CA2346152A1 - Promotion of self-renewal and improved gene transduction of hematopoietic stem cells by histone deacetylase inhibitors - Google Patents

Abstract

A method of promoting stem cell self-renewal is disclosed which comprises exposing a population of stem cells, particularly hematopoietic stem cells, to an effective dose of a histone deacetylase inhibitor, particularly trichostatin A, trapoxin, or chlamydocin. The invention is also directed to the use of histone deacetylase inhibitors to increase the number of transduc ed stem cells.

Description

WO 00/23567 ' PCT/EP99/07741 PROMOTION OF SELF-RENEWAL AND IMPROVED GENE
TRANSDUCTION OF HEMATOPOIETIC STEM CELLS
BY HISTONE DEACETYLASE INHIBITORS
This invention relates to the field of self renewal divisions and transduction of stem cells, particularly hematopoietic stem cells (HSCs).
The maintenance of the hematopoietic system relies on primitive pluripotent hematopoietic stem cells that have the capacity to self renew and repopulate all the blood cell t.o lineages with relevant progenitor cells. Due to their capacity for self renewal and their pluripotent, long term reconstituting potential, HSCs have long been considered ideal for transplantation to reconstitute the hematopoietic system after treatment for various hematologic disorders or as a target for the delivery of therapeutic genes.
Additionally, human HSCs have potential applications in restoring the immune system in autoimmune diseases and ;t5 in the induction of tolerance for alogenic solid organ transplantation.
Early in hematopoiesis, a pluripotent stem cell differentiates and gives rise to either lymphoid, myeloid, or erythroid restricted cells. These cells in turn differentiate into progenitor cells that are committed to a specific cell type. Evidence indicates these progenitor ?0 cells have lost the capacity for self-renewal and contribute little if anything to engraftment following transplantation. It appears that infused HSCs are necessary for short term and sustained engraftment. Furthermore, the kinetics of engraftment is proportional to the number of infused HSCs. This is particularly true in allotransplantation settings.
Therefore attempts to improve graft quality may be accomplished if there is an increase in HSC
number.
:zs Additionally, increase in HSC number by culturing in vitro will allow improved therapeutic approaches for the treatment of many diseases including cancer, autoimmune and certain genetic diseases.
HSCs are generally obtained from bone marrow, mobilized peripheral blood and 3o umbilical cord blood. Presently large volumes of bone marrow must be extracted and frozen to ensure that sufficient number of stem cells are present to reconstitute the hematopoietic system. Additionally, single cord blood donors provide insufficient numbers of hematopoietic stem cells for adult therapeutic use. Therefore, methods developed for promotion of HSC
self renewal as distinguished from stem cell expansion could provide a tremendous advantage for therapeutic uses.
Since the 1980's, numerous discoveries have been made concerning the influence of growth factors, such as interleukins and cytokines, on hematopoietic cells.
These hormones exert a profound effect on the growth and differentiation of hematopoietic cells. Some of these molecules include 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). In an attempt to achieve optimal production of progenitors during in vitro culture, these factors have been tested in many combinations. A number of major breakthroughs in stem cell replication came with the discovery of c-kit ligand and a ligand for the fetal liver kinase receptor, fik2/flk3 ligand (FL). Both of these cytokines synergize with other cytokines in vitro and have the effect of sustaining viability and recruiting very primitive hematopoietic cells into cycle without increasing their differentiation. Additionally, the molecule thrombopoietin (TPO) which promotes megakaryocytopoiesis was discovered to have a potent effect on the viability 2o and replication of both human and murine primitive hematopoietic progenitor cells in vitro.
Other culture conditions and additives have been studied with the hope of optimizing HSC
replication. These aspects of t:he culture system include the culture media, the surface for adhesion, a.nd schedules for replacement of gases and nutrients. The role of stroma cells has also been investigated in stem cell self renewal technology. Cell adhesion molecules expressed on the surface of primitive cells, such as VLA-4, VLA-5 and L-selectin have been explored for effects on stem cell cycling. Specific receptor-ligand interactions between stromal cells and HSCs have been identified that appear to be critical for stem cell maintenance.
While the manipulation of culture conditions is one approach to promote HSC
self 3o renewal, the present invention is concerned with the transcriptional regulation of genes involved in self renewal through l:he modulation of histone acetylation.
In eukaryotic cells, DNA molecules are tightly complexed with histone proteins to form chromatin, and changes :in chromatin structure are known to be of fundamental importance in regulation of gene. expression. Histones are rich in arginine and/or lysine, and are classified in five major groups designated H1(lysine-rich), H2A and H2B
(slightly lysine rich), and H3 and H4 (arginine rich). These proteins have two domains, the histone fold domain and the amino tail domain. The amino tail domain interacts with DNA at specific sites between the positively charged arginine/lysine residues and the negatively charged phosphate groups of the DNA. At specific: points in the cell cycle, the amino tail domain undergoes transient modifications including acetylation. The histone-modifying enzymes, particularly acetyltransferases and deacetylases, are known to play active roles in the transcription and assembly of chromatin. While not meant to be limiting, the prevailing scientific view is that a correlation exists between the level of histone acetylation and deacetylation and 't5 transcriptional activity, and that histone acetylation leads to a decondensation of chromatin which facilitates the access of transcriptional factors and components of the basal transcriptional machinery to DNA. (Grunstein, Michael, Nature 389:349 - 352, {1997); Wade, P. et al., TIBS 22:128 - 132, (1997)). It is also believed that some transcriptional cofactors (activators) have histone acetyl transferase activity, and therefore these coactivators could z0 direct local destabilization of repressive histone-DNA interactions.
Conversely, a growing number of transcriptional repressors have been found to act through the recruitment of histone deacetylases (HDACs). Several repressors of transcription are involved in the mechanisms that govern cellular differentiation and proliferation and have 25 been implicated in normal hematopoiesis or leukemogenesis. Some of these repressors are briefly mentioned.
Retinoid receptors are involved in myeloid maturation but may also play a critical role 30 in the development of pluripotent hematopoietic stem cells (Tsai, et al.
Genes & Dev. 8: 2831 (1994)). While these receptors l~~have as transcriptional activators when bound to their ligand, in the non-ligand state they repress the basal transcriptional activity by associating with the transcriptional corepressors SMRT and N-CoR. Nagy, et al. has shown that the SMRT
transcriptional corepressor exerts its activity by forming a multisubunit repressor complex in which the histone deacetylase 1 (HDAC1) is recruited. (Nagy, et al. Cell, 89:

(1997)) It was confirmed that lnistone deacetylation is fundamental for retinoid signaling by showing that the deacetylase inhibitor trichostatin A (TSA) could greatly enhance the biological activity of retinoic acid. This was demonstrated using the HL60 promyelocytic cell line that undergoes myeloid maturation in response to retinoic acid. In the presence of sub-optimal doses of retinoic acid, a full differentiative response of the cells was achieved by simultaneous treatment with TS.A.
Promyeiocyctic leukemia zinc finger (PLZF) protein is predominantly expressed in the most primitive hematopoietic compartment and has been shown to inhibit cell cycle of hematopoietic cells (Mol. Cell Biol.18: 5533( 1998)). PLZF is a DNA binding protein that represses transcription by interacting with either SMRT or N-CoR which recruit histone deacetylases (Guidez, et al. Blood 91: 2634 {1998)) and Lin, et al. Nature 391: 811 (1998)).
Activation of myc by genomic alterations resulting from chromosomal rearrangements or retroviral insertion occurs iti human, rodent and avian leukemias and lymphomas. MYC
proteins are expressed in proliferating cells and are down regulated upon cell-cycle withdrawal or differentiation. MYC is part of a transcriptional network that involves other factors. MYC
can dimerize with a protein called MAX which can bind to DNA and to the protein MAD.
MYC-MAX dimers are transcriptionally active, whereas MAD-MAX dimers repress transcription. It has been shown that MAD-MAX recruits histone deacetylases and that this interaction is the basis for the IV4AX-MAD repressive activity. TSA has been shown to abolish the repression. (Laherty, et aL t:ell 89:349 - 356 (1997)).
Another co-repressor whose activity can be modulated by histone deacetylase inhibitors is the mammalian CBFI/RBP-Jx which switches from a transcriptional repressor to an activator upon Notch activation. In the absence of Notch activation CBF1/RBP-Jx represses transcription by bindvig to SMRT/HDACI, while Notch activation disrupts the formation of the repressor complex. TSA treatment can induce the transcription of CBFl regulated genes. (Genes and Dev., I2: 2269-2277 ( 1998)). Notch signaling has been shown to influence the development of primary primitive hematopoietic precursor cells in vitro (Blood 1998, 91: 4084-4091) and overexpression of the activated Notchl in the 32D
myeloid cell line inhibits differentiation and perrruts expansion of undifferentiated cells, findings consistent with the known function of Notch in Drosophila (PNAS 1996, 93:13014-9).
In a recent review article, it was disclosed that the HDAC inhibitor, trichostatin A
(TSA) induced a variety of biological responses in cells including induction of differentiation and cell cycle arrest. (Yoshid.a, et al., BioEssays 17:42-429( 1995)).
Additionally various inhibitors of HDAC have been implicated in triggering terminal differentiation of malignant cells. (Richon, et al., Proc. Natl. Acad. Aca. USA, 95:3003-3007 (1998)).
While the literature di.~;closes the use of HDAC inhibitors in studies to reverse the effects of HDAC and HDAC complexes in the repression of transcription, the literature does not disclose a method of using HDAC inhibitors to promote hematopoietic stem cell cycling, to promote HSC self renewal divisions (or replication), nor to enhance maintenance of primitive cell function during gene integration over gene integration in the absence of histone deacetylase inhibitors.
In one aspect of the invention, a method of promoting self renewal division of hematopoietic stem cells is provided, comprising obtaining a population of hematopoietic cells from a source of hematopoietic cells, wherein the obtained hematopoietic cells include a subpopulation of hematopoietic stem cells; culturing the hematopoietic cells under growth supporting conditions; exposiing the cultured cells to an effective amount of a histone deacetylase inhibitor wherein self-renewal divisions of the stem cells is promoted; and obtaining a composition of thc: self-renewed hematopoietic stem cells. The method may also include a further step comprising obtaining a population of enriched hematopoietic stem cells from the hematopoietic cells prior to culturing the stem cells. Preferably the method includes selection of enriched human hematopoietic stem cells from the phenotype group consisting of CD34+; Thy-1+; CD34'Thy-I+; CD34-Thy-1+; CD34+EM''; CD3f'Thy-1+Liri ;
CD3f"CD38'°'-; CD34'Thy-1+CD381°~- ; CD34'Thy-1~'EM+; and CD3f'Thy-1+CD381°~-EM+. In one embodiment, the invention includes the composition of self renewed cells produced by the method defined above.
1. In another aspect of the invention, a culture is provided, comprising isolated 1o mammalian hematopoifaic cells which include a subpopulation of engrafting cells capable of self renewal, an effective amount of one or more histone deacetylase inhibitors, and effective amounts of growth and self renewal supporting cytokines wherein the effective amount of the histone deacetylase inhibitor promotes self renewal division of the engrafting cells in the culture. In one preferred embodiment, the hematopoietic cells are human. In another prefered embodiment, the histone deacetylase inhibitor is selected from the group trichostatin A, trapoxin, chlamydocin, sodium butyrate or dimethyl sulfoxide.
In a further aspect of the invention, a method of promoting self renewal division of 2o stem cells is provided, comprising treating a population of stem cells with an effective amount of one or more histone deacetylase inhibitors and allowing the stem cells to self renewal. In one embodiment the stem cells are enriched hematopoietic stem cells characterized as CD34'Thy-1+.
In an additional aspect, the invention provides a method of generating transduced mammalian stem cells comprising treating stem cells in a culture with an effective amount of a histone deacetyiase inhibitor, :introducing a gene into the cultured stem cells using retroviral mediated transfer, and allowing transduction of the stem cells wherein the number of transduced stem cells is increased over the number of transduced stem cells exposed to 3o substantially the same conditions but in the absence of treatment with the histone deacetylase _7_ inhibitor. In a preferred aspect, the stem cells are hematopoietic stem cells, preferably human hematopoietic cells, and the gene is a therapeutic gene. The method is further comprised of administering an effective amount of a population of the transduced stem cells to a mammalian subject, preferably, a human subject. In one embodiment the stem cells are allogeneic or xenogeneic to the subject anti in another embodiment the stem cells are autologous to the subject.
In yet a further aspect, the invention provides a method of genetically modifying stem cells by contacting a gene delivery vehicle comprising a polynucleotide with a population of l0 stem cells cultured in the presence of an effective amount of a histone deacetylase inhibitor, and obtaining genetically modified stem cells. In a preferred embodiment, the delivery vehicle is a retroviral vector, a lentivira vector, an adenoviral vector or a liposome delivery vehicle.
In yet a further aspect., the invention provides a method of restoring hematopoietic capability in a subject, comprising contacting a population of hematopoietic stem cells with an effective amount of a histone deacetylase inhibitor; transducing the hematopoietic stem cells by exposing the hematopoietic stem cells to a vector including a nucleic acid sequence encoding a therapeutic gene; and administering an effective amount of a population of the transuded hematopoietic stem cells to a subject wherein hematopoietic capability is restored.
In another aspect, the invention provides a method of improving engraftment of genetically modified mammalian stem cells comprising exposing stem cells in a culture to an effective amount of a histone deacetylase inhibitor, introducing a heterologous gene into the cultured stem cells, generating an increase in the number of genetically modified stem cells over that in the absence of exposure to a histone deacetylase inhibitor, and administering the modified cells to a subject. In one embodiment, the stem cells are allogeneic and in another embodiment, the stem cells are autogolous. Preferably the heterologous gene is a therapeutic gene.
A further aspect of the invention provides a method of transducing stem cells WO 00/Z3567 PCT/EP9910774i _g_ comprising exposing a population of stem cells to an effective amount of a histone deacetylase inhibitor; transducing the stern cells with a foreign gene; and obtaining transduced cells wherein the number of transduced stem cells is increased over the number of transduced stem cells grown under substantially the same conditions but in the absence of exposure to an effective amount of a histone deacetylase inhibitor. In one embodiment the cells are transduced in culture and in a second embodiment the cells are transduced in vivo. In a further embodiment the stem cells arc: hematopoietic stem cells, particularly mice or human. In yet another embodiment, the cells are transduced with a retroviral vector derived from Moloney murine leukemia virus or murvae stem cell virus.
l0 In yet an additional aspect, the invention provides a culture comprising a population of stem cells; an effective amount of one or more histone deacetylase inhibitors;
an effective amount of one or more growth supporting cytokines; and a gene delivery vehicle including a polynucleotide encoding a marker gene or therapeutic gene. In one embodiment the gene delivery vechicle is a vector derived from a retrovirus, adenovirus or adeno-associated virus.
Figure 1 illustrates the increased number of human hematopoiefic progenitor cells expressing the Thy-1 antigen from a culture of MPB CD34+ cells exposed to HDAC-inhibitors.

Figures 2A and 2B illustrate that a culture of human MPB CD34+ cells exposed to the HDAC inhibitor chlamydocin increases total CAFC activity within the culture relative to cultures without chlamydocvi. Figure ZA shows the total CAFC activity among Thy-1+ cells and Figure 2B shows the tota CAFC activity among cells.
Figure 3 illustrates that a larger proportion of human CD34+ hematopoietic progenitor cells cultured with chlamydocin retain expression of the Thy-1 antigen throughout 4 divisions in culture (bottom) than cells cultured without HDAC-inhibitors (top).
Figure 4 illustrates that the proportion of Thy-1+ cells expressing the myeloid lineage antigen CD 15 is not upregulat~ed on human CD34+ MPB cells cultured with HDAC-inhibitors.
Figures SA, 5B and 5C: illustrate the profile of Lin, Thy-1, c-kit, and Sca-1 expression among viable murine HSCs analyzed four days after growth in media alone (A) or media supported with TSA (B) or chlamydocin (C).
Figure 6 illustrates engraftment in NOD SCID marrow of MPB CD34+ cells cultured in TPO, FL, and KL with 24 hour incubation with Chlamydocin.
1o The following abbreviations used through out the disclosure are listed herein below:
FITC = fluorescein TPO = thrombopoietiti FL = Flt3 ligand KL = c-kit ligand IL = Interleukin LIF = leukemia inhibitory factor MPB = mobilized peripheral blood CFSE = carboxyfluorescein-diacetate succinimidylester HSC = hematopoietic stem cell TSA = Trichostatin A
DMSO = dimethyl sulfoxide FBS = fetal bovine serum IMDM = Iscove's modified Dulbecco's medium HDAC = histone deacetylase HDAC-I = histone deacetylase inhibitor CAFC = cobblestone-area-forming cell PE = phycoerythrin CM = culture medium 3o APC = allophycocyansn EPO = erythropoietin FN = fibronectin fragment CH296 FACS = fluorescence-activated cell sorter As used herein the term "stem cell" includes stem cells of various cell types, such as, muscle, epithelial, neural and bone stem cells. More particularly the invention is concerned with hematopoietic stem cells. The term "hematopoietic stem cell" (HSC) refers to mammalian and avian hematopoietic stem cells and means a population of hematopoietic cells containing the engrafting potential for in vivo therapeutic application.
Hematopoietic cells encompass not only I3SCs, but ;also erythrocytes, neutrophils, monocytes, platelets, mast cells, eosinophils and basophils, B and T lymphocytes and NK cells as well as the respective lineage progenitor cells. Stem cells ma.y also be defined in vitro by the presence of CAFC activity.
Animal models for long term engrafting potential of candidate human hematopoietic stem cell populations include the SCID-hu bone model (Kyoizumu, et al., Blood 79:1704 (1992); and Murray et al., Blood 85: 368 - 378 (1995) ), the in utero sheep model (Zanjani, et al., J. Clin.
Invest. 89:1179 ( 1992)), NOD SCID (Larochelle, et al., Nature Medicine 2:

(1996) and Conneally, et af, Proc. Natl. Acad. Sci. 94:9836 - 9841 (1997)) and primate models (Kiem, et al., Blood 90:4630- 4645 (1997) and Srour, et al., J.
Hematother 1:143 -153 (1992)). A mouse hematopoietic stem cell has been obtained in at least highly concentrated form where fewer than about 30 cells obtained from bone marrow were able to reconstitute all of the lineages of the hematopoietic system of a lethally irradiated mouse. Each assayed cell was multipotent for all hematopoietic lineages.
An engrafting cell mews any cell that can lodge in a hematopoietic tissue and function to repopulate blood cell linea,ges. Additionally, as used in the specification and claims, the singular form "a", "an", and "l:he" include plural references unless the context clearly dictates otherwise. For example, a stem cell includes a plurality of cells. The term "self renewal" or "self renewal divisions" also referred to as "replication", is defined herein to mean cell division without apparent cell differentiation or without loss of cell engrafting capacity. The term "expansion" is intended to mean allowance of progenitor cells to increase in number and differentiate from the pluripotE:nt stem cells used to initiate the culture.
As used herein the term "cytokine" refers to any one of the numerous factors that exert a variety of effects on cells, for example, inducing growth or proliferation. The cytokines may be human in origin, or may be derived from other species when active on the cells of interest. Included within the scope of the definition are molecules having similar biological activity to wild type or purified cytokines, for example produced by recombinant means; and molecules which bind to a cytolcine factor receptor and which elicit a similar cellular response as the native cytokine factor.

Non-limiting examples of cytokines which may be used alone or in combination in the practice of the invention include, but are not limited to, IL-2, IL-3, IL-6, IL-12, IL-la, IL-11, KL, (designated interchangeably with c-kit ligand, stem cell factor (SCF), steel factor (Stl) and mast cell growth factor (MCGF)), granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), TPO, LIF, FL, and MIP-la.
As combinations, fused proteins may be employed, where the two factors are fused together or a cytokine is fused to its soluble receptor. The order of fusion will not be critical so long as the two portions of the molecules act independently and provide for their biological function.
l0 A non-limiting example includes combinations of IL-3 and IL-6 (Broxmeyer, et al, Exp.
Hematol. 18:615 ( 1990). The present invention also includes culture conditions in which one or more cytokines is specifically excluded from the medium. Cytokines are commercially available from many vendors such ass R & D Systems Inc., (Minneapolis, MN), Genzyme Diagnostics (Cambridge, MA) and Genentech (South San Francisco, CA).
The term "culturing" refers to the propagation of cells or organisms on or in media of various kinds.
An "effective amount" of a cytokine is an amount sufficient to promote survival, 24 growth, and/or division of stem cells, particularly hematopoietic stem cells. "Survival" is defined herein as the ability to continue to remain alive or function.
As used herein the term "mammal" includes but is not limited to humans, mice, monkeys, farm animals, sport animals, pets, and other laboratory rodents and animals.
Preferably the term refers to humalns.
"Histone deacetylase activity" or " the activity of a histone deacetylause protein" refers to the biochemical activity associated with histone deacetylase proteins. The biochemical activity includes binding to and optionally catalyzing the deacetylation of am acetylated histone; this may result in mediating the activity of transcriptional co-repressors such as, but not limited to, SMRT and N-CoR. In general, compounds that inhibit HDAC have been shown to result in activation of gene expression.
An effective dose or amount of a histone deacetylase inhibitor is defined as the amount of a HDAC-I which increases the number of self renewal divisions of stem cells within a population of cells, particularly hematopoietic cells wherein overall cell viability may or may not be reduced and as a result increases the number of stem cells available for genetic modification as compared to stem cells within a popluation of cells grown under essentially the same conditions but in the .absence of HDAC-I. .
As used herein, the term "genetic modification" refers to any addition, deletion or disruption to a cell's normal nucleotides. The method of this invention is intended to encompass any genetic modification method of exogenous or foreign gene transfer (or nucleic acid sequence transfer) into stem cells (preferably into hematopoietic stem cells). The term ~5 "genetic modification" encompasses use of a gene delivery vehicle and includes but is not limited to transduction (viral mediated transfer of nucleic acid to a recipient, either in vivo or in vitro), transfection (uptake by cells of isolated nucleic acid), liposome mediated transfer and others means well known in the art.
20 A "therapeutic gene" is defined herein as an entire gene or only the functionally active fragment of the gene. The therapeutic gene may be capable of compensating for a deficiency in a patient that arises from a defective or absent endogenous gene.
Additionally, a therapeutic gene may be one that antagonizes production or function of an infectious agent, antagonizes pathological procEases, improves a host's genetic makeup, facilitates engraftment, 25 or a stem cell's therapeutic potency.
As used herein, the term "retroviral mediated gene transfer" and "retroviral transductian" are used interchangeably.
3o Methods of obtaining hematopoietic cells and stem cells are well known in the art and include isolation of the stem cells from other cells in hematopoietic tissues of the body and particularly bone marrow. Ste;m cells from bone marrow appear to be in the range of about 0.01 to about 0.1 % of the bone marrow cells. Bone marrow cells may be obtained from ilim, sternum, tibiae, femora, spine or other bone cavities. Other non-limiting sources of s hematopoietic stem cells include embryonic yolk sac, fetal liver, fetal and adult spleen, blood, including adult peripheral blood and umbilical cord blood. (To, et al., Blood 89:2233-2258 (1997)).
For the isolation of bane marrow an appropriate solution may be used to flush the bone, including but not limited to, salt solution, supplemented with fetal calf serum or other naturally occurring factors in conjunction with an acceptable buffer at low concentrations, generally about 5 to 25 mM. Buffers include but are not limited to HEPES, phosphate and lactate buffers. Bone marrow can also be aspirated from the bone in accordance with conventional techniques.
1s The manner in which stem cells may be separated or selected from other cells is not critical to this invention. Various procedures which may be employed include magnetic separation using antibody coated magnetic beads, affinity chromatography, and cytotoxic agents joined to a monoclonal antibody. Also included is the use of fluorescence activated cell sorters (FACS) wherein the ce;lls can be separated on the basis of the level of staining of the particular antigens. These techniques are well known to those skilled in the art and reference is made to U.S. Patent Nos. 5,()61,620; 5,409,8213; 5,677,136; and 5,750,397;
and Yau, et al., ( 1990) Exp. Hematol. 18:219-222.
The order of separation is not critical to this invention. However, preferably cells are initially separated by a coarse separation followed by separation using positive and/or negative selection. In such positive selE:ctions, up to about 15%, usually not more than about 10%, preferably not more than about 5% and most preferably not more than about 1%
of the total cells in the retained cell population will lack the marker used for separation. In some instances it may be desirable to directly treat a hematopoietic cell population with an effective dose of a histone deacetylase inhibitor without first separating a subpopulation of HSCs from the hematopoietic cells.
Stem cells, which constitute only a small percentage of the total number of hematopoietic cells are characterized by both the presence of markers associated with specific epitopic sites identified by antibodies (positive selection) and the absence of certain markers as identified by the lack of bindnng of specific antibodies. All of the monoclonal antibodies (mAbs) that react with a particular membrane molecule are grouped together as a cluster designator (CD). Lin~ cells refer to a cell population selected on the basis of the lack of 1o expression of at least one lineage specific marker. For example, those human cells which lack markers associated with T cells (such as CD2, CD3, CD4, and CD8), B cells (such as CD10, CD 19 and CD20), myeloid cells; (such as CD 14, CD 15 and CD33), natural killer cells (such as CD2, and CD56), and the like:.
Human stem cells may be characterized by the following non-limiting phenotypes:
CD2 , CD3 , CD4 , CD7 , CD8 , CD 10 , CD 14 , CD 15 , CD 19 , CD20 , CD33 , CD34 , CD34+, CD38j°/ , CD45RA , C:D59+/ , CD71 , CDW109+, glycophorin , Thy-1''(CD90), HLA-DR+/ , AC133+, rhodamine 123 (rho123~°) or a combination thereof.
Monoclonal antibodies to the molecule Thy-1 in combination with mAbs to CD34 have been used to isolate murine, non-human primate and human HSCs. USP 4,714,680 describes a population of cells expressing the CD34 marker. More recently other mAbs have been identified for positive selection of human stern cells. These are designated EM, specifically, EMS, EM10 and EM16 (Chen, et al., Immunological Reviews, 157: 41 - 51, 1997). As used herein EM+
means an EM phenotype that expresses any EM marker.
:!5 In one preferred embodiment the surface antigen expression profile of an enriched hematopoietic stem cell population will be selected from the following group, CD34+; Thy-1'';
CD10-; CD19-; CD15~; CD33-; CD34''Thy-1+; CD34 Thy-1+; CD34''EM+;
CD34+CD381°~-;
CD34''Rhol2s; CD33 Thy-1+; CDl9~Thy-1+; CD34''Thy-1'Liri ; CD34''Thy-1'EM+;

WO 00/23567 ' PCT/EP99/07741 CD34+CD45RA CD71 ; HLA-DR+/-CD34'' Thy-1+I,iri ; CD34iThy-1+CD381°~'; and CD34;'Thy-1+CD381°~~EM+. In .a more preferred embodiment the expression profile of an enriched population will be selected from CD34''; Thy-1+; CD34~Thy-1+; CD34 Thy-1+;
CD34~EM+; CD34+CD381°/-; C'D34'Thy-1+Liri ; CD34'"Thy-1+EM+; CD34''Thy-1+CD381°~-;
and CD34+'Thy-1+CD381°~-EM+, particularly when EM+ is EM10+. The combination of markers used to isolate and define an enriched HSC population may vary as other markers become available.
Generally the number of cells obtained after positive selection will be fewer than about 20% of the original cells, more frequently fewer than about 10%, generally fewer than 5%, most frequently fewer than 1.0 io, and may be as low as 0.2% or less.
Compositions having greater than 80%, usually greater than about 90% of human stem cells may be achieved by the enumerated separation techniques when the desired stem cells are identified by being CD34i"Thy-1 ~''L,iri .
is Murine (m)HSCs are included in the Lin°°gn° population which corresponds to cells that express at the most dim levels of a lineage specific marker. For example those cells which lack expression of, or express love levels of T lymphocytes markers (such as CD2, CD3, CD4, CDS
20 or CD8), B lymphocytes markers (such as CD19 or B220), myeloid cells markers (such as Gr-1 or Mac-1=CDllb), natural killer markers (such as NK1.1) or erythroid markers (such as Ter119).
Murine HSCs may be ch~uacterized by the following non-limiting phenotypes: CD2-;
2.5 CD3-; CD4' ; CD4'°'"; CDS- ; CD8- ; CD19- ; B220-; Gr-1-; Mac-1-;
Mac-1'°; NK1.1-;
Ter119~; Ter119'°; Sca-1+;Thyl.l''; c-Kit - or c-Kit~"~"; CD34-; CD34+;
CD38 ~'~'; CD43~'~';
H-2K ~'~'; or AA4.1+. The combined use of monoclonal antibodies and fluorescent vital dyes such as the mitochondria-binding dye Rhodamine 123 (Rho-123) or the DNA-binding dye WO 00/23567 ~ PCT/EP99/07741 Hoechst 33342 can increase the enrichment of HSC in a sorted population. The most primitive cells are enriched in the Rho-12:3'° fraction as well as in the Hoechst 33342'° fraction.
In a preferred embodiment, an enriched murine HSC population is characterized by Thy-1'°; Liri "°; Sca-1+; and c-lit+. In a more preferred embodiment the enriched population is sorted by or selected from the phenotypes Thy-1'°""Lin"° Sca-1+; c-Kit'Lin"°Sca-1+; c-Kit+Sca-1+or c-Kit+Thy-llowL~..n°Sca-1+.
In mice HSC can be largely separated, in vitro or in vivo, from more mature to progenitors by their differential sensitivity to cytotoxic drugs such as 5-fluoruracil. Further separation of murine HSC can be achieved using counterflow centrifugal elutriation (CCE) on the basis of cell size and density; the small cell subset obtained by CCE at a flow rate of 25mL./min contains HSC with long term repopulating activity and very few progenitors (Jones, et al. Nature 347:188 ( 1990)).
:l5 Once hematopoietic cells, are harvested, and HSCs optionally separated, the cells are cultured in a suitable medium under growth supportive conditions which includes a combination of growth factors that are sufficient to maintain the growth of hematopoietic cells. Any suitable container, flack, chamber, bag, bioreactor with perfusion or appropriate 2 0 vessel such as a 24 well plate or the like can be used. Culture containers are readily available from commercial vendors. While the seeding level is not critical and will depend on the type of cells used, in general the seeding level will be at least about 10 cells per ml, more usually at least about 100 cells per ml and generally not more than about 2 x 106 cells per ml and usually not more than 105 cells per ml when the cells are CD34+.
Various culture media ca.n be used and non-limiting examples are Iscove's modified Dulbecco medium (IMDM), :K-vivo 15 (serum free), and RPMI-1640. These are commercially available from various vendors for example JRH BioSciences. The formulations can be supplemented with a variety of different nutrients, growth factors, cytokines and the 3~D like. The medium can be serum free or supplemented with suitable amounts of serum such as WO 00/23567 ' PCT/EP99/07741 fetal calf serum or autologous serum or plasma. Preferably, if the cells or cellular products are to be used in humans, the medium will be serum free or supplemented with autologous serum or plasma. In general, the medium will include effective amounts of cytokines, particularly TPO, KL, FL and ILs. One skniled in the art is aware of the various growth supporting culture media and culturing techniques for HSC's and reference is made to Lansdorp, et al., J. Exp.
Med. 175:1501 (1992) and Petzer, et al., PNAS 93:1470 (1996).
In one aspect the medium formulation is supplemented with TPO. A preferred concentration range is from albout 0.1 ng/mL to about 500 p.g/mL, more preferred is from about 1.0 ng/mL to about 1000 ng/mL, even more preferred is from about 5.0 ng/mL to about 300 ng/mL, and most preferred is from about 10.0 ng/mL to about 100 ng/mL.
Additionally, the medium will include Flt3 ligand (FL) and c-kit ligand (KL) each individually at a preferred concentration range from about 0.1 ng/mL to about 1000 ng/mL, more preferred from about 1.0 ng/mL to about 500 ng/ml,, and even more preferred from about 10 ng/mL to about 300 is ng/mL.
While various interleukins may be used to supplement the medium, IL-6 is preferred.
A preferred concentration range is from about 0.1 ng/mL to about 500 ng/mL, more preferred from about 1.0 ng/mL to about 100 ng/mL, and most preferred from about 5 nglmL
to about 2o SO ng/mL. Hyper IL-6, a high ;affinity or covalent complex of IL-6 and soluble IL-6 receptor, may also be used and reference is made to Conneally, et al., Ann. Meeting of the International Soc. for Exper. Hematology, Vancouver, Abst. #60 (1998).
Other cytokines may be added individually or in combination and include but are not 25 limited to IL-1, IL-2, IL-3, Il.-6, IL-12, IL-11, stem cell factor, G-CSF, GM-CSF, Stl , MCGF, LIF and MIP-la. When murine stems cells are cultured, a preferred non-limiting medium includes mIL-3, mIL-6 and mSCF. A preferred concentration range is from about O.I
ng/mL to about 1000 ng/mL, more preferred is from about 1.0 ng/mL to about 500 ng/mL, and most preferred is about 5.0 ng/mL to about 200 ng/mL.

In one embodiment, thc: cytokines are added to the media and replenished by media perfusion. Alternatively the cytokines may be added separately, without media perfusion, as a concentrated solution through separate inlet ports, for example when a bioreactor system is used.
Other molecules can be added to the culture media, for instance, adhesion molecules, such as fibronectin or RetroNc:ctinT"" (commercially produced by Takara Shuzo Co., Otsu Shigi, Japan). The term fibronec;tin refers to a glycoprotein that is found throughout the body and its concentration is particularly high in connective tissues where it forms a complex with collagen. RetroNectinT"" may bE; used at a concentration range from about 0.1 p g/ml to about 1000 pg/ml. A more prefered range is about 0.2 pg/ml to S00 pg/ml and a most prefered range is about U.4 pg/ml to 100 ~cg/ml.
HDAC-I compounds arc: also included in the culture medium and these compounds may be added individually or v1 combination, either prior to gene delivery or during gene delivery in an amount and under suitable conditions to allow stem cell self renewal divisions.
The following compounds are r~spresentative examples of HDAC-I that may be employed in the present invention.
The microbial metabolite known as trichostatin A (TSA) is a HDAC-I (Yoshida, et al., :?0 BioEssays, 17:42 - 429 ( 1995)). See EP 0 331 524 B 1 for methods of preparation of TSA.
Other compounds related to TSA include butyrate, particularly sodium n-butyrate; and hybrid polar compounds (HPCs), such as suberoylanilide hydroxamic acid (SAHA) and m-carboxycinnamic acid bishydroxamide (CBHA). These compounds have two polar groups separated by an apolar S- to 6- carbon methylene chain. Additionally the inhibitors appear to :!5 cause the accumulation of acetylated histone H4 in cells in culture.
(Richon et al., Proc. Natl.
Acad Sci. 95:3003 - 3007 (1998)).
Another known microbial metabolite which functions as a HDAC-I is trapoxin, a microbially derived cyclic tetrapeptide containing two L-phenylalanines, specifically Trapoxin 30 A (TPX). (See Yoshida, et al., BioEssays, 17:42 - 429 (1995)). Not only is the chemical WO 00/23567 ~ PCT/EP99/07741 structure of TPX completely different from TSA, the mode of inhibition is totally different from TSA and butyrate.
Cyclic tetrapeptide related compounds to TPX having the amino acid - 2-amino-8-oxo-9, 10-epoxy-decanoic acid in their molecules are included as histone deacetylase inhibitors. The following compounds are mentioned: chlamydocin (Closse, et al., Helv. Chim.
Acta 57: 533 - 545 ( 1974)), HC-toxin (Liesch, et al., Tetrahedron 38:45 - 48 ( 1982)); Cyl-2;
and WF-3161 (Umehara, K. J. Antibiot 36: 478 - 483 ( 1983). The fungal metabolite depudecin, (See Kwon, et aL, Proc. Natl. Acad Sci. USA, 95:3356 - 3361 ( 1998); and t0 radicicoI are also mentioned as histone deacetylase inhibitors. W097/35990 discloses many histone deacetylase inhibitor compounds and these are incorporated by reference.
In a preferred embodiment, the HDAC-I is selected from the group of TSA, TPX, chlamydocin, butyrate, particularly sodium n-butyrate, DMSO and HDAC-I analogs thereof.
i.5 While the concentration range of the HDAC-I used will vary and will depend on the specific inhibitor, a preferred concentration range will be 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 deactylase inhibitor is (1) TSA
provided at a 2,0 concentration range from about 0.01 ng/mL to 1000 ng/mL, more preferably from about 0.1 ng/mL to about 100 ng/mL, and most preferably from about 1.0 ng/mL to about 10 ng/mL, (2) TPX, provided at a concentration range from about 0.001 nM to about 100 nM, more preferably from about 0.01 nM to about 50 nM, and most preferably from about 0.1 nM to about 1.0 nM; and (3) chlamydocin, provided at a concentration range from about 0.001 nM
25 to about 100 nM, more preferably from about 0.01 nM to about 50 nM, and most preferably from about 0.1 nM to about 1.0 nM.
While specific histone deacetylase inhibitors have been defined and numerated herein, the scope of the invention includes a broad class of histone deacetylase inhibitors including 30 compounds not enumerated herein. One means of determining whether a compound not WO 00/23567 ' PCT/EP99/07741 enumerated herein falls within the class of histone deacetylase inhibitors includes but is not limited to using standard enzymatic assays derived from measuring the ability of an agent to inhibit catalytic conversion of a substance by the subject protein. In this manner, inhibitors of the enzymatic activity of histone deacetylase proteins can be identified. (See Yoshida, et al., J
Biol Chem. 265:17174-17179 (1990)). Compounds may be isolated by screening for detransforming activity using oncogene-transformed cells ( Kwon, et al., Proc.
Natl. Acad Sci. USA, 95:3356 - 3361 ( 1998). Furthermore, reference is made to Waterborg, et al., Analytical Biochem. 122:313-318 {1982) for an assay that measures acetate released by deacetylation of lysine from a peptide derived from a N-terminal sequence of histone H4.
This assay involves the extraction of tritiated acetyl groups with organic solvent. A more recent reference for HDAC assays is Kolle, et al., "Biochemical Methods for Analysis of Histone Deacetylases", Methods: A Companion to Methods of Enrymology 15:323-(1998). Reference is also made t:o W097/35990 for disclosure of an assay used to determine if a compound is an HDAC-I. Inhibitors of HDAC may function by different mechanisms and mention is made of TSA and TP:~C. In one embodiment of the invention, an effective amount of a HDAC-I is used to promote self renewal division of stem cells and particularly hematopoietic stem cells in culture as compared to stem cells cultured under essentially the same conditions, but in the absence of HDAC-I in the culture. "Grown or cultured under essentially the same conditions" means the cells are exposed to the same concentration of 2n growth supporting factors, the sane culture medium, and the same culture period.
Once the cells are exposed to HDAC-Is, the culture period will vary and will depend on the culture conditions. Under i:he appropriate conditions cells potentially could be cultured indefinitely. However, in general, cells will be cultured from about 1 to about 28 days, 2~> preferably from about 1 to about 14 days, more preferably from about 1 to about 7 days.
However, cells may be exposed for 1 to about 5 days or for 1 to about 3 days.
Mention is made that the culture period could be less than 1 day. While not meant to be a limitation of the invention, it takes in general cultured stem cells approximately 18 to 24 hours to undergo self renewal divisions. However, after 5 days some of the cultured cells may still be in the first 3G division. Therefore, cultured cells after 5 days may have completed various numbers of self WO 00/23567 ' PCT/EP99/07741 renewal divisions ranging from 1 to 7. After 5 days in culture the cells may divide at the approximate rate of one division per day or may return to a quiescent state.
In one embodiment, the cells will be cultured for about 1 to about 5 days prior to exposure to an effective amount of a HDAC-I. However, the HDAC-I may be added upon the initial culturing. During this time frame, a gene delivery vehicle such as a vector comprising a nucleic acid sequence encoding a therapeutic gene or a marker gene may be introduced into the culture by means well known in the art.
Ex vivo stem cell self renewal may be measured by various different means.
Three 1o general ways include in vivo assays; in vitro assays; and analysis of surface antigens. In vivo assays include the use of the SCID-hu mouse system. {McCune, et al., Science 241: 1632 -1639, 1988). In this system, human fetal bone fragments are implanted subcutaneously into severe combined immune deficient (SCID) mice. Once the implanted human fetal bone fragments are vascularized in the ;iCID-hu mouse, the marrow cavity can be injected with a test human cell population and analyzed for multilineage engraftment of the donor cells.
Recently other models have been developed using the beige/nude/XID(bnx) mouse and the nonobese diabetic SCID (NOD-SCID) mouse. (Greiner, et al., Stem Cells 16:166-177 (1998) and Nolta et al., Proc. Natl. AcaGl. Sci. USA 93:2414-2419 (1996)). The preferred way to evaluate mammalian non-human stem cell activity is by transplantation into irradiated hosts by 2o means well known to those skilled in the art.
In vitro systems for measurement of mammalian stem cell activity includes the long term culture initiating cell assay {LTCIC) and the cobblestone-area-forming cell (CAFC) assay. (Pettengell, et al., Blood 8~L:3653 ( 1994); Breems, et al., Leukemia 8:1095 ( 1994);
Reading, et al., Exp. Hem. 22:786 (Abst # 406) ( 1994); and Ploemacher, et al., Blood 74:2755 ( 1989)). In the CAFC assay a sparsely plated cell population is simply tested for its ability to form distinct clonal outgrowths (or cobblestone areas) on a stromal cell monolayer over a period of time. This assay gives frequency readouts that correlate with LTCIC and are predictive of engraftment in in vivo assays and patients. A particularly preferred CAFC assay 3o is described in Young, et al., Blood 88:1619 ( 1996).

WO 00/23567 ' PCT/EP99/07741 Flow cytometry can be used to subset hematopoietic cells from various tissue sources by the surface antigens they express. A combination of these assays may be used to test for HSCs. It is preferred that the SCII7-hu mouse model and the CAFC assay be used to confirm cells with stem cell function.
In one embodiment the invention concerns a model for measuring self renewal in vitro which includes labeling hematopoiietic cell populations particularly enriched populations of HSC with a division tracking dye. Division tracking dyes are fluorescently tagged molecules that bind stably to subcellular structures without apparent interference with cellular function.
With each cell division, fluorescence intensity of the daughter cells is halved relative to the parent cell, allowing cell division to be tracked by flow cytometry. Many dyes may be used including but not limited to carbo~;yfluorescein diacetate succinimidyl ester (CFSE); PKH26 (Young, et al., Blood 88:1619 ( 1996)); and PKH2 (Nordon, et al., British J.
Hematol. 98:528 - 539 {1997) and Traycoff, et al., ~;xp. Hematolo. 26:53-62 (1998)). Most preferred is CFSE.
Antibodies conjugated to other fluorochromes and directed against surface antigens characteristic of HSCs are then used to allow simultaneous measurement of cell division and primitive phenotypes.
To test stem cell function after each division cycle in vitro, individual populations can be purified by flow cytametry and SCID-hu, and CAFC assays can be performed on each subset. The above list of assays used to measure functional compositions of hematopoietic cell populations is not meant to be limiting in any manner. One skilled in the art may use other known assays or combinations thereof.
In another preferred embodiment, the invention concerns a method of genetically modifying stem cells including contacting a population of stem cells with a gene delivery vehicle including a polynucleotide in the presence of an effective amount of a HDAC-I. Gene delivery or transfer can be mediated by methods well known in the art including viral mediated transfer of DNA or RNA; liposome mediated transfer; and other methods as mentioned WO 00/23567 ' PCT/EP99/07741 below. The term polynucleotide should be understood to include equivalent terms such as nucleotide sequence, nucleic acids and the like.
Retroviral vectors are partivcularly prefered gene delivery vehicles.
Retroviral vectors enter the cell via its normal mechanism of infection or they can be modified such that the vector binds to a different cell surface receptor or ligand to enter the cell.
Additionally, recombinant derived adenovirus (AD) vectors, particularly those that reduce the potential for recombination and generation o1E wild-type virus, have been constructed (see and W095/11984). ADs are a relatively well characterized homogenous group of viruses including over 50 stereotypes (~~V095/27071; and Frey, et al., 1998 Blood 8:2781-2792).
ADs are easy to grow and do not require integration into the host cell genome.
Adeno-associated virus (AAV) has also been used as a gene delivery or transfer system. (U.S. Pat. No. 5,693,531 and U.S. Pat. No. 5,691,176). They are small, single-stranded DNA viruses that can integrate into the genome of infected cells.
Recombinant AVV
vectors have been produced in hiigh titers which can transduce target cells at high efficiency.
Herpes simplex virus (HSV) vectors are being considered by researchers for gene therapy in the transfer of gene to neural tissues. Various commercially available vectors include: pSG, pSV2CAT, and pXtl available from Stratagene and pMSG, pSVL, and pSVK3 available from 2o Pharmacia.
Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, CA) and Promega Biotech. (Madison, WI). In order to optimize expression and/or in vitro transcription, it may be necessary to remove, add or alter 5' and/or 3' untranslated portions of the vectors to eliminate potentially inappropriate alternative translation initiation codons or other sequences that nnay interfere with or reduce expression, either at the level of transcription or translation. AltE:rnatively, consensus ribosome binding sites can be inserted 3o immediately 5' of the start codon to enhance expression. Other non-limiting elements which WO 00/23567 ~ PCT/EP99/07741 may be added to the vectors are enhancer elements, scaffold attachment regions (SAR) and matrix attachment regions (MAR), (See W097146687). Examples of these vectors are viruses, such as baculovirus, retroviruses, bacteriophages, cosmids, plasmids, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts and may be used for gene therapy as well as for simple protein expression.
As mentioned above, preferred vectors are retroviral vectors, and particularly preferred are amphotropic retroviral vectors. These vectors may include a therapeutic gene or lD a marker gene and a polynucleotide comprising the retroviral genome of part thereof.
Reference is made to Coffin, et al., "Retroviruses", (1997) Chapter 9 pp: 437-473 Cold Springs Harbor Laboratory Press.. Retroviral vectors useful in the methods of this invention are produced recombinantly by procedures already taught in the art.
W094/29438, W097/21824 and W097/21825 describe the construction of retroviral packaging plasmids and packaging cells lines. Retroviruses are subdivided into seven groups. Five of these groups represent retroviruses with oncogenic potential and the other two groups are the lentiviruses and spumaviruses. The most common retroviruses are those based on the Moloney murine leukemia virus I;MoMLV-vector). Other MoMLV derived vectors include, LMiLy, LINGFER, MINGFR anti MINT. Further vectors include those based on Gibbon ape 2~D leukemia virus (GALV); Moloney murine sarcoma virus (MoMSV);
myeloproliferative sarcoma virus (MPSV); murine embryonic stern cell virus (MESV), for example MESV-MiLy; murine stem cell virus (MSCV); and spleen focus forming virus (SFFV).
(Agarwal et al., J. of Virology, 72:3720 ( 1998)). Non-limiting examples of 1enliviro1 derived vectors include vectors based on human immunodeficiency virus (HIV-1 and HIV-2). New vector systems are continually being developed to take advantage of particular properties of parent retroviruses such as host range, usage of alternative cell surface receptors and the like.
Particularly preferred vectors include DNA from a murine virus corresponding to two long terminal repeats and a package signal. In one embodiment the murine viral vector is derived from a MoMLV or a MSCV. Optionally, the vector will include one or more SAR
elements.
3~D However, the present invention is not limited to a particular retroviral vector, but includes WO 00123567 ' PCT/EP99/07741 any retroviral vector wherein ~transduction of a population of HSCs is enhanced in the presence of HDAC-I over transduction of a population of HSCs under essentially the same conditions but in the absence of IHDAC-1 in the culture media.
In producing retroviral vectors constructs, the viral gag, pol and env sequence will generally be removed from the virus, creating room for insertion of foreign or heterologous DNA sequences. Genes encoded by foreign DNA are usually expressed under the control of a strong viral promoter in the long terminal repeat (LTR). Selection of appropriate control regulatory sequences is dependent on the host cell used and selection is within the skill of one in the art. Numerous promoters are known in addition to the promoter of the LTR. Non-limiting examples include the phage lamda PL promoter, the human cytomegalovirus (CMV) immediate early promoter; the jJ3 region promoter of the Moloney Murine Sarcoma Virus (MMSV), Rous Sarcoma Virus (RSV), or Spleen Focus Forming Virus (SFFV);
Granzyme A
promoter; CD34 promoter; and the CD8 promoter. Additionally, inducible or multiple control i.5 elements may be used. Plasmids containing retroviral genomes are widely available from the American Type Culture Collection (ATCC) and other sources known to those in the art.
Such a construct can be packaged into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Therefore, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virions that are secreted into the culture medium. The virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences.
Most of the packaging cell lines currently in use have been transfected with separate plasmids, i;5 each containing one of the necessary coding sequences so that multiple recombination events are necessary before a replicatiion competent virus can be produced.
Alternatively, the packaging cell line harbors a provirus. (The DNA form of the reverse-transcribed RNA once it integrates into the genomic DIVA of the infected cell). The provirus has been crippled so that although it may produce all the proteins required to assemble infectious viruses, its own RNA can not be packaged into virus. RNA produced from the recombinant virus is packaged WO 00/23567 ' PCT/EP99/07741 -2b-instead. Therefore, the virus stock released from the packaging cells contains only recombinant virus.
The range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein. The recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable incorporation of the foreign gene product. In general, murine ecotropic env of MoMLV
allows infection of rodents cells, whereas amphotropic env allows infection of rodent, avian 1o and some primate cells including human cells. Amphotropic packaging cell lines for use with MoMLV systems are known :in the art and are commercially available. Packaging cell lines include but are not limited to, PA12, PE501, PA317, PG 13, 'l'CRIP, RD 114, GP7C-tTA-G10, ProPak-A (PPA-6), PT67and FLYA13. (See Miller, et al., (1985) Mol. Cell Biol.
5:431-437; Miller, et al., (1.986) Mal. Cell Biol. 6:2895-2902; Miller et al., (1989) Biotechniques 7:980; Danos, et al. ( 1988) Proc. Natl. Acad. Sci. USA 85:b4b0-6464; Rigg et al., ( 1996) Virology 218:290 - 295; and Finer et al., ( 1994) Blood 83:43 -50. Also reference is made to WO 97121825 which discloses a method for obtaining a retrovirai packaging cell capable of producing retroviral vectors, particularly supernatants produced from the packaging cell lines ProPak are. taught. Recently, the G-glycoprotein from vesicular stomatitis 2o virus (VSV-G) has been substiituted for the MoMLV env protein. (See Burns, et al., (1993) Proc. Natl. Acad Sci USA 90:8033-8037; and W092/14829). Xenotropic vector systems also exist which allow infection of human cells.
The vectors will contain at least one and preferably two heterologous genes or gene sequences; (i) a marker gene and (ii) a therapeutic gene to be transferred.
The following list of potential genes that may be :incorporated into the vectors is given by way of example and is not meant to limit the inventior.~ in any manner.
A marker gene may be iinciuded in the vector for the purposes of monitoring successful 3o transduction and for selection of cells into which the DNA has been integrated. Non-limiting WO 00/23567 ° PCT/EP99/07741 examples of marker genes include antibiotic resistance markers, such as resistance to 6418 or hygromycin. Additionally, negative selection may be used, for example, wherein the marker is the HSV-tk gene. This gene will make the cells sensitive to agents such as acyclovir and gancyclovir. Selection could also be made by use of a stable cell surface marker to select for transgene expressing stem cells by FACS sorting. The Neon (neomycin/G418 resistance) gene is commonly used but any convenient marker gene whose sequences are not already present in the recipient cell can lne used. Further, non-limiting examples include NGFR (nerve growth factor receptor), GFP (the bacterial green fluorescent protein), DHFR
(a dihydrofolate reductase gene which confers resistance to methotrexate), the bacterial hisD
gene, murine 1o CD24 (HSA), murine CDBa (l~~t), bacterial genes which confer resistance to puromycin or phleomycin, and ~i-galactosidase;.
The therapeutic gene may include genes or gene sequences effective in the treatment of adenosine deaminase deficiency (ADA); sickle cell anemia; recombinase deficiency;
recombinase regulatory gene deficiency; HIV such as an antisense or trans-dominant REV
gene or a gene carrying a herpes simplex virus thymidine kinase (HSV-tk)). The therapeutic gene may include gene sequences expressing the LDL (low-density lipoprotein) receptor.
The therapeutic gene may also encode new antigens or drug resistant genes.
Further, the therapeutic gene may encode a toxin or an apoptosis inducer effective to specifically kill 2o cancerous cells, or a specific suicide gene to cancerous hematopoietic cells may be included.
Therapeutic genes also encompass antisense oligonucleotides or ribozyme genes useful for translational suppression. The vector will usually comprise one therapeutic gene. However, more than one gene may be necc;ssary for the treatment of a particular disease, and the vectors used in the present invention may include more than one gene. Alternatively, more than one gene can be delivered using several compatible vectors. Depending on the genetic defect, the therapeutic gene can include rel;ulatory and untranslated sequences. For human patients, the therapeutic gene will generally 'be of human origin, although genes of closely related species that exhibit high homology and biologically identical or equivalent function in humans may be used if the gene does not produce an adverse immune reaction in the recipient.

WO 00/23567 ' PCT/EP99/07741 Nucleotide sequences far the therapeutic gene will generally be known in the art or can be obtained from various sequence databases such as GenBank. One skilled in the art will readily recognize that any therapeutic gene can be excised as a compatible restriction fragment and placed in a vector in such a. manner as to allow proper expression of the therapeutic gene in hematopoietic cells.
Methods of transduction include direct co-culture of cells with producer cells (Bregni, et al., (1992) Blood 80:1418-1422) or culturing with viral supernatant alone with or without appropriate growth factors and polycations (Xu, et al., Exp. Hemat. 22:223-230). After viral transduction, the presence of the viral vector in the transduced stem cells or their progeny may be verified by methods such as 1?CR. These techniques are well known in the art.
The application of gene therapy using HSC is well known and the following provides a non-exhaustive list of diseases afar which gene transfer into HSCs is potentially useful. These diseases include bone marrow disorders, erythroid cell defects, metabolic disorders and the like. Particularly bone marrow transplantation should be enhanced by the methods claimed herein.
Genetically modified cells obtained by the methods described herein may be further used in autologous, xenogeneic: or allogenic settings. Autologous cells are derived from an individuals own tissue; xenogeneic cells are derived from a different species, and allogenic cells are derived from a genetically different individual of the same species.
The modified cells may be administered in any physiologically acceptable vehicle, normally intravascularly, although they may also be introduced into bone or other convenient site which cells may find an appropriate site for regeneration and differentiation. Usually at least 1 x 105 cells may be administered. Preferably, at l~;ast 1 x 106 or more cells may be introduced by injection, catheter or the like. If desired, factors such as TPO, IL-2, IL-3, IL-6, IL-11, GM-CSF, G-CSF, interferons, EPO and the life may also be included.
3o The methods provided by the present invention overcome deficiencies of prior art WO 00/23567 ~ PCT/EP99/07741 methods of gene transfer by enhancing the transfer of genes into stem cells.
Gene integration into stem cells by using vectors, particularly retroviral vectors requires cell division. In general, stem cell division results in differentiation. It is believed that HDAC-Is may inhibit differentiation during cell division, thereby increasing the CD34'Thy-1+ cell content of the transduced product.
The cells obtained as described above may be used immediately, expanded by means known in the art, or frozen at liquid nitrogen temperatures and stored for long periods of time, being thawed and capable of being used. The cells will usually be stored in 10% DMSO, 50% FCS, and 40% RPMI 1640 medium. Once thawed, the cells may be further expanded.
Methods of expansion of HSCs by use of growth factors and/or stromal cells associated with stem cell proliferation and differentiation are well known to those skilled in the art. (US Pat.
No. 5,744,36 i ) The stem cell composition produced by the methods herein disclosed may be used for a variety of therapeutic means as disclosed in the literature. They can be used to fully reconstitute an immuno-compromised host such as an irradiated host and/or a host subject to chemotherapy. The cells may also be used for the treatment of genetic disease.
The cells may be used in bone marrow transplants, where the cells may be freed of neoplastic cells or other :ZO cells that are pathogenic. The use of stem cells will minimize graft-versus-host disease, and may also be used to induce donor-specific immunlogic tolerance in the host.
Therefore an embodiment of the invention vncludes a method of improving engraftment of genetically modified mammalian hematopoietic cells, particularly stem cells, including exposing the hematopoietic cells in a culture to an effective amount of a HDAC-I, introducing a :?5 polynulceotide encoding a therapeutic gene or a marker gene into the cultured cells, generating an increase in the number of genetically modified cells over that in the absence of exposure to the HDAC-I and ad,~ministering an effective amount of the population of modified cells to a subject. Preferably the; mammal and subject are human, and the cells are enriched hematopoietic stem cells, particularly CD34''Thy-1+ cells. In a preferred embodiment, the ao cells are transduced with a retroviral vector selected from MoMLV and MSCV
derived WO 00/23567 ' PCT/EP99/07741 vectors and optionally include one or more SAR elements.
Additionally, a method a~f restoring hematopoietic capability in a subject is claimed comprising contacting a population of hematopoietic stem cells with an effective amount of a HDAC-I, transducing the HSCs .and administering an effective amount of a population of the transduced cells to the subject.
The practice of the present invention will employ, unless otherwