNase free (GI BCO-BRL) were added, and the mixture was incubated at 37 ° C for 30 minutes to digest and degrade the mixed genomic DNA.
  • RNA was obtained. From this MA, cDNA was synthesized using oligo dT as a primer. That, RNA corresponding to 10 5 cells to prepare a reaction liquid so as to correspond to 20 micro liters reaction.
  • the composition of the reaction solution used was that recommended in the instruction manual for reverse transcriptase (Superscript IK GIBCO-BRL). The reaction was carried out at 42 ° C. for 60 minutes, and then the reverse transcriptase activity was inactivated by keeping the temperature at 72 ° C. for 10 minutes.
  • sequences and annealing temperatures of the various primers used in the PCR reaction are as follows.
  • Table 2 shows the results of evaluating the expression level based on the intensity of the amplified band.
  • the symbols in the table are as follows.
  • Notch- and Notch-2 were expressed in all of the examined hematopoietic cells.
  • Notch-3 is confirmed to be cell type-specific expression in immature T cells, mature T cells, and B cells.
  • Notch-4 was not expressed in hematopoietic cells.
  • HES-1 HES-3 which is present downstream of the Notch / Delta signaling system, was expressed in all the cells examined.
  • HES-5 was expressed in a cell type-specific manner in cells other than hematopoietic stem cells and hematopoietic progenitor cells.
  • HES-1 Monitor expression and localization of HES-1 in cells transfected with retrovirus
  • a vector was constructed so that HES-1 and a fluorescent protein, EGFP (Enhanced green fluorescent fluorescence protein, Clontech) were expressed as a fusion protein (see FIGS. 1 to 4).
  • EGFP Enhanced green fluorescent fluorescence protein, Clontech
  • PSV2CMVHES-1 (Kyoto University, distributed by Dr. Ryuichiro Kageyama, see Sasai, Y., Genes Dev., 6: 2620, 1992) is digested with EcoRI and EcoRI digested with pFLAG-CMV2 vector (Kodak)
  • a subcloned PFLAG-CMV2-HES-1.1 (Fig. 1) was prepared in the same direction as the FLAG transfer direction.
  • the translation initiation codon of HES-1 was changed, and the translation initiation codon of HES-1 was modified so that EGFP and HES-1 were expressed as a fusion protein, and changed to the Bglll site.
  • a synthetic oligonucleotide (SEQ ID NO: 15) having a sequence that changes the translation start point ATG to Bglll, and an antisense oligonucleotide at a site located downstream from the Pstl site in the HES-1 gene. (SEQ ID NO: 16) was used as a primer, and PCR was performed using PSV2CMVHES-1 as type II. A fragment containing the HES-1 gene obtained by digesting this PCR product with Bglll and Pstl was cloned into Bglll and Pst-1 sites of pFLAG-CMV2-HES-1.1 described above. This brassmid was named PFLAG-CMV2-HES-1.2 (Fig. 2).
  • a fragment containing HES-1 obtained by digesting PFLAG-CMV2-HES-1.2 with Bgll I and EcoRI was cloned into the Bglll / EcoRI site of pEGFP-CI (Clontech), and pEGFP-CI-HES- 1 ( Figure 3).
  • a gene was constructed in which EGFP and HES-1 were on one transcription unit.
  • a fragment containing HES-1 obtained by digesting pEGFP-CI-HES-1 with Eco47III and Sail was extracted from Hpal and Xhol using a PMSCV2.1 vector (obtained from Dr. R. Hawley, University of Toronto, Hawley , RG, Gene Ther., 1: 136, 199).
  • This vector was named pMSCV-EH (Fig. 4). Thereafter, this plasmid was used for producing a retrovirus for HES-1 infection.
  • a retrovirus vector into which only the EGFP gene was transferred was constructed.
  • a fragment containing EGFP obtained by digesting pEGFP-CI with Eco47III and Sail was inserted into a PMSCV2.1 vector digested with Hpal and Xhol. This vector was pMSCV-E.
  • mice Pregnant C57B1 / 6 mice were purchased from Nippon Charls River Inc. On the 14th day of pregnancy, the mouse was laparotomized and the fetus was aseptically removed. After carefully separating the fetal liver from contaminating other tissues, the cells were dispersed with a syringe fitted with a 21-gauge injection needle and layered on Lymphoprep (Nycomed). This was centrifuged at 1500 rpm at 25 ° C. for 30 minutes to collect cells collected at the interface. After washing the cells twice with PBS, the cells were suspended in a staining buffer (PBS, 5% FCS ⁇ 0.05% NaN 3 ).
  • a staining buffer PBS, 5% FCS ⁇ 0.05% NaN 3
  • the cell suspension was incubated with the differentiation antigen, which was biotinylated, that is, anti-CD8 antibody, anti-B220 antibody, anti-GII-l antibody, and anti-Terll9 antibody. And left on ice for 30 minutes. Then, after washing twice with a staining buffer, avidin magnet beads were added, and the mixture was left on ice for 30 minutes. After washing twice with the staining buffer again, remove the cells presenting the differentiation antigen using a magnet. Then, differentiated antigen-negative cells (Lin-) were obtained.
  • the differentiation antigen which was biotinylated, that is, anti-CD8 antibody, anti-B220 antibody, anti-GII-l antibody, and anti-Terll9 antibody.
  • FITC-labeled anti-CD34 antibody PE-labeled anti-Sca-1 antibody, Texas Red-labeled avidin, and APC-labeled anti-c-kit antibody were added to the differentiation antigen-negative cell solution, and left on ice for 30 minutes. After washing twice with the staining buffer, Lin-Sea-1 + c-kit + cells were selected on a cell saw.
  • the cells were collected and examined for colony forming ability (the colony forming ability was determined by adding 0.9% methylcellulose, 30% FCS, 0.1% serum albumin, and 10 ng / ml SCF
  • the culture was performed in the presence of I-3, IL-6, EP0, TP0, and 0.5 mg / ml G418.
  • the morphology and number of emerging colonies were detected.
  • the various hematopoietic factors used in the above are all recombinant and pure.
  • Table 3 shows the results under the above conditions.
  • the numbers in the table are the number of colonies per 2,000 infected hematopoietic stem cells.
  • Table 3 Morphology of infected lettuce wiwi colonies (/ 2,000 hematopoietic stem cells)
  • the number of colonies shown in the table is a colony resistant to G418, and indicates only the EGFP gene alone or only the cell into which the EGFP-HES-1 fusion gene has been introduced. Furthermore, the expression of the target gene in these colonies was also confirmed by observing the expression of EGFP under a fluorescence microscope. In cells transfected with the EGFP gene alone, the entire cytoplasm fluoresced, and no localization in the nucleus was confirmed. On the other hand, in the strain into which the EGFP-HES-1 fusion gene was introduced, it was confirmed by fluorescence microscopy that the nucleus emitted specific fluorescence, and this HES-1 was normally expressed and functioned in the nucleus. Was speculated to be.
  • hematopoietic stem cells differentiate and only hematopoietic progenitor cells GM-CFC, G-CFC, and M-CFC appear.
  • HES-1 hematopoietic progenitor cells
  • undifferent hematopoietic cells were maintained, and the differentiation of hematopoietic stem cells was successfully regulated by HES-1 gene transfer.
  • INDUSTRIAL APPLICABILITY According to the present invention, hematopoietic stem cells and / or hematopoietic progenitor cells can survive and proliferate in a state where differentiation is suppressed.

