JP2002527101A - Improved transduction of hematopoietic stem cells and promotion of self-renewal by histone deacetylase inhibitors - Google Patents

Abstract

  (57) [Summary] A method of promoting self-renewal of stem cells comprising exposing a stem cell population to an effective amount of a histone deacetylase inhibitor, particularly trichostatin A, trapoxin, or chlamydosin. The present invention also relates to the use of histone deacetylase inhibitors to increase the number of transduced stem cells.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention relates to techniques for transducing stem cells, especially hematopoietic stem cells (HSCs), and for self-renewing division.

[0002]

[Prior art]

Maintenance of the hematopoietic system relies on primordial pluripotent hematopoietic stem cells, which repopulate all blood cell lineages through self-renewal and associated progenitor cells.
Have the ability to Due to such self-renewal ability, long-term reconstitution ability and pluripotency, HSCs can be used to transplant or regenerate therapeutic genes to reconstitute the hematopoietic system after treatment of various blood diseases. It has long been ideal as a transport target. Therefore, human HSCs could be used in restoring the immune system in autoimmune diseases and in inducing tolerance to allogeneic solid organ transplantation.

[0003] In the early stages of hematopoiesis, pluripotent stem cells differentiate and give rise to lymphocytes, myeloid cells or erythroid limited cells. These cells then differentiate into progenitor cells associated with the specific cell type. The available evidence indicates that these progenitor cells have lost their ability to self-renew and contribute little to engraftment after transplantation. Infusion of HSCs may be necessary for short-term, sustained colonization. Furthermore, the kinetics of engraftment is proportional to the number of HSCs injected. These are especially true for allograft cases. Thus, as the number of HSCs increases, attempts to improve the quality of the transplant will be achieved. Thus, increasing the number of HSCs by in vitro culture will provide an improved therapeutic tool for the treatment of many diseases, including cancer, self-immunization, and certain genetic diseases.

[0004] HSCs are usually obtained from bone marrow, mobilized peripheral blood, and cord blood.
At present, to ensure the presence of a sufficient number of stem cells to reconstitute the hematopoietic system,
A large amount of bone marrow must be extracted and frozen. In addition, a single cord blood donor does not provide a sufficient number of hematopoietic stem cells for adult treatment. Therefore, if a method was developed to promote the self-renewal of HSCs, as distinct from stem cell proliferation,
It would be very beneficial for therapeutic use.

[0005] Since the 1980's, numerous discoveries have been made regarding the effects of growth factors such as interleukins and cytokines on hematopoietic cells. These hormones have potent effects on the growth and differentiation of hematopoietic cells. These molecules include, for example, interleukin (IL) -1, IL-2, IL-3, IL-4, IL-5, IL
-6, IL-7, erythropoietin (EPO), granulocyte colony stimulating factor (G
-CSF), macrophage colony stimulating factor (M-CSF), and granulocyte macrophage colony stimulating factor (GM-CSF). Many of these factors have been tested in combination in an attempt to achieve optimal production of progenitor cells during in vitro culture. Numerous major advances in stem cell replication are due to c-
Flk2 / f, a kit ligand and a ligand for the fetal liver kinase receptor
Related to the discovery of lk3 ligand (FL). Both of these cytokines have a synergistic effect with other cytokines in vitro, maintain survival, and have the effect of leading the very primitive hematopoietic stem cells to their cycle without increasing differentiation. In addition, thrombopoietin (TP) promotes megakaryocytopoiesis.
O) The molecule was found to have potent effects on the survival and replication of human and mouse primordial hematopoietic progenitor cells in vitro. Other culture conditions and additives were studied for the possibility of optimizing HSC replication. These points in the culture system include the culture medium, the surfaces to adhere to, and the schedule of gas and nutrient exchanges. The role of stromal cells has been investigated in stem cell self-renewal technology. The effects of cell adhesion molecules, such as VLA-4, VLA-5 and L-selectin, expressed on the surface of progenitor cells on the stem cell cycle have been studied. A specific receptor-ligand interaction between stromal cells and HSCs cells has been identified and appears to be essential for stem cell maintenance.

[0006] One approach (means) for manipulating culture conditions to promote HSC self-renewal
However, the present invention relates to the transcriptional regulation of genes involved in self-renewal through modulation of histone acetylation.

[0007] In eukaryotic cells, DNA molecules are tightly complexed with histone proteins to form chromatin, and changes in chromatin structure are known to be most important in regulating gene expression. Histones are rich in arginine and / or lysine, and it is known that changes in chromatin structure are fundamentally important in regulating gene expression. Histones are classified into five major groups called H1 (lysine-rich), H2A and H2B (slightly lysine-rich), and H3 and H4 (arginine-rich). These proteins have two domains, a histone fold domain and an amino tail domain. The amino tail domain interacts with the DNA at a specific site between its arginine and / or lysine and the negatively charged phosphate group of the DNA. At specific points in the cell cycle, the aminotail domain undergoes transient modifications, including acetylation. Histone modifying enzymes, particularly acetyltransferases and deacetylases, are known to play an active role in transcription and chromatin assembly. A strong, but not limiting, scientific view is that there is a correlation between histone acetylation and deacetylation levels and transcriptional activity, and that histone acetylation causes chromatin to unfold (dilution). : Decondensation), which leads to transcription factors and basal transcriptional machinery
The access of DNA components to DNA (Grunstein, Michael, Nature 389: 3
49-352, (1997); Wade, P. et al., TIBS 22: 128-132, (1977)). Some transcription cofactors (activators) have histone acetyltransferase activity, and it is therefore thought that these coactivators are also indicative of local destabilization of the inhibitory histone-DNA interaction. I have.

[0008] Conversely, many transcription repressors that function through the action of histone deacetylases (HDACs) have become known. Some transcriptional repressors govern cell differentiation and proliferation and are involved in mechanisms involved in normal hematopoiesis or leukemogenesis. Some of these repressors are briefly described.

[0009] Retinoid receptors are involved in bone marrow cell mutations but may play an important role in the development of pluripotent hematopoietic stem cells (Tsai, et al., Genes & Dev., 8: 283).
1 (1994)). These receptors function as transcription activators when bound to a ligand, while in the non-ligand state, the transcription corepressors SMRT and N-
Suppresses basic transcriptional activity by associating with CoR. According to Nagy, et al., The SMRT transcriptional corepressor is a histone deacetylase 1 (HDAC1).
By forming a multi-subunit repressor complex incorporating
It was shown to exhibit that activity (Nagy, et al., Cell, 89: 373-380 (1997)).
Histone deacetylation was confirmed to be fundamental to retinoid signaling because the deacetylase inhibitor trichostatin A (TSA) can significantly enhance the biological activity of retinoic acid. This is because HL60 promyelocytic cells that mutate myeloid cells in response to retinoic acid
) System was demonstrated. In the presence of suboptimal amounts of retinoic acid, co-treatment with TSA achieved a complete differentiation response of the cells.

[0010] The promyelocytic leukemia zinc finger (PLZF) protein is predominantly expressed in most primordial hematopoietic compartments and has been shown to inhibit the cell cycle of hematopoietic cells (Mol. Cell Biol. 18: 5533). (1998)). PLZF is a DNA binding protein, SMRT incorporating histone deacetylase 1 (HDAC1).
Or suppresses transcription by interacting with N-CoR (Guidez, et al., B
lood 91: 2934 (1998); Lin, et al. Nature 391: 811 (1998)).

Myc activation by genomic alterations resulting from chromosomal rearrangements or retroviral insertions causes leukemia and lymphoma in humans, rodents, and birds. MYC
The protein is expressed in proliferating cells and is negatively regulated upon cell cycle withdrawal or differentiation. MYC is part of a transcription network that includes other factors. MYC can form a dimer with a protein called MAX that can bind to DNA and the protein MAD. MYC-MAX dimer activates transcription, while MAD-MAX suppresses transcription. MAD-MAX incorporates histone deacetylase, indicating that this interaction is the basis for the inhibitory activity of MAD-MAX. It was shown that suppression was extinguished by TSA (Laherty,
et al. Cell 89: 349-356 (1997)).

Other corepressors whose activity is modulated by histone deacetylase inhibitors include mammalian CBF1 / RBP-Jκ, which is switched from a transcriptional repressor to an activator by Notch activation. Can be done. In the absence of Notch activation, CBF1 / RBP-Jκ is SMRT / HDAC
Binding to 1 represses transcription and Notch activation prevents the formation of repressor complexes. The transcription of a gene regulated by CBF1 can be induced by TSA treatment (Genes and Dev., 12: 2269-2277 (1998)). Notch signaling has been shown to affect the development of primary primordial hematopoietic progenitor cells in vitro (Blood 1998, 91: 4084-4091). Overexpression of activated Notch1 in 32D myeloid cells inhibits differentiation and allows undifferentiated cells to proliferate,
Consistent with known functions of Notch in Drosophila (PNAS
1996, 93: 13014-9).

In a recent journal article, the HDAC inhibitor trichostatin A (TS
A) has been described to induce various biological responses in cells such as differentiation induction and cell cycle arrest (Yoshida, et al., Bio
Essays 17: 42-429 (1995)). Various other HDAC inhibitors have been implicated in causing terminal differentiation of malignant cells (Richon, et al., Proc. N
atl.Acad.Sci. USA, 95: 3003-3007 (1998)).

[0014]

The document discloses the use of HDAC inhibitors in studies to reverse the effects of HDAC and the HDAC complex on transcriptional repression, but the document discloses that HDAC inhibitors To enhance the maintenance of progenitor cell function during repeated gene integration in the absence of HDAC inhibitors, to promote the hematopoietic stem cell cycle, to promote HSC self-renewal division (or replication) Is not disclosed.

[0015]

[Means for Solving the Problems]

 In one embodiment of the present invention, a) a hematopoietic cell comprising a hematopoietic stem cell subpopulation from a hematopoietic cell source.
Obtaining a cell population, b) culturing the hematopoietic cells under growth-supporting conditions, c) culturing the cultured cells in an effective amount.
Exposed to a histone deacetylase inhibitor to promote self-renewal division of the stem cells.
D) obtaining a composition of self-renewing hematopoietic stem cells,
A method for promoting regenerative division is provided. The method further includes culturing the stem cells.
Obtaining an enriched hematopoietic stem cell population from the hematopoietic cells prior to
You. Preferably, the method comprises CD34⁺, Thy-1⁺, CD34⁺Thy-1 ⁺ , CD34⁺EM⁺, CD34⁺Thy-1⁺Lin⁻, CD34⁺CD38 ^{lo /-} , CD34⁺Thy-1⁺CD38^{lo /-}, CD34⁺Thy-1⁺ EM⁺, And CD34⁺Thy-1⁺CD38^{lo /-}EM⁺Of the phenotype consisting of
Selecting from the group an enriched human hematopoietic stem cell population. One embodiment of the present invention
And the composition of self-renewing cells obtained by the above method.

Further, in one aspect of the invention, a) an isolated mammalian hematopoietic cell containing a subpopulation of self-renewing engrafted cells; b) an effective amount of one or more histone deacetylase inhibitors. And c) a culture comprising an effective amount of a growth and self-renewal supporting cytokine, wherein said effective amount of a histone deacetylase inhibitor promotes self-renewal division of said engrafted cells in said culture. I will provide a. In a preferred embodiment, the hematopoietic cells are human. In another preferred embodiment, the histone deacetylase inhibitor is selected from trichostatin A, trapoxin, chlamidosin, sodium butyrate, or dimethyl sulfoxide.

Further, in one aspect of the present invention, a self-renewing division of the stem cells comprises treating the stem cell population with an effective amount of one or more histone deacetylase inhibitors, and allowing the stem cells to self-renew. Provide a way to promote In one embodiment, the stem cell is C
Hematopoietic stem cells characterized as enriched D34 ⁺ Thy-1 ⁺ .

Further, in one aspect of the invention, a) treating the stem cells in the culture with an effective amount of a histone deacetylase inhibitor, b) introducing a gene into the stem cells by retrovirus-mediated transfer, and c) A method for producing transduced mammalian stem cells, comprising transducing said stem cells, wherein the number of transduced stem cells is exposed to substantially the same conditions except that the number of transduced stem cells is not treated with the histone deacetylase inhibitor. Provided above, wherein the number of transduced stem cells has been increased. In a preferred embodiment, the stem cells are hematopoietic stem cells, preferably human hematopoietic stem cells, and the gene is a therapeutic gene. The method further comprises administering an effective amount of the transduced stem cell population to a mammalian subject, preferably a human subject. In one embodiment, the stem cells are allogeneic or xenogeneic to the subject, and in another embodiment, the stem cells are autologous to the subject.

Further, in one aspect of the present invention, a) a gene delivery vehicle containing the polynucleotide is contacted with a stem cell population cultured in the presence of an effective amount of a histone deacetylase inhibitor; and b) the genetically modified A method of genetically modifying a stem cell, comprising obtaining a stem cell. In a preferred embodiment, the delivery vehicle is a retrovirus vector, a lentivirus vector, an adenovirus vector or a liposome delivery vehicle.

Further, in one aspect of the invention, a) contacting a population of hematopoietic stem cells with an effective amount of a histone deacetylase inhibitor, and b) exposing the hematopoietic stem cells to a vector containing a nucleic acid sequence encoding a therapeutic gene. And c) administering to the subject an effective amount of the transduced hematopoietic stem cell population to restore hematopoietic potential.

Further, in one embodiment of the present invention, a) exposing a stem cell population in culture to an effective amount of a histone deacetylase inhibitor; b) introducing a heterologous gene into the cultured stem cells; Engrafting the genetically modified mammalian stem cells, comprising increasing the number of genetically modified stem cells compared to the absence of an exposure to an enzyme inhibitor, and c) administering the modified cells to a subject. Provide improvement methods. In one embodiment, the stem cells are allogeneic, and in another embodiment, the stem cells are autologous. Preferably, the heterologous gene is a therapeutic gene.

Further, in one aspect of the invention, a) exposing a population of stem cells to an effective amount of a histone deacetylase inhibitor; b) transducing the stem cells with an exogenous gene; and c) transducing the transduced cells. A method of transducing stem cells, comprising: obtaining a number of transduced stem cells grown under substantially the same conditions except that the number of transduced stem cells is not exposed to an effective amount of the histone deacetylase inhibitor. The above method is provided which is increased. In one embodiment, the cells are transduced in culture, and in a second embodiment, the cells are transduced in vivo. In yet another embodiment, the stem cells are hematopoietic stem cells, particularly mouse or human. In yet another embodiment, the cells are transduced with a retroviral vector derived from Moloney murine leukemia virus or mouse stem cell virus.

Further, in one aspect of the invention, a) a stem cell population, b) an effective amount of one or more histone deacetylase inhibitors, c) an effective amount of one or more growth-supporting cytokines, and d. A) providing a culture comprising a gene delivery vehicle containing a polynucleotide encoding a marker gene or a therapeutic gene; In one embodiment, the gene delivery vehicle is a vector derived from a retrovirus, adenovirus or adeno-associated virus.

Abbreviations used throughout this specification are described below. FITC = fluorescein. TPO = Thrombopoietin. FL = F13 ligand. KL = c-kit ligand. IL = interleukin. LIF = leukemia inhibitory factor. MPB = mobilized peripheral blood. CFSE = carboxyfluorescein-diacetate succinimidyl ester. HSC = hematopoietic stem cells. TSA = trichostatin A. DMSO = dimethyl sulfoxide. FBS = fetal calf serum. IMDM = Iskov-modified (denatured) Dulbecco's medium. HDAC = histone deacetylase. HDAC-I = histone deacetylase inhibitor. CAFC = Coblestone region forming cells. PE = phycoerythrin. CM = culture medium. APC = allophycocyanin. EPO = erythropoietin. FN = fibronectin fragment CH296. FACS = fluorescence activated cell sorter.

As used herein, the term “stem cell” includes various cell types such as muscle, epithelium, nerve, osteostem and the like. More specifically, the invention relates to hematopoietic stem cells. By "hematopoietic stem cells (HSC)" is meant mammalian and avian hematopoietic stem cells, and a population of hematopoietic cells that have the potential to engraft (potentially) in in vivo therapeutic applications. H for hematopoietic cells
In addition to SCs, erythrocytes, neutrophils, monocytes, platelets, mast cells, eosinophils, basophils, B and T lymphocytes, NK cells, and precursor cells of each of these lineages are included. Stem cells can also be defined in vitro by the presence or absence of CAFC activity. Animal models for long-term viability of human hematopoietic stem cell population candidates include SCI
D-hu bone model (Kyoizumi et al. (1992) Blood 79: 1704; Murray et al. (1
995) Blood 85 (2) 368-378), in utero sheep model (Zanjani
et al. (1992) J. Clin. Invest. 89: 1179), NOD SCID (Larochelle, et.
al., Nature Medicine 2: 139-1337 (1996) and Conneally, et al., Proc. Nat.
l.Acad.Sci. 94: 9836-9841 (1997)) and a primate model (Kiem, et al., Blood 9).
0: 4630-4645 (1997) and Srour, et al., J. Hematother 1: 143-153 (1992)))
There is. Mouse hematopoietic stem cells were obtained at least in an enriched form, where less than about 30 cells obtained from bone marrow were able to reconstitute the entire lineage of the hematopoietic lineage of lethal irradiated mice . Each assay cell was pluripotent for all hematopoietic systems.

As used herein, “engrafting cell” means any cell that can remain in hematopoietic tissue and repopulate a blood cell lineage. "Self-renewal" or "self-renewal division" is also referred to as "replication" and as used herein means cell division without loss of apparent cell differentiation or cell viability. The term "proliferation" refers to the differentiation from pluripotent stem cells used to increase the number of progenitor cells and initiate culture.

[0027] As used herein, the term "cytokine" refers to any one of a number of factors that have a variety of effects on a cell, such as, for example, proliferation or differentiation. The source of the cytokine is human or may be from another species if it is active on the cell of interest. Included within this definition are molecules having biological activity similar to wild-type or purified cytokines, for example, molecules produced by recombinant means, and cellular responses that bind to cytokine receptors and resemble natural cytokine factors. Are included.

Non-limiting examples of cytokines used alone or in combination in the practice of the present invention include interleukin 2 (IL-2), interleukin 3 (IL
-3), interleukin 6 (IL-6), interleukin 12 (IL-12)
), Interleukin 1α (IL-1α), interleukin 11 (IL-11
), KL (used interchangeably with "c-kit ligand"), stem cell factor (S
CF), steel factor (Stl), mast cell growth factor (MCGF), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (G
M-CSF), thrombopoietin (TPO), leukemia inhibitory factor (LIF), F
L, and MIP-1α. Combinations of these, ie, fusion proteins in which the two factors are fused or where the cytokine is fused to its soluble receptor, can also be used. The order of fusion is not important, as long as the two proteins of the fusion molecule can function independently and provide their biological function. Non-limiting examples include combinations of IL-3 and IL-6 (Broxmeyer, et al.
al., Exp. Hematol. 18: 615 (1990)). The invention includes culture conditions in which one or more cytokines have been specifically removed from the medium. Cytokines are available from R & D Systems (Minneapolis, MN), Genzyme Dyanostic (Cambridge, MA) and Genentech (Ssouth San F)
rancisco, CA).

The term “culture” refers to the growth of cells or organs on or in various types of media.

The term “effective amount” means an amount sufficient to promote the survival, proliferation and / or division of stem cells, especially hematopoietic stem cells. The term "surviving" refers to the ability to remain alive or active.

The term “mammal” includes, for example, humans, mice, monkeys, livestock animals, sport animals, companion animals (pets), and other experimental rodents and animals. Preferably it means human.

“Histone deacetylase activity” or “histone deacetylase protein activity” refers to a biological activity associated with a histone deacetylase protein. Said biological activity includes binding activity and, where appropriate, activity of catalyzing the deacetylation of acetylated histones. This mediates the activity of transcriptional corepressors such as, for example, SMRT and N-CoR. In general, compounds that inhibit HDAC have been shown to activate gene expression.

An effective amount or dose of a histone deacetylase inhibitor (HDAC-I) increases the number of self-renewing divisions of cells, particularly stem cells, within a hematopoietic cell population, while reducing total cell viability. As a result, the number of stem cells available for genetic modification can be compared to the number of stem cells in a cell population cultured under substantially identical conditions except for the absence of HDAC-I. Is defined as the amount of HDAC-I that increases.

As used herein, the term “genetic modification” refers to the addition, deletion, or disruption of a cell to normal nucleotides. The methods of the invention include any genetic modification that transfers a heterologous or exogenous gene (or nucleic acid sequence) to a stem cell, preferably a hematopoietic stem cell. The term "genetic modification" includes the use of gene delivery vehicles, including, but not limited to, transduction (in vivo or in vitro, virus-mediated transfer of nucleic acid to a recipient), transfection (simple There are cells (incorporation of isolated nucleic acids), liposome-mediated transfer, and other means well known in the art.

As used herein, “therapeutic gene” is defined as the entire gene or only a functionally active fragment of the gene. A therapeutic gene is one that can compensate for a disease caused by a lack or defect of an endogenous gene. In addition, therapeutic genes may antagonize the production or infection of infectious agents, neutralize pathological progression, promote genetic make-up of the host, facilitate engraftment, or It can also improve therapeutic capacity.

“Retrovirus-mediated (mediated) gene transfer” and “retroviral transduction” are synonymous.

[0037] Methods for obtaining hematopoietic cells and stem cells are known in the art. For example, there is a method of isolating stem cells from other cells in the body's hematopoietic tissue, particularly bone marrow. Stem cells from the bone marrow may represent about 0.01 to about 0.1% of the bone marrow cells. Bone marrow cells
Obtained from bone marrow sources including the iliac (from the hip through the iliac crest), the sternum, the (varus) tibia, the femur, the spine, and other bone cavities. Other sources of hematopoietic stem cells include, but are not limited to, blood including embryonic yolk sac, fetal liver, fetal and adult spleen, adult peripheral blood and umbilical cord blood (To, et al., Blood 89: 2233-2258). (1997)).

For the isolation of bone marrow, by way of non-limiting example, fetal serum (FCS) or saline supplemented with natural factors may be added to a low concentration of an acceptable buffer, usually about 5-25 mM. Rinse the bone with the appropriate solution in combination with Non-limiting examples of conventional buffers include, for example, HEPES, phosphate buffer, lactate buffer, and the like.
Alternatively, bone marrow can be aspirated from bone in a conventional manner.

The method of separating or selecting stem cells from other cells is not critical to the invention.
There are various operations such as magnetic separation using magnetic beads coated with antibodies, affinity chromatography, and cytotoxic agents conjugated to monoclonal antibodies. Furthermore, there is a fluorescence activated cell sorter (FACS), which allows cells to be selected according to the degree of staining with a specific antigen. These techniques are well known to those skilled in the art and include U.S. Patent Nos. 5,061,620; 5,409,8213, 5,677,136; and 5,750,397 and Yau
, et a., (1990) Exp. Hematol. 18: 219-222.

The order of separation is not important in the present invention. Preferably, however, the cells are first separated by coarse separation and then by positive (positive) and / or negative (negative) selection. In such a positive selection, about 1% of all cells in the retained cell population
5% or less, usually about 10% or less, preferably about 5% or less, most preferably about 1% or less.
Less than% lack the marker used for separation. In some cases, the hematopoietic cell population is treated directly with an effective dose of a histone deacetylase inhibitor without first separating the HSCs subpopulation from the hematopoietic cells.

Stem cells, which make up only a small percentage of the total number of hematopoietic cells, are identified by the presence of a marker associated with the specific epitope site identified by the antibody (positive selection) and lack of binding to the specific antibody Characterized by the absence of certain markers. All of the monoclonal antibodies (mAbs) that react with a particular membrane molecule are grouped together as a cluster display (CD: differentiation antigen). Lin ^- cells are selected based on the lack of expression of at least one lineage-specific marker. For example, markers relating to T cells (such as CD2, CD3, CD4 and CD8), markers relating to B cells (such as CD10, CD19 and CD20), markers relating to osteoblasts (such as CD14, CD15 and CD33), Human cells lacking markers associated with natural killer cells (such as CD2 and CD56).

Human stem cells can be characterized by the following non-limiting phenotypes: CD2 ⁻ , CD3 ⁻ , CD4 ⁻ , CD7 ⁻ , CD8 ⁻ , CD10 ⁻ , CD14 ⁻ , CD15 ⁻ , CD19 ⁻ , CD20 ⁻ , CD33. ⁻ , CD34 ⁻ , CD34 ⁺ , CD38 ^{lo / −} , CD45RA ⁻ , CD59 ^+/− , CD71 ⁻ , CDW1
09 ^+, glycophorin ^{-, Thy-1 + (CD90} ), HLA-DR +/-,
AC133 ⁺ and rhodamine 123 (Rho123 ^lo ) or a combination thereof. Conjugation of mAbs to CD34 with monoclonal antibodies to the Thy-1 molecule has been used to isolate mouse, non-human primate and human HSCs. U.S. Pat. No. 4,714,680 discloses a cell population that expresses the CD34 marker. Recently, other mAbs have been identified for positive selection of human stem cells. These have been named EM, especially EM5, EM10 and EM16 (Chen, et al., Immunological Reviews, 157: 41-51, 1997). As used herein, EM ⁺ means a phenotype that expresses any EM marker.

In one preferred embodiment, the surface antigen expression profile of the enriched hematopoietic stem cell population is selected from the following group: CD34 ⁺ , Thy-1 ⁺ , CD10 ⁻ , CD19 ⁻ , CD15 ⁻ , CD33 ⁻ , CD34. ⁺ Thy-1 ⁺ , CD34 ^- Thy-1 ⁺ , CD34 ⁺ EM ⁺ , CD
^{34 + CD38 lo / -, CD34} + Rho 123, CD33 - Thy-1 +,
CD19 ⁻ Thy-1 ⁺ , CD34 ⁺ Thy-1 ⁺ Lin ⁻ , CD34 ⁺ Thy
-1 ⁺ EM ⁺ , CD34 ⁺ CD45RA ⁻ CD71 ⁻ , HLA-DR ^+/− CD
34 ⁺ Thy-1 ⁺ Lin ⁻ , CD34 ⁺ Thy-1 ⁺ CD38 ^{lo / −} , and CD34 ⁺ Thy-1 ⁺ CD38 ^{lo / −} EM ⁺ .

In a more preferred embodiment, the surface antigen expression profile of the enriched hematopoietic stem cell population is selected from the following group: CD34 ⁺ , Thy-1 ⁺ , CD34 ⁺ Thy-1 ⁺ , CD34 ^- Thy-1. ⁺ , CD34 ⁺ EM ⁺ , CD34 ⁺ CD38 ^{lo / −} , CD34 ⁺ Thy-1 ⁺ L
in ⁻ , CD34 ⁺ Thy-1 ⁺ EM ⁺ , CD34 ⁺ Thy-1 ⁺ CD38 ^{lo / −} and CD34 ⁺ Thy-1 ⁺ CD38 ^{lo / −} EM ⁺ , particularly EM ⁺ is EM1
0, which is the ^+. The combination of markers used to separate and define the enriched HSC population may vary as other markers become known.

In general, the number of cells selected as a result of positive selection is less than about 20% of the original cells,
More preferably less than 10%, generally less than 5%, most preferably less than 1.0%
Yes, and can be as low as 0.2%. The desired stem cells can be transferred to CD34⁺Thy-1 ⁺ Lin⁻80% or more, usually about 90% or more of human
A composition having stem cells can be obtained.

Mouse (m) HSCs are included in the Lin ^{neg / lo} population corresponding to cells expressed at the most ambiguous (dim) level of lineage specific markers. For example, a T lymphocyte marker (eg, CD2, CD3, CD4, CD5, or C
D8), expressing a B lymphocyte marker (eg, CD19 or B220), a bone marrow cell marker (eg, Gr1 or Mac-1 = CD11b), a natural killer cell marker (eg, NK1.1), or an erythrocyte marker (Ter119) Cells that do not or express at low levels.

Mouse (m) HSCs are characterized by the following non-limiting phenotype: CD2⁻, CD3⁻, CD4⁻, CD4^low, CD5⁻, CD8⁻, CD19 ⁻ , B220⁻, Gr-1⁻, Mac-1⁻, Mac-1^lo, NK1.1⁻,
Ter119⁻, Ter119^lo, Sca-1⁺, Thy1.1^lo, C-K
it⁻Or c-Kit^bright, CD34⁻, CD34⁺, CD38^{hi gh} , CD43^high, H-2K^highOr AA 4.1⁺It is. Mito
Chondria-binding dye rhodamine 123 (Rho-123) or DNA-binding dye
Fluorescent activating dyes such as Kist 33342 and monoclonal antibodies are used in combination.
To increase the enrichment of HSCs in the sorted population
It is possible. The most primitive cells are Rho-123^loOr DNA binding dye
Hoechst 33342^loConcentrated in fractions.

In a preferred embodiment, the enriched mouse HSC population is characterized by: Thy-1 ^lo , Lin ^{− / lo} , Sca-1 ⁺ , and c-Kit ⁺ . In a more preferred embodiment, the enriched population was sorted or selected from the following phenotypes: Thy-1 ^low Lin ^{− / lo} , Sca-1 ⁺ , c-Kit ⁺ Lin ^{− / lo} Sca-1 ⁺ , c -Kit ⁺ Sca-1 ⁺ , or c-Kit ⁺ Thy-1 ^low Lin- ^{/ lo} Sca-1 ⁺ .

In mice, HSCs can be isolated in large amounts from more mature precursor cells in vitro or in vivo, with different sensitivities to cytotoxic drugs such as 5-fluorouracil. Counterflow centrifugal elutriation: C based on cell size and cell density
CE) can further separate mouse HSCs. 25m at CCE
The small cell population obtained at a flow rate of L / min contained HSCs with long-term reproductive activity and very few precursor cells (Jones, et al., Nature 347: 188 (199
0)).

Once the hematopoietic cells have been collected and the HSCs have been appropriately separated, the cells are cultured in a suitable medium under growth-supporting conditions containing a combination of growth factors sufficient to maintain the growth of the hematopoietic cells. Any suitable container, flask, chamber, bag, bioreactor with perforation or suitable container such as a 24-well plate can be used. Culture vessels are readily available from commercial suppliers. The seeding level is not critical, but depends on the type of cells used, and in general, the seeding level is at least about 10 cells / ml, more usually at least about 100 cells / ml.
ml, typically less than about 2 × 10 ⁶ cells / ml, and usually less than 10 ⁵ cells / ml when the cells are CD34 ⁺ .

[0051] Various types of media can be used. As non-limiting examples, Iskov modified Dulbecco's medium (IMDM), X-vivo15 (serum free), and RPMI-1640
Can be mentioned. These are commercially available from various sources such as, for example, JRH BioSciences. Various kinds of nutrients, growth factors and cytokines can be added to the preparation. The medium may or may not contain an appropriate amount of serum such as fetal calf serum, autologous serum or plasma. However, when proliferating cells or cell populations are used for human therapy, the medium is preferably serum-free or autologous serum or plasma. Generally, the medium will contain an effective amount of cytokines, especially TPO, KL, F
Contains L and ILs. Those skilled in the art are aware of various types of growth support media and culture techniques for HSCs. Lansdorp, et al., J. Exp. Med. 175: 150
1 (1992) and Petzer, et al., PNAS 93: 1470 (1996).

[0052] In one embodiment, the media preparation is supplemented with TPO. Preferably about 0.1
ng / mL to a concentration of about 500 μg / mL, more preferably about 1.0 ng / m
L to about 1000 ng / mL, more preferably about 5.0 ng / mL to about 300
Added at a concentration of ng / mL. Further, the medium contains Flt3 ligand (FL) and c-kit ligand at a suitable concentration of about 0.1 ng / mL to about 10 ng / mL, respectively.
00 ng / mL, more preferably from about 1.0 ng / mL to about 500 ng / mL
, More preferably at a concentration of about 10 ng / mL to about 300 ng / mL.

While various types of cytokines can be added to the medium, IL-6 is preferred. Suitable concentrations are from about 0.1 ng / mL to about 500 ng / mL, more preferably, from about 1.0 ng / mL to about 100 ng / mL, even more preferably, about 5 ng / m2.
L to about 50 ng / mL. HyperIL-6 (IL-6 and soluble IL-
6 high affinity or covalent complex with the 6 receptor) can also be used. Conneall
y, et al., Ann.Meeting of the Intenational Soc. for Exper.Heamatology,
See Vancouver, Abst. # 60 (1998).

Other cytokines can be added to the medium individually or in combination,
Non-limiting examples include IL-1, IL-2, IL-3, IL-6, IL-1
2, IL-11, stem cell factor, G-CSF, GM-CSF, Stl, MCGF,
LIF and MIP-1α can be mentioned. When culturing mouse stem cells, suitable non-limiting media include mIL-3, mIL-6, and mSCF. Their preferred concentration is from about 0.1 ng / mL to about 1000 ng / mL, more preferably from about 1.0 ng / mL to about 500 ng / mL, even more preferably about 5 ng / mL.
. 0 ng / mL to about 200 ng / mL.

In one embodiment, the cytokine is contained in the medium and sprinkled with the medium (media pe
rfusion). For example, in another example using a bioreactor system, cytokines are not supplied as media supplements, but rather to a separate input port (
It is added separately as a concentrated solution from the inlet port).

Other molecules that can be added to the culture medium include adhesion molecules such as fibronectin and / or RetroNectin ^™ (Takara Shuzo, Otsu, Shiga, Japan). The term "fibronectin" refers to a glycoprotein found in the body and particularly high in connective tissue that forms a complex with collagen. RetroNecti
^nTM is from about 0.1 μg / mL to about 1000 μg / mL, more preferably about 0 μg / mL.
. It can be used at a concentration of 2 μg / mL to about 500 μg / mL, more preferably about 0.4 μg / mL to about 100 μg / mL.

HDAC-I compounds can also be included in the culture medium, and these compounds
Before or during gene transfer, they can be added separately or in combination under appropriate conditions that allow for the self-renewing division of the stem cells. The compounds described below are
It is a representative example of HDAC-I that can be used in the present invention.

A microbial metabolite known as trichostatin A (TSA) is HDAC-I (Yoshida, et al., BioEssays, 17: 42-429 (1995)).
. See EP 0 315 524 B1 for the preparation of TSA. Examples of other compounds related to TSA include butyrate, especially sodium n-butyrate, and hybrid polar such as suberoylanilide hydroxamic acid (SAHA) and m-carboxynamate bishydroxamide (CBHA). There are compounds (HPCs). These compounds have two polar groups separated by a non-polar 5- or 6-carbon methylene chain. Furthermore, these inhibitors appear to cause the accumulation of acetylated histone H4 in cells in culture (Richon, et al., Proc. Natl. Acad.
Sci. 95: 3003-3007 (1998)).

Other compounds known as microbial metabolites having the function of HDAC-I include:
Trapoxin, a cyclic tetrapeptide derived from a microorganism containing two L-phenylalanines, particularly trapoxin A (TPX) (Yoshida, et al.,
BioEssays, 17: 42-429 (1995)). TPX is completely different from TSA and butyrate not only in its chemical structure, but also in its mode of inhibition.

[0060] HDAC-I also includes a cyclic tetrapeptide compound having 2-amino-8-oxo-9,10-epoxy-decanoic acid in the molecule related to TPX. The following compounds can be mentioned. Chlamydocin: Closse, et al., He
lv. Chim. Acta 57: 533-545 (1974)), HC-toxin (Liesch, et al., Tetra
hedron 38: 45-48 (1982)), Cyl-2, and WF-3161 (Umehara, KJ
Antibiot 36: 478-483 (1983)). Depudecin, a bacterial metabolite (depudecin, Kwon,
Natl. Acad, Sci. USA, 95: 3356-3361 (1998)) and radicicol are also included in HDAC-I. WO 97/35990 discloses a number of histone deacetylase inhibitors, the contents of which are included herein.

In a preferred embodiment, HDAC-I is selected from TSA, TPX, chlamydosine, butyrate, especially sodium butyrate, DMSO, and their HDAC-I analogs. The concentration of HDAC-I used will vary and depends on the particular inhibitor, but is preferably from about 0.001 nM to about 10 mM, more preferably from about 0.01 nM to
About 1000 nM.

In a preferred embodiment, the histone deacetylase inhibitor is (1) TSA, in a concentration range of about 0.001 ng / ml to about 1000 ng / ml, more preferably about 0.01 ng / ml. To about 100 ng / ml, most preferably about 1.0 ng / ml.
ng / ml to about 10 ng / ml; (2) TPX, whose concentration range is about 0
. 001 nM to about 100 nM, more preferably about 0.01 nM to about 50 nM, most preferably about 0.1 nM to about 1.0 nM; and (3) chlamydosin,
The concentration ranges from about 0.001 nM to about 100 nM, more preferably about 0.01 nM.
nM to about 50 nM, most preferably about 0.1 nM to about 1.0 nM.

Although defined herein with reference to specific histone deacetylase inhibitors,
The scope of the present invention includes a wide range of histone deacetylase inhibitors not listed here. One example of a method for determining whether a compound not listed here is a class of histone deacetylase inhibitors is to measure the ability of a reagent to inhibit the catalytic conversion of a substrate of a protein of interest. A standard enzyme assay derived from In this method, inhibitors of the enzyme activity of the histone deacetylase protein are identified (Yoshida, et al., J. Biol. Chem. 265: 17174).
-17179 (1990)). Compounds can be isolated by screening for detransforming activity using cells transformed with the oncogene (oncogene) (Kwon, et al., Proc. Natl. Acad. Sci. USA
, 95: 3356-3361 (1998)). In addition, for assays measuring acetic acid release by deacetylation of lysine from peptides derived from the N-terminal sequence of histone H4, Wa
terborg, et al., Analytical Biochem. 122: 313-318 (1982).
This assay involves extraction of the tritylated acetyl group with an organic solvent. HD
A more recent reference on AC assays can be found in Kolle, et al., "Biochemicl Met
hods for Analysis of Histone Deacetylases ", Methods: A Companion to Meth
ods of Enzymology 15: 323-332 (1998). In addition, WO97 / 3 describes an assay used to determine that a compound is HDAC-I.
5990 is also disclosed. HDAC inhibitors are thought to function by different mechanisms and are described for TSA and TPX. In one embodiment of the present invention, compared to stem cells cultured under substantially the same conditions except for the absence of HDAC-I in culture, the present invention is more effective for promoting self-renewal division in the culture of stem cells, particularly hematopoietic stem cells. An amount of HDAC-I is used. "Culture or growth under substantially the same conditions" means that the cells are treated with the same concentration of growth supporting factor, the same medium, and the same culture period.

The duration of the culture once the cells have been exposed to HDAC-I varies and depends on the culture conditions. Under appropriate culture conditions, cells have the ability to grow indefinitely. However, generally, the cells are cultured for about 1 to about 28 days, preferably for about 1 to about 14 days, more preferably for about 1 to about 7 days. However, the cells may remain for 1-5 days or 1
Exposure for ~ 3 days. The cultivation period can be less than one day. Although not a limitation of the present invention, self-renewal division takes about 18 to 24 hours in typical cultured stem cells. However, some cultured cells are still in initial division even after 5 days of culture. Therefore, the cultured cells after 5 days of culture have undergone various numbers of self-renewal divisions ranging from 1 to 7. After 5 days in culture, the cells are dividing at approximately one division rate per day or have returned to the stationary phase. In one embodiment, the cells are cultured for about 1-5 days before being exposed to an effective amount of HDAC-I. However, HDAC-I may be added at the start of the culture. In such a time frame,
A gene delivery vehicle, such as a vector containing a nucleic acid sequence encoding a therapeutic or marker gene, is introduced into the culture by well known means.

In vitro (ex vivo) self-renewal of stem cells can be measured by various means. The three main methods are in vivo assays, in vitro assays, and surface antigen analysis. The in vivo assay uses the SCID-hu mouse system (McCune,
et al., Science 241: 1632-1639, 1998). In this system, human fetal bone fragments are implanted subcutaneously in severe combined immunodeficiency (SCID) mice. After the transplanted human fetal bone fragments are vascularized in SCID-hu mice, the bone marrow cavity is injected with the test human cell population and analyzed for multilineage engraftment of donor cells. Recently, Beige / nude
/ XID (bnx) mice and other models using non-obese diabetic SCID (NOD-SCID) mice have been developed (Greiner, et al., Stem Cells 16: 166-177 (
1998); Nolta et al., Proc. Natl. Acad. Sci. USA 93: 2414-2419 (1996)).
A preferred method of assessing mammalian non-human stem cell activity is to implant into an irradiated host by means well known in the art.

In vitro assays for mammalian stem cell activity include the long-term culture initiation assay (L
TCIC) and Cobblestone Area Formation Assay (CAFC) (Pettenge
ll, et al., Blood 84: 3653 (1994); Breens, et al., Leukemia 8: 1095 (1994);
Reading, et al., Exp.Hem. 22: 786 (Abst # 406) (1994); Ploemacher, et al.
, Blood 74: 2755 (1989)). In CAFC, a sparsely populated cell population is simply reduced in its ability to form distinct clonal outgrowths (or cobblestone regions) on a stromal cell monolayer over a period of time. Tested. This assay provides a frequency readout associated with LTCIC and can predict in vivo assays and engraftment in patients. A particularly suitable CAFC assay is described in Young, et al., Blood 88: 1619 (1
996).

Using flow cytometry, hematopoietic cells from various tissue sources can be subset by the surface antigens on which they are expressed. These assays may be used in combination to test HSCs. Using the SCID-hu mouse model and the CAFC assay, it can be confirmed that the cells have a stem cell function.

In one embodiment of the present invention, it relates to a model for measuring in vitro self-renewal, comprising labeling a hematopoietic cell population, particularly a population enriched in HSCs, with a division tracking dye. A mitotic tracking dye is a fluorescently labeled molecule that binds stably to its subcellular structure without apparently interfering with cell function. With each cell division, the fluorescence intensity of the daughter cells is reduced to half that of the parent cells, and cell division can be followed by flow cytometry. As non-limiting examples, carboxyfluorescein diacetate succinimidyl ester (CFSE), PKH26 (Young,
et al., Blood 88: 1619 (1996)), and PKH2 (Norton, et al., British J.
Hematol. 98: 528-539 (1997); Traycoff, et al., Exp.Hematolo. 26: 53-62 (19
98)). Most preferred is CFSE. Using antibodies against surface antigens characteristic of HSCs coupled to other fluorochrome sources, both cell division and primordial phenotype can be measured simultaneously.

To test the function of stem cells after each division cycle in vitro, CAFC assays are performed on each subset, purified by flow cytometry and SCID-hu. The above list of assays used to determine the functional composition of the hematopoietic cell population is not limiting in any way. One skilled in the art can use other known assays or a combination thereof.

In one embodiment of the present invention, a method of genetically modifying stem cells comprising contacting a stem cell population with a polynucleotide-containing gene delivery vehicle in the presence of an effective amount of HDAC-I. Gene delivery or transfer is mediated by methods known to those of skill in the art, including, for example, virus-mediated (mediated) transfer of DNA or RNA, liposome-mediated transfer, and other methods described below. The term "polynucleotide" should be understood to include terms synonymous with nucleotide sequence or nucleic acid and the like.

[0071] Retroviral vectors are particularly suitable gene delivery vehicles. A retroviral vector can enter a cell by its usual mechanism of infection, or it can be modified to allow it to bind to another differentiated cell receptor or ligand and enter the cell. Furthermore, recombinant derived adenovirus (AD) vectors, in particular,
See, for example, WO95 / 00655 and WO95 / 11984 which have constructed vectors which have reduced the potential for the appearance of recombinant and wild-type viruses. AD is a group of relatively well-characterized homogeneous viruses of viruses containing more than 50 serotypes (WO
95/27071 and Frey, et al. 1998 Blood 8: 2781-2792). AD is easy to propagate and does not need to be integrated into the genome of the host cell.

Adeno-associated virus (AAV) has also been used in gene transfer systems (see US Pat. Nos. 5,693,531 and 5,691,176). They are small, single-stranded DNA viruses that integrate into the genome of infected cells. Recombinant AVV vectors are produced at high titers, which transduce target cells at high rates. Herpes simplex virus (
HSV) vectors have been considered by researchers as gene therapy for gene transfer into neural tissue. Various commercially available vectors include pSG, pSV2CAT, and pXtl available from Stratagene, and pMSG, pSVL, and pSVK3 available from Pharmacia.

Vectors having both a cloning site and a promoter to which the polynucleotide is operably linked are well known to those skilled in the art. Such vectors are capable of transcribing RNA in vitro or in vivo and include Stratagene (LaJolla, CA) and
It is commercially available from Promega Biotech (Madison, WI). Removing 5 ′ and / or 3 ′ untranslated portions of the clone to optimize expression and / or in vitro transcription;
Additions or modifications need to be made to remove alternative translation initiation codons that may be inappropriate or other sequences that interfere with or reduce expression at the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted 5 'immediately prior to the start codon to enhance expression. Other non-limiting elements that can be added to the vector include enhancer elements, scaffold attachment regions (SAR) and matrix attachment regions (MAR) (see WO 97/46687).
Examples of vectors are bacteriophages, such as baculoviruses and retroviruses, such as baculoviruses and retroviruses, which have been described for expression in various eukaryotic and prokaryotic hosts and have been used for gene therapy and simple protein expression. , Cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art.

As mentioned above, suitable vectors are retroviral vectors, especially omnidirectional retroviral vectors. These vectors include a polynucleotide containing a therapeutic or marker gene and a portion of the retroviral genome. Coff
in, et al., "Retroviruses," (1997) Chapter 9, pp: 437-473 Cold Spring Har
See bor Laboratory Press. Retroviral vectors useful in the methods of the present invention are produced recombinantly by procedures already known in the art. WO
/ 94/29438, WO97 / 21824, WO97 / 21825 describe the construction of retroviral packaging plasmids and packaging cell lines. Retroviruses are divided into seven groups. Five of these groups are carcinogenic (on
cogenic potential), the other two being lentiviruses and foam viruses. The most common retrovirus is Moloney murine leukemia virus (
MoMLV-vector). Other MoMLV-derived vectors include LMiLy
, LINGFER, MINGFR and MINT. In addition,
Gibbon ape) Leukemia virus (GALV); Moloney mouse sarcoma virus (Mo
MSV); Osteoproliferative sarcoma virus (MPSV); for example, mouse embryonic stem cell virus (MESV) such as MESV-MiLy; mouse stem cell virus (MSCV)
); Spleen focus-forming virus (SFFV) (Agarwal, et al., J. of Virolo)
gy, 72: 3720 (1998)). Non-limiting examples of lentivirus-derived vectors include human immunodeficiency virus (HIV-1 or HIV-2) derived vectors. The development of new vector constructions utilizing the specific properties of the parent retrovirus, such as the host range and alternative cell surface receptors, continues. Particularly preferred vectors include the two long terminal repeats (LTRs) and the DNA from the mouse virus corresponding to the package signal. In one embodiment, the mouse viral vector is M
Derived from oMLV or MSCV. Optionally, the vector contains one or more SARs. However, the present invention is not limited to a particular retroviral vector, and transduction of a population of HSCs is performed under substantially the same culture conditions except that HDAC-I is not contained in the medium. HD for population transduction
Any retroviral vector, if enhanced in the presence of AC-I, is included.

In preparing retroviral vector constructs, the viral gag, pol, and env sequences are generally removed from the virus, leaving room for insertion of foreign or heterologous DNA sequences. The gene encoded by the exogenous DNA is usually expressed under the control of a strong viral promoter in the LTR. Appropriate regulatory sequences are selected according to the host used by those skilled in the art. Many promoters are known in addition to the LTR promoter. As non-limiting examples, the phage lambda PL promoter, human cytomegalovirus (CMV) immediate early promoter, Moloney mouse sarcoma virus (MoMSV), Rous sarcoma virus (
RSV) and spleen focus-forming virus (SFFV) U3 region promoter, granzyme A promoter, CD34 promoter, and CD8 promoter. Furthermore, it is also possible to use inductive or multi-regulatory elements. Plasmids containing the retroviral genome are available from the American Type Culture Collection (ATCC), and other sources are known in the art.

Such constructs can be efficiently packaged within viral particles if gag, poll and env functions are provided in trans by the packaging cell line. That is, the vector construct is introduced into a packaging cell,
The gag, poll and env proteins produced by the cells are combined with vector RNA to produce infectious virions, which are secreted into the culture medium. The virus thus produced infects the target cell and integrates into its DNA, but does not produce infectious virus particles due to the lack of essential packaging sequences. Most of the currently available packaging cell lines have been transfected with separate plasmids, each containing one of the necessary coding regions, so that replication-competent virus can be produced. Multiple recombination events are required. In another method, the packaging cell line already has an integrated provirus (the DNA form of the reverse transcribed RNA, which is incorporated into the genome of the infected cell). In this case, the cells produce all the proteins needed to make up the infectious virus, but are cripple
Cannot package its own RNA into the virus. Instead, the RNA produced by the recombinant virus is packaged. Thus, the virus stock released from the packaging cells contains only the recombinant virus.

The range of host cells infected with a retrovirus or retroviral vector depends on the envelope protein of the virus. The recombinant virus can actually infect any type of cell recognized by the env protein provided by the packaging cell, integrates the virus genome into the transduced cells, and stably produces the foreign gene product I do. In general, MoMLV mouse homotropy
v can infect rodent cells, but the amphotropic env can infect some primate cells, including rodents, bird cells, and human cells. Mo
Amphoteric packaging cell lines that can be used in the MLV system are known in the art and are commercially available. Non-limiting examples include PA12, PE50
1, PA317, PG13, @CRIP, RD114, GP7C-tTA-G1
0, ProPak-A (PPA-6), PT67 and FLYA13 (Miller et al. (1985) Mol. Cell. Biol. 5: 431-437; Miller et al.
(1986) Mol. Cell. Biol. 6: 2895-2902; Miller et al. (1989) Biotechniques
7: 980; Danos et al. (1988) Proc. Natl. Acad. Sci. USA 85: 6460-6464; Ri
gg, et al., (1996) Virology 218: 290-295; Finer et al., (1994) Blood 83: 4
3-50). Furthermore, WO97 / 21825 discloses a retroviral packaging cell line capable of producing a retroviral vector, in particular, a packaging cell line ProPak.
Are disclosed for supernatants produced from Recently, the G-glycoprotein (VSV-G) of vesicular stomatitis virus has been replaced by the MoMLVenv protein (Burns, et al., (1993) Proc. Natl. Cad. Sci. USA 90: 8033-8037; And WO92 / 14829). There are also heterotrophic vector systems capable of infecting human cells.

Usually, the vector contains at least one, preferably two, foreign genes or genetic sequences; (i) a marker gene, and (ii) a therapeutic gene to be introduced.
The following list of genes that may be incorporated into the vector is given by way of example and does not limit the invention in any way.

[0079] Marker genes can be included in vectors for the purpose of selecting integrated cells relative to cells into which the DNA construct has not been integrated, and tracking whether the transduction was successful. For example, as non-limiting examples of various marker genes,
Mention may be made of resistance markers for antibiotics such as G418 or hygromycin. Negative selections are also possible, such as using the HSV-tk gene, which sensitizes cells to drugs such as acyclovir or ganciclovir, as a marker. Alternatively, selection can be made by employing an appropriate cell surface marker for selecting a stem cell expressing the transgene by FACS sorting. The NeoR (neomycin / G418 resistance) gene is commonly used, but any conventional marker gene with sequences not present in the recipient cell can be used. Further, as non-limiting examples, NGFR (Nerve Growth Factor Donor),
GFP (bacterial green fluorescent protein), DHFR (dihydrofolate reductase gene conferring methotrexate resistance), bacterial hisD, mouse CD24
(HSA), a bacterial gene that confers resistance to mouse CD8a (lyt), puromycin or pheleomycin, and β-galactosidase.

Therapeutic genes can include the following therapeutically effective genes or gene sequences: adenosine deaminase deficiency (ADA), sickle cell anemia, recombinase deficiency, recombinase-regulated gene deficiency, antisense or HIV, such as the transdominant REV gene, or the herpes simplex thymidine kinase gene (
HSV-tk). The therapeutic gene can also include a gene sequence that expresses the LDL (low density lipoprotein) receptor. The therapeutic gene can further encode a new antigen or drug resistance gene. In addition, the therapeutic gene can include a toxin or apoptosis-inducing agent effective to specifically kill cancer cells, or a suicide gene specific for cancerous hematopoietic cells. Therapeutic genes may also include antisense oligonucleotide or ribozyme genes that are useful for suppressing translation.
Vectors usually contain one therapeutic gene. However, the treatment of certain diseases may require one or more genes, and the vectors used in the present invention can contain one or more genes. Alternatively, one or more genes can be delivered to a plurality of compatible vectors. Depending on the genetic defect, the therapeutic gene can contain regulatory and untranslated sequences. In human patients, the therapeutic gene is generally of human origin, but exerts high homology and biologically identical or equivalent functions in humans when no malignant immune response is produced in the patient. A gene of a neighboring species can also be used.

The nucleotide sequence of a therapeutic gene is known in the art or
Available from various sequence databases such as Those of skill in the art will readily recognize that any therapeutic gene can be cleaved into compatible restriction enzyme fragments and incorporated into the vector in a manner that allows the therapeutic gene to be appropriately expressed in hematopoietic cells.

Transduction methods include direct co-culture of cells with producer cells (Bregni, et a
l., (1992) Blood 80: 1418-1422) or a method of culturing with appropriate growth factors and polycations or virus supernatant alone (Xu, et al., Exp. Hemat. 22: 223-230)
There is. After viral transduction, the presence of the viral vector in the transduced stem cells or precursor cells thereof can be confirmed by means such as PCR.
These techniques are well-known in the art.

Gene therapy using HSCs is well known in the art, and the following example is a non-limiting list of diseases for which gene transfer into HSCs may be useful. These diseases include bone marrow disease, erythrocyte deficiency, and metabolic disease. In particular, bone marrow transplantation is improved by the present invention.

The genetically modified cells obtained by the method of the present invention may further comprise autologous, xenogeneic,
Alternatively, it can be used for the same kind of setting. Autologous cells are obtained from each individual's own organ, heterologous cells are obtained from different species, and allogeneic cells are generally obtained from different individuals of the same species. The modified cells are any physiologically acceptable vehicle, usually administered intravascularly. However, they can also be introduced into bone, or other convenient site where a suitable site for regeneration and differentiation of the cells is obtained. Usually, at least 1 × 10 ⁵ cells will be administered. Preferably, at least 1x1
0 ⁶ cell number or more is introduced by injection or catheter or the like. If desired, TPO, IL-2, IL-3, IL-6, IL-11, GM-CSF, G-
Factors such as CSF, interferon and EPO may also be included.

The method provided by the present invention overcomes the shortcomings of conventional gene transfer methods by increasing (enhancing) the genes to be transferred into stem cells. Integration of genes into stem cells using vectors, especially retroviral vectors, requires cell division. In general, stem cell division causes differentiation. HDAC-I is thought to inhibit this differentiation during cell division. as a result,
The content of CD34 ⁺ Thy-1 ⁺ cells in the transduction product can be increased.

The cells thus obtained can be used immediately, or grown by a method known in the art, or frozen at liquid nitrogen temperature, stored for a long time, and then thawed. It is also possible. The cells are usually 10% DMSO, 50% FCS,
And stored in 40% RPMI 1640 medium. Once thawed, cells can be grown further. Methods of expanding HSCs utilizing growth factors and / or stromal cells in connection with stem cell growth and differentiation are well known to those skilled in the art (US Pat.
5,744,361).

The stem cells prepared by the methods disclosed herein can be used for various therapeutic means described in the literature. The cells can be used to completely reconstitute an immunocompromised host, such as an irradiated host and / or a host to be treated for chemotherapy. The cells can also be used for treating a genetic disease. The cells are free of neoplasms or other pathogenic cells and can be used for bone marrow transplantation. By using stem cells, graft-versus-host disease can be minimized and donor-specific immune tolerance can be induced in the host. Therefore, as a specific example of the present invention,
Exposing a population of hematopoietic cells in culture to an effective amount of a histone deacetylase inhibitor, introducing a polynucleotide encoding a therapeutic gene or a marker gene into the cultured cells, and exposing no exposure to the histone deacetylase inhibitor A method for improving the engraftment of genetically modified mammalian hematopoietic cells, particularly stem cells, comprising increasing the number of genetically modified stem cells and administering an effective amount of the modified cell population to a subject. I will provide a. Preferably, the mammal and the subject are human and the cells are enriched hematopoietic stem cells, especially CD34 ⁺ Thy-1 ⁺ cells. In a preferred embodiment, the cells are transduced with a retroviral vector selected from MoMLV and MSCV-derived vectors, and optionally contain one or more SAR elements.

Further, the present invention provides that the method comprises contacting a population of hematopoietic stem cells with an effective amount of a histone deacetylase inhibitor, transducing HSCs, and administering to the subject an effective amount of the transduced cell population. The present invention provides a method for restoring hematopoietic ability in a subject.

In practicing the present invention, unless otherwise indicated, conventional techniques of cell biology, molecular biology, cell culture, and immunology included in the art are used. For example, Sambrook, Fritsch, Mmaniatis, MOLECULAR CLONING: A LABORATORY MANUAL
^{, 2 nd edition (1989);} CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, (FM Ausu
bel et al. Eds., 1987); the series METHODS IN ENZYMOLOGY (Academic Press
, Inc.); PCR 2: A PRACTICAL APPROACH (MJ McPherson, BD Hames and GR
Taylor eds., 1995); ANIMAL CELL CULTURE (RI Freshney. Ed., 1987); AN
See TIBODIES: A LABORATORY MANUAL (Harlow et al., Eds., 1987).

Various publications, patents and published patent specifications are cited. These disclosures are:
In order to more completely describe the state of the art to which this invention pertains, it is incorporated herein by reference.

The present invention described generally above has been described in more detail by reference to the following examples. These examples are included only for the purpose of describing certain embodiments of the invention and are not intended to limit the invention in any way.

[0092]

【Example】Example 1-Human cells  Cells: 7.5- or 10 μg / kg / day G-cells with informed consent
Leukaphers from normal donors mobilized with CSF for 5-6 days
is) A sample was obtained. Isolex300SA or 300 (Baxter Healthcare Corp., Deerfiel
d, Illinois) on CD3 from S. leukaeparsis samples in SyStemix
4⁺The cells were concentrated.

CD34 from fresh MPB⁺Thy-1⁺Cell Purification: CD34 by Flow Cytometry⁺Thy-1⁺To select cells
Directly links anti-CD34 (PR20, SyStemix Inc., Palo Alto CA) to CY-5
And anti-human Thy-1 (PR13, SyStemix Inc., Palo Alto CA)
It was directly bound to phycoerythrin (PE). Purified mouse IgG
1 (Sigma, St. Louis, MO) conjugated to PE or Cy5 and used as isotype control.
Used. MPBCD34 selected by Isolex⁺Transfer cells to phenyl red (JRH Bi
osciences, Lenexa, KS), 2% fetal bovine serum (FBS) (Gemini Bioproducts, C
alabasas, CA), 0.1% heat-inactivated human gamma globulin (Gamimune: Miles
Inc., Elkhart, IN) and 10 mM HEPES (JRH Biosciences, Lenexa, KS)
Staining buffer (SB) consisting of Iscove's unmodified Dulbecco's medium (IMDM)
To 10⁷Resuspended at cells / ml. Cells were treated with anti-CD34-CY5 (5 μg / mL) and
And anti-Thy-1-PE (25 μg / mL) or the appropriate isotype control IgG1-
Stain with CY5 and IgG1-PE at 4 ° C. for 30 minutes, wash and 5 × 10 5 in cold SB
⁶Resuspended at cells / mL. Propidium iodine (PI) (Molecular Probes Inc
., Eugene, OR) was added at 1 μg / ml to detect non-viable cells.
CD34⁺Thy-1⁺Cells and CD34⁺Thy-1⁻Cell population of 5W algo
Becton Dickinson FACStar Plus with integrated laser^TM:( Becton Dickinson, S
an Jose, CA). The software for forward versus side scatter.
Cell region, PI, CD34 and Thy-1 to establish and survive CD34⁺Th
y-1⁺Cells and CD34⁺Thy-1⁻A cell population was selected. Selected cells
The population was analyzed again to confirm that the cell population was cleanly separated.

Cytokines and cell culture: Recombinant human thrombopoietin (TPO) was obtained from R & D Systems, Minneapolis, MN.
Purchased from. Recombinant Flt3 ligand (FL) and c-kit ligand (K
L) was manufactured by SyStemix Inc. IL-6 and LIF are from Novartis Inc., Bas
purchased from el Switzerland. Cells are about 112 hours, 2 × 10 ⁵ cells / mL
(CD34 ⁺ cells) or 5 × 10 ⁵ cells / mL (CD34 ⁺ Thy-1 ⁺ cells) at a humidification chamber (Corning Costar Corp., Cam) with a saturation humidity of about 100%.
The plate was cultured at 37 ° C. on a 6- or 24-well flat bottom plate in a bridge, MA). Cells were cultured in a culture medium (CM) (X) containing 1% bovine serum albumin (BSA) (Sigma) supplemented with the cytokines: TPO (50 ng / mL); FL (100 ng / mL); and KL (100 ng / mL). −
Vivo-15 medium) (Bio Whittaker, Walkerville, MD). TSA
Was added at 5 ng / mL, TPX was added at 0.25 nM and 0.5 nM, and chlamidosin was added at 0.25 nM and 0.50 nM. TSA was purchased from Wako Bioproducts, Richmond VA and included in 1
Stored at −20 ° C. at a concentration of mg / mL. TPX and Chlamidosin are from Novartis Pharmac
euticals Corporation, East Manover, NJ and stored at -20 ° C at a concentration of 1 mM in DMSO.

As shown in FIG. 1, when CD34 ⁺ cells were cultured for 5 days with cytokines (TPO, FL and KL) as growth support factors and TSA, TPX or chlamydosin as HDAC inhibitors, HDAC inhibitor The number of Thy-1 ⁺ cells was substantially increased compared to cultures containing no. Of the HDAC inhibitors tested, chlamidosin showed the largest increase, averaging 7-fold. These results are the average of four experiments, and the error bars indicate the standard deviation from the average.

Isolation of cell population after culture: Cells after culture were harvested from tissue culture wells, counted, and stained with anti-CD34-CY5 and anti-Thy-1-PE, or the appropriate isotype control as described above. FACStar Pl
Using a us flow cytometer, the cell population was isolated as if the cells before culture were sorted. The sorted cell populations were re-analyzed and the events (ev
ents) was found to be contained within each sorted area, and less than 5% of the Thy-1 ⁻ population contaminated Thy-1 ⁺ cells.

[0097] Cobblestone area-forming assay: Cells are limited on a preformed mouse stromal monolayer by the method described in Young et al., Blood 88: 1619 (1996). Seed at dilution (100-0.78 cells per well, 24 wells per dilution). The culture medium was 1 mM sodium pyruvate (JRH Bio
Sciences), 5 × 10 ⁻⁵ M2-mercaptoethanol (Sigma, St. Louis, MO)
, 10% FBS (Hyclone, Logan, UT) and cytokine LIF (50ng / mL)
And a 1: 1 IMDM / RPMI mixture containing IL-6 (10 ng / mL). 1
Half of the medium was changed at weekly intervals, and the cobblestone area formation was measured after 5 weeks. Holes containing cobblestone regions were counted under a microscope and their frequency was estimated statistically as shown below.

The frequency readout information is obtained by SAS statistical analysis using maximum likelihood estimation and linearity χ
Two assays (Biostatics consulting, Palo Alto, CA) were used to determine the number of positive wells (wells) in each row of the CAFC assay limiting dilution series. The readout by limiting dilution is considered to fit a linear model if the conservative χ2 test is greater than 0.05. Significant differences in CAFC frequency readouts were determined using Nova (Microsoft) and a P less than 0.05
Was determined to be significant.

[0099] As shown in FIG. 2A, ^{Thy-1 +} cell number and total CAFC activity of ^{Thy-1 +} a fraction of cells, 2-3 in Chlamydocin containing medium compared without chlamydocin
Increased by a factor of two. Total cell numbers were the same for both. However, total CAFC activity in the chlamydosin-containing medium was increased 2-fold (FIG. 2B). From these results, stem cell replication or self-renewal, as measured by Thy-1 ⁺ phenotype and function, is increased by exposing the cells to HDAC-I during culture.

SCID-hu bone assay: The SCID-hu bone assay was performed as described in Luens, et al., Blood 91: 1206 (1998). C. B-17. Scid / scid mice were implanted with human fetal bone fragments (mean gestational age 22 weeks) 8-10 weeks prior to use in the assay. The starting donor cell population for the SCID-hu bone assay was CD34, selected as described above. The cultured donor cell population was purified by flow cytometry for Thy-1 antigen expression as described above. The host received total body irradiation (400 rads) immediately prior to injection, and Thy- at doses ranging from 5,000 to 15,000 per human fetal bone graft.
¹⁺ cells were injected. Eight weeks after injection, mice are sacrificed, bone marrow cells are harvested from the grafts, and anti-W6 / 32-PE for detection of pan-human leukocyte antigen HLA class I major histocompatibility complex and HLA allotype Stained with a FITC-conjugated antibody against Cells were analyzed using a FACScan analyzer (Becton Dickinson Immunocyt
(Geometry Systems). Grafts containing at least 1% of hematopoietic cells with the donor HLA antigen were considered positive. The survival rate is the number of grafts positive for donor cells relative to the number of grafts injected. Table 1 shows that the stem cell function is chlamydosin (CH
L) shows that Thy-1 ⁺ cells from the containing cultures were maintained on an individual cell basis. In addition, the dose (dose) was determined by using Thy-
1 ⁺ number of cells.

[0101]

[Table 1]

NOD SCID Reproduction Assay: Trafficking of mouse bone marrow with injected (IV) human hematopoietic cells
afficking) and engraftment thereon were measured with the NOD SCID repopulation assay. 6-10 week old NOD SCID mice (SyStemix purchased from Jackson
) Were irradiated with 350 rads of radiation. Human MPB was injected into the tail vein or orbital sinus. In this engraftment assay, T
No re-purification of hy-1 ⁺ cells. Instead, the same number of cells were injected from all cultures. Six weeks later, the mice were sacrificed and bone marrow cells were collected from the long bones of the hind limb. Cells were fluorescently labeled with anti-CD45-APC capable of detecting human cells, and FA
Analyzed with CS Calibur ^™ . Table 6 shows the results. Cell engraftment was small and engraftment was improved when chlamidosin was added in a 24 hour incubation.

Division Tracking Dye Labeling: Isolex Selected CD34 ⁺ cells (concentration 3-5 × 10 ⁶ / mL) were converted to carboxyfluorescein diacetate succinimidyl ester (CFSE) dye (Mole
(Molecular Probes Inc., Eugene, OR) at a concentration of 1.25 μM in IMDM without phenol red in the dark for 10 minutes at room temperature. The labeling reaction was stopped by adding 1/5 volume of FBS and 10 volumes of cold CM. Aliquots of dye-labeled cells were fixed with 1% paraformaldehyde, stored at 4 ° C., and used as markers for fluorescence intensity of non-dividing cells after culture. Analysis was performed by FACS Calibur (Be
cton Dickinson, Immunocytometry System).

As shown in FIGS. 3A, B, and C, Thy-1 expression after 5 days of culture in the presence of chlamydosin was retained even after at least four cell divisions, while chlamydosin was not expressed. Cell division in culture in the presence was rapidly lost.

Analysis of cell phenotype after culture: For expression of CD34 and Thy-1 antigen, anti-cD34-CY5 and anti-Th
Evaluation was performed as above using y-1-PE. Human CD2, CD14, CD1
5, FITC-conjugated monoclonal antibodies against CD33 and CD19 (Becton
Dickinson, San Jose CA) was used to assess expression of the series antigens. Analysis was performed by FACS Ca
Made in libur.

Using the above method, the surface lineage antigens CD2, CD14, and CD19 were negligible both before and after 5 days of culture, in the presence or absence of HDAC inhibitors. Expression of CD15 and CD33 increased at a similar rate after culture in the presence or absence of HDAC inhibitors. A, B, C, and D in FIG. 4 show that the percentage of Thy-1 ⁺ cells expressing CD15 is about 30% after 5 days of culture in the presence or absence of an HDAC inhibitor. . These results indicate that the expression of progenitor antigens such as CD34 and Thy-1 was increased in cultures containing HDAC inhibitors, but the expression of other surface antigens associated with differentiation was not. I have.

[0107]Example 2 mouse cells  Isolation of mouse stem cells: 4 weeks old BA. Bone marrow was collected from one mouse (C57BL / Ka.AKR / Jsys) and
For competitive Sca-1 antibody (Pharmingen) and magnetic column (Miltenyi Biotec Inc)
Sca-1 expressing cells were enriched using positive selection. Sca-1 concentration
Cells are further c-Kit-APC, Thy1.1-FITC, streptavidin
-Texas Red and Mac-1, Gr-1, B220, CD2, CD4
, CD5, and CD8 labeled with a series of PE-linked series (Lin) antibodies (all
Antibodies purchased from Pharmingen). 1 μg / m propidium iodide (PI)
Non-viable cells were excluded by addition at 1 l. C-Kit with high concentration of stem cell activity⁺
Thy-1^loLin^{lo /-}Sca-1⁺Subset the Vantage sorter (Becto
n Dickinson, San Jose CA).

Culture conditions: The selected cells were mouse interleukin (mIL) -3 (10 ng / ml), mI
Culture medium (XVivo-15 medium) supplemented with L-6 (10 ng / ml) and mouse stem cell factor (mSCF) (100 ng / ml) (purchased from R & D) (Bio Whittaker, Walkervi)
(lle, MD) (cell density 5 × 10 ⁴ cells / ml). TSA (Wako Biop
roducts, Richmond VA) 0.5 ~ 10ng / ml, Chlamidosin (Novartis Pharmaceuti)
cals) was added to the medium at 0.25 nM to 2 nM. Cells were cultured for 4 days at 37 ° C. under 5% CO ₂ in a multiwell flat bottom tissue culture plate (96,48 depending on the number of cells initially seeded).
, Or 24 wells).

Cell analysis after culture: After 4 days of culture, the cells were collected, and a certain amount thereof was stained with trypan blue to obtain a neat cell.
Viable and non-viable cell counts were determined with an ubauer hemocytometer. At least 20 in total
0 cells were recorded. Some of the cultured cells (corresponding to at least 40000 viable cells) were stained as described above. c-Kit, Thy-1, Lin and Sca-
¹⁺ expression was measured by FACS with a Vantage sorter (Becton Dickinson, San Jose CA). The limits of the gate used to determine the percentage of cells falling into the c-Kit ⁺ Thy-1 ⁺ Lin ^{lo / −} Sca-1 ⁺ subset are the boundaries that separate the bimodal population for each individually observed staining. Defined as These limits are mIL
Set for cells treated with -3, mIL-6, and mSCF alone. The number of c-Kit ⁺ Thy-1 ⁺ Lin ^{lo / −} Sca-1 ⁺ cells in the culture was c-Kit ⁺
The percentage of cells in the Thy-1 ⁺ Lin ^{lo / −} Sca-1 ⁺ subset was multiplied by the total number of viable cells in culture. Table 2 summarizes the results obtained in representative experiments. 5A, 5B and 5C.

[0110]

[Table 2]

[0111] In this experiment, 50 × 10 ³ cells pieces of sorted c-Kit ^{+ Thy-1 l
o} Lin ^{lo / −} Sca-1 ⁺ (KTLS) cells were placed in a 24-well plate at mlI
The cells were cultured in an XVivo-15 medium supplemented with L-3, mIL-6 and mSCF. TSA 3 ng / ml and chlamidsin 1 nM were added to the medium. Four days later, cells were harvested and subjected to FACS analysis. The number of viable cells recovered after 4 days of culture was H
The addition of DAC-I to the growth medium reduced 2-4 fold. HDAC-I
As compared to cell growth in the absence, a very major portion of cells treated with TSA or chlamydosin remained Lin negative (93 or 88% vs. 55%), with higher levels of Thy-1 (95%). -96% vs. 30%) and Sca-1 (94 or 88% vs. 46%). Cells grown on any of the HDAC-Is
A higher percentage of positives for c-Kit remained compared to the absence of -I (67
Or 73% vs. 51%). When grown in the absence of HDAC-I, only 8% of the cultured cells have a stem cell phenotype (c-Kit ⁺ Thy-1 ^lo Lin ^{lo /} -Sca
Whereas was left at -1 ^+), the cells cultured in TSA or chlamydocin, respectively, 58% and 57% showed the phenotype. 4 days culture period, HDA
C-Kit ⁺ Thy-1 ^lo Lin ^{lo / −} Sca- grown in the absence of CI
¹⁺ cells increased 3.1 fold, whereas cells cultured with TSA or chlamydosin showed 7.2 or 5.0 fold growth, respectively.

FIGS. 5A, 5B and 5C show the growth medium alone (A) or the addition of 3 ng / ml TSA (
B) or Lin, which was analyzed after culturing for 4 days with addition of 1 nM chlamydosin (C),
7 shows profiles of Thy1, cKit, and Sca-1. The area limits used to calculate the percentage belonging within each gate are represented by dotted lines. Non-viable cells have been excluded from the analysis.

[0113]Example 3-RetroNectin ^TM Transduction  Retroviral Infection After obtaining consent, CD34 as described in Example 1⁺Thy-1⁺In cells
CD34⁺Enriched cells (10⁶Cells / mL) in 5 mL of X-Vivo-15 medium
(Bio Whittaker, Walkerville, MD) for 48 hours at 37 ° C, CO₂Cultured under
Was. The medium contains cytokines: TPO (100 ng / mL; R & D SyStems, Minneapolis, MN)
FL (100 ng / mL); and KL (100 ng / mL) were added. Chlamydosin is added to cultures at 0.
It was added at a concentration of 50 nM / mL for 72 hours. Chlamidosin is Novartis Pharmaceutical
s Corporation, East Manover, NJ, at -1 mM in DMSO.
Stored at ° C.

Non-tissue culture treated plates (Falcon, Lincoln Park, NJ) were coated with 2 μg / cm ² fibronectin fragment CH-296 (FN) (BioWhittaker, Walkersville, MD) and incubated for 2 hours at 37 ° C. Plates were blocked for 30 minutes with phosphate buffer (HSA / PBS) containing 2% human serum albumin. After this, 2.5
% Hepes buffer and Hanks' adjusted salt solution (HBSS).

After 48 hours, the cultured cells were centrifuged for 10 minutes at 1200 rpm at 4 ° C.
Resuspended in the same buffer. Cells are coated with FN containing the same amount of retroviral supernatant
Add to the plate and add polybrene or protamine for 20 hours at 37 ° C, CO ₂ (Lishan SU et al., 1997, Hematopoietic Stem Cells-Based G
ene Therapy for Acquired Immunodeficiency Syndrome: Efficient Transducti
on and Expansion of RevM10 in Myeloid Cells In Vivo and Vitro. Blood: Apr
. 2283-2290).

A Moloney murine leukemia virus (MoMLV) vector was prepared from the ProPAk (PP-A.6) packaging cell line (Forestell, et al., 1997, Nov.
el Retrovirus Packaging Cell Lines: Complementary Tropisms nd Improved V
(ectorProduction For Efficient Gene Transfer, Gene Therapy, 4: 600-610)
. The vector comprises -LTR-NGFR-SV40Neo-LTR-, where LTR is the viral long terminal repeat, NGFR is truncated and shortened human nerve growth factor receptor, SV40 is the SV40 promoter, and Neo. Is G418
Resistant (Miller et al., 1989, Improved Retroviral Vectors for Gene Tra
nsfer and Expression. BioTechniques. 7: 980-990).

Cells were harvested from plates by gentle pipetting and centrifugation (as described above). The cell pellet was resuspended in X-Vivo-15 medium containing 1% BSA. Surviving cells were counted by trypan blue staining. 1:10 against trypan blue
After being diluted at a ratio of 1, the suspension was placed in a hemacytometer in a suspended state. Dead cells were judged by their blue color. These methods are well known to those skilled in the art.

FACS analysis of gene expression in CD34 ⁺ , Thy-1 ⁺ and whole cells: After transduction, cells were analyzed by FACS as described below. A subset of the above cells was prepared using GM-SCF (10 μg / mL), EPO (2U), IL-3 (10 ng / mL).
mL), IL-6 (10 ng / mL), LIF (100 ng / mL), and KL (100 ng / mL). Culture (1.0 ml) was performed on a 24-well tissue culture plate (
Falcon). After 72 hours, the cultured cells were collected and the anti-CD34-A
PC (Becton Dickinson, San Jose, CA), anti-Thy-1-PE (SyStemix Inc.
, Palo Alto CA) and anti-NGFR-FITC (Boehringer Mannheim, Indiana
polis, IN) or the appropriate isotype control (Becton Dickinson, San Jose, CA). Fluorescence was analyzed on a FACS Calibur (Becton Dickinson) using standard techniques. Anti-NGFR-FITC was bound with SyStemix. According to the results summarized in Table 3, not only the number of cells expressing the Thy-1 antigen but also the number of Thy-1 ⁺ NGFR ⁺ cells were increased by using chlamidone.

[0119]

[Table 3]

Footnotes in Table 3: Each value for (+) and (−) is the average (AVE) of five experiments. Each value for CHL + BSA is the average (AVE) of three experiments. SD = standard deviation. CHL is chlamydosin added at 0.5 nM / mL. BSA is bovine serum albumin and was added at 1.0%. Magnification = (+) average CHL / (−) average CHL.

In vitro assay for transduction of primordial PHP: After approximately 72 hours of transduction, cells were counted and 20,000 cells per culture condition (+ or-chlamydosin) were plated in 24-well plates (Coning Science Products, Actoscience).
n MA) in two wells of Sys1 mouse stromal cells (Young et al., Bl
ood, Vol. 87: 545 1996) for 5 weeks in the presence of exogenous human IL-6 (20 ng / mL) and LIF (100 ng / mL). Half of the medium was changed weekly. Medium is 10
50/50 RPMI / IMDM mixture containing 50% fetal calf serum. After 5 weeks in stromal culture,% NG in CD34 ⁺ cells with and without chlamydosin
No significant differences were noted for FR (data not shown).

F of gene expression in CD34 ⁺ and whole cells cultured on Sys1 stroma
ACS analysis: The above cells were collected and filtered through a 70 mm cell strainer (Falcon) to remove stromal cells. Viable hematopoietic cells were counted and the cells were replaced with GM-SCF (10 μg /
mL), EPO (2U), IL-3 (10 ng / mL), IL-6 (10 ng / mL), LIF (100 ng / mL
) And KL (100 ng / mL) in X-vivo15 medium for 3 days. Cells were purified using anti-NGFR-FITC and anti-CD34-APC (Becton Dickinson, San Jo
se, CA). Cells were analyzed on a FACS Calibur (Becton Dickinson).

Further LTC-CFC assay for SV40Neo in LTC-CFC: Three 40,000 cells / ml from C were subjected to GM-SCF (10 μg / mL), EPO
(2U), IL-3 (10 ng / mL), IL-6 (10 ng / mL), and KL (100 ng / mL) in a methylcellulose colony assay (MethoCult, StemCell Technologies, Vanc
ouver, Canada). After 12-14 days, hematopoietic colonies were scored according to standard standards known in the art.

Each colony (64) was placed in 50 μl of lysis buffer as shown below. The lysate (cell lysate) was incubated at 37 ° C. overnight and at 95 ° C. for 15 minutes.
Heat inactivated and stored at -20 ° C. Perform a PCR assay and run the SV40 Neo
Was tested for transgene markers. B-globulin was used as a positive control for the presence of DNA. PCR lysis buffer A and B mixture in 1: 1 ratio with 14
4 mg proteinase K was mixed. Lysis buffer A was 100 mM KCL, 1
Contains 0 mM Tris HCl and 2.5 mM MgCl ₂ . Lysis buffer B contained 10 mM Tris HCl, 2.5 mM MgCl ₂ and 1% Tween,
And 1% NP40. Final concentration is 100 mg / ml proteinase K
Met. For PCR analysis, 10 μl (1,000 cells) and 5 μl (500 cells)
Aliquots were collected on thermocycle plates. Known specific primers and probes were used for PCR.

The PCR was performed using 10 mM Tris-HCl (pH 8.3), 1.5 mM MgC
l _2, 50mM KCl, dATP of each 0.1666mM, dCTP, dGTP
, DTTP, SvNeo forward primer 1 and SvNeo reverse (r
everse) Primer 2 (0.833 μM), B-globulin forward primer 1 and B-globulin reverse primer 2 (0.227 μM), and Taq D
Performed in a 30 μl volume containing NA pomerase (1 unit). The PCR program was performed at 95 ° C for 5 minutes using a Perkin-Elmer 9600 thermocycler.
Cycles, 95 ° C. for 30 seconds, 62 ° C. for 30 seconds, and 72 ° C. for 1 minute
Zero cycles; and one cycle at 72 ° C. for 10 minutes. PCR products were analyzed by 3% agarose gel electrophoresis. These techniques are well-known in the art.

[0126]Example 4-Spinoculation transduction:  Obtained from healthy volunteers using the standard procedure and the standard procedure described in Example 1.
Hematopoietic stem cells derived from G-CSF mobilized peripheral blood are collected by an immunoaffinity system.
CD34⁺Purified by cell selection.

Culture purified CD34 ⁺ cells (3 × 10 ⁶ ) in Teflon® cell bags (American Fluoroseal, Inc., Gaitherburg, Md.) In 1.5 ml of serum-free X-vivo 15 medium for 48 hours did. The size of the Teflon cell bag used in the experiment depends on the number of cells to be cultured and transduced. The medium contains TPO (200 ng / mL), FL (200 ng / mL), and IL-3 (40 ng / mL) in the presence or absence of the histone deacetylase inhibitor, chlamidosin (0.50 nM / mL). , IL-6 (40 ng / mL), and LIF (200 ng / mL).

After 48 hours of culture, an equal volume (1.5 ml) of retroviral supernatant was added to the cultured cells. The retrovirus vector and supernatant used in this experiment are described in Example 3. The cells were centrifuged at 3,400 rpm (Soval RD6000) for 4 hours,
Arranged along the flat side of the bag placed at the bottom of the centrifuge bucket. The cells were resuspended by gently pressing the bag and returned to the incubator. 3
After incubation for 20 hours at 7 ° C., 5% CO ₂ , the cells were harvested from the bags and the stem cell content (CD34 ⁺ cells and CD34 ⁺ Th as described in Example 3).
y-1 ⁺ cells).

[0129]

[Table 4]

As shown by the data in Table 4, the cell harvest procedure after transduction was not affected by chlamydosin.

[0131]

[Table 5]

The mean is the average of six experiments.

Using the data in Tables 4 and 5, the absolute number of HSCs (CD34 ⁺ Thy-1 ⁺ ) was calculated. Table 6 summarizes the results. The yield is per 1 × 10 ⁶ cells at the start of transduction. These results suggested that about twice as many HSCs in the population were transduced as compared to cells transduced without chlamidsin. Cells transduced for 3 days in the presence or absence of chlamydosin showed an equivalent content (99 +/- 1.4%) for CD34 ⁺ cells.

[0134]

[Table 6]

[0135]

[Table 7]

[0136]

[Table 8]

Footnotes to Table 8: 1.75 x 1.71 are Thy expressing transgene in infusion products.
This means an approximately 3-fold increase in # of -1 ⁺ cells. The average is the average of six experiments.

[0138]Example 5 Dose Dependence for Chlamidosin:  CD34⁺The selected cells were subjected to spinoculation using the MoMLV vector.
And transduced as described in Example 3. According to the results shown in Table 9,
Midosin dose-dependently stimulated ex vivo transduced CD34 from mobilized peripheral blood.⁺Fine
Vesicle stem cells (CD34⁺Thy-1⁺) The content was increased. This data is
The optimal concentration for cells incubated for 3 days is 0.5 nM to 1.0 nM.
It indicates that there is. Furthermore, the results show that in the presence of 0.5 nM chlamydosin
Starting CD34⁺Explain that the stem cell content for the population was increased.

[0139]

[Table 9]

Footnotes to Table 9: Results are the average of two experiments. CD34 ⁺ Thy at the start of culture (day 0)
The -1 ⁺ cell content was 37%.

[Brief description of the drawings]

FIG. 1 shows the increase in the number of human hematopoietic progenitor cells expressing Thy-1 antigen from cultures of MPB CD34 ⁺ cells exposed to HDAC inhibitors.

FIG. 2. Human MPB CD exposed to the HDAC inhibitor chlamidsin
34 ⁺ cell cultures were Thy-1 ⁺ intracellular versus cultures without chlamydosin (
FIG. 2A) or increased total CAFC activity in the culture (FIG. 2B).

FIG. 3. When cultured with chlamydosin (FIG. 3B), 4 times of the culture was observed compared to when cultured without chlamydosin, an HDAC inhibitor (FIG. 3A).
This shows that a higher percentage of human CD34 ⁺ hematopoietic progenitor cells retain expression of Thy-1 ⁺ antigen throughout division.

FIG. 4. Percentage of bone marrow antigen CD15 Thy-1 ⁺ cells is up-regulated in human CD34 ⁺ MPB cultured with the HDAC inhibitor chlamydosin.
gulated).

FIG. 5A shows the profile of Lin, Thy-1, c-Kit, and Sca-1 expression in viable mouse HSCs analyzed after 4 days of culture in medium alone.

FIG. 5B shows the profile of Lin, Thy-1, c-Kit, and Sca-1 expression in surviving mouse HSCs analyzed after 4 days of culture in medium supplemented with TSA.

FIG. 5C shows the profile of Lin, Thy-1, c-Kit, and Sca-1 expression in surviving mouse HSCs analyzed after 4 days of culture in chlamydosin supplemented media.

FIG. 6 shows engraftment of NODSCID bone marrow of MPB CD34 ⁺ cells incubated for 24 hours in TPO, FL and KL with chlamydosin.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 15/09 C12N 5/00 E // A61K 35/12 B (C12N 5/06 15/00 A C12R 1 : 91) (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA ( BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY , CA, CH, CN, CR, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Hill Beth Louise United States of America California 94087 Sunnyvale Quintile Way 981 F-term (Reference) 4B024 AA01 CA04 DA03 EA02 EA04 GA11 HA17 4B065 AA93X AB01 AC20 BA02 BA24 BB19 BD25 BD41 CA24 CA44 4C084 AA13 ZA51 ZB07 4C087 AA01 ZA07 ZA07 Z

Claims (34)

[Claims] 1. a) obtaining a population of hematopoietic cells containing a subpopulation of hematopoietic stem cells from a source of hematopoietic cells; b) culturing the hematopoietic cells under growth-supporting conditions; A method for promoting self-renewing division of hematopoietic stem cells, comprising exposing the stem cells to self-renewing division, and exposing the stem cells to a self-renewing hematopoietic stem cell composition. 2. The method of claim 1, further comprising, prior to step b), obtaining an enriched hematopoietic stem cell population. 3. The method of claim 1, further comprising growing the self-renewing cell composition. 4. The method of claim 1, further comprising selecting a hematopoietic stem cell subpopulation from the cultured cells.
A method according to any one of claims 1 to 3. 5. The method according to claim 1, further comprising genetically modifying the hematopoietic stem cells by contacting the gene delivery vehicle containing the polynucleotide with hematopoietic stem cells exposed to a histone deacetylase inhibitor. The method according to any one of the preceding claims. 6. The method according to claim 1, wherein the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, trapoxin, chlamydosin, sodium butyrate, and dimethyl sulfoxide. Method. 7. A self-renewing cell composition obtainable by the method according to claim 1. 8. An isolated mammalian hematopoietic cell containing a subpopulation of self-renewing engrafted cells; b) an effective amount of one or more histone deacetylase inhibitors;
And c) a culture comprising an effective amount of a cytokine that supports growth and self-renewal, wherein the effective amount of a histone deacetylase inhibitor promotes self-renewal division of the engrafted cells in the culture. 9. The culture according to claim 8, wherein the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, trapoxin, chlamydosin, sodium butyrate, and dimethyl sulfoxide. 10. A method of promoting self-renewal division of stem cells, comprising treating a stem cell population with an effective amount of one or more histone deacetylase inhibitors, and allowing the stem cells to self-renew. 11. The method of claim 10, wherein the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, trapoxin, chlamydosine, sodium butyrate, and dimethyl sulfoxide. 12. The method according to claim 10, wherein the stem cell is a hematopoietic stem cell characterized as C-enriched D34 ⁺ Thy-1 ⁺ . 13. m) treating the stem cells in culture with an effective amount of a histone deacetylase inhibitor; n) introducing a heterologous gene into said cultured stem cells by retrovirus-mediated transfer; and o) said stem cells. A method for producing transduced mammalian stem cells, comprising exposing the transduced stem cells to substantially the same conditions except that the number of transduced stem cells is not treated with the histone deacetylase inhibitor. The above method, wherein the number is increased. 14. The method according to claim 13, wherein the stem cells are hematopoietic stem cells. 15. The method of claim 13, further comprising isolating the transduced stem cell population. 16. The method of claim 13, 14, or 15, further comprising administering to the mammalian subject an effective amount of the transduced stem cell population. 17. The method of claim 16, wherein said stem cells are allogeneic to said subject. 18. The method of claim 16, wherein said stem cells are autologous to said subject. 19. A method comprising the steps of: s) contacting a gene delivery vehicle containing a polynucleotide with a stem cell population cultured in the presence of an effective amount of a histone deacetylase inhibitor, and t) obtaining a genetically modified stem cell. How to genetically modify stem cells. 20. The method according to claim 19, wherein the gene delivery vehicle is a retrovirus vector, a DNA virus vector, live DNA or a liposome delivery vehicle. 21. The method of claim 19, wherein the polynucleotide encodes a therapeutic gene. 22. The method of claim 19, 20 or 21, further comprising expanding a genetically modified stem cell population. 23. The method of claim 19, 20 or 21, further comprising isolating a genetically modified stem cell population. 24. The method of claim 19, 20 or 21, further comprising administering an effective amount of the genetically modified stem cell population. 25. Transducing the hematopoietic stem cells by contacting a) a population of hematopoietic stem cells with an effective amount of a histone deacetylase inhibitor and b) exposing the hematopoietic stem cells to a vector containing a nucleic acid sequence encoding a therapeutic gene. And c) administering a transduced hematopoietic stem cell population in an effective amount to the subject to restore hematopoietic ability. 26) exposing the stem cells in culture to an effective amount of a histone deacetylase inhibitor; aa) introducing a therapeutic gene into said cultured stem cells; bb) exposing no histone deacetylase inhibitor Increase the number of genetically modified stem cells
cc) and a method for improving engraftment of genetically modified mammalian stem cells, comprising administering an effective amount of the modified cells to a subject. 27. The method according to claim 26, wherein the cells are hematopoietic stem cells. 28. The method of claim 27, wherein the stem cells administered to the subject are allogeneic. 29. The method of claim 27, wherein the stem cells administered to the subject are autologous. 30. A stem cell trait comprising dd) exposing a population of stem cells to an effective amount of a histone deacetylase inhibitor, ee) transducing said stem cells with a heterologous gene, and ff) obtaining transduced cells. The method of introduction, wherein the number of transduced stem cells is greater than the number of transduced stem cells grown under substantially the same conditions except that the transgenic stem cells are not exposed to an effective amount of the histone deacetylase inhibitor. Method. 31. The method of claim 30, wherein the cells are transduced in a medium that supports the survival and growth of the stem cells. 32. The method of claim 30, wherein the cells are exposed in vivo to a histone deacetylase inhibitor. 33. A medium comprising IL-3, IL-6, soluble IL-6 receptor, LI
32. The method of claim 31, wherein the method is selected from the group consisting of F, TPO, KL, and FL alone, and combinations thereof. 34. hh) a stem cell population, ii) an effective amount of one or more histone deacetylase inhibitors, jj) an effective amount of one or more growth-supporting cytokines, and kk) a marker gene or a therapeutic gene. A culture comprising a gene delivery vehicle containing the encoding polynucleotide.