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Abstract

La présente invention se rapporte à un procédé de régulation de la différenciation et de la prolifération des cellules souches hématopoïétiques mammaliennes et/ou des cellules parents hématopoïétiques différenciées à partir de celles-ci, ce procédé consistant à stimuler l'expression des gènes de répression de la différenciation dans ces cellules souches hématopoïétiques, par exemple, à transférer ensuite un gène de répression de la différenciation incorporé dans un vecteur de virus, et parallèlement, à traiter ces cellules avec des stimulants de cellules sanguines.
PCT/JP1998/004884 1997-10-28 1998-10-28 Procede de regulation de la differenciation/ proliferation des cellules souches hematopoietiques WO1999022001A1 (fr)

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JP29604197A JP3962459B2 (ja) 1997-10-28 1997-10-28 造血幹細胞の分化・増殖調節方法

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US7238711B1 (en) 1999-03-17 2007-07-03 Cambridge University Technical Services Ltd. Compounds and methods to inhibit or augment an inflammatory response
WO2004072264A2 (fr) * 2003-02-12 2004-08-26 Johns Hopkins University School Of Medicine Determination de la destinee par hes1 dans des cellules souches et progenitrices hematopoietiques et utilisation
EP1613742A2 (fr) * 2003-04-08 2006-01-11 YEDA RESEARCH AND DEVELOPMENT Co. LTD. Cellules souches presentant une sensibilite accrue a un agent chimio-attractif, methodes de production et d'utilisation associees
JP2010193879A (ja) 2009-01-27 2010-09-09 Jms Co Ltd 臍帯血造血幹細胞の増殖制御方法およびその用途

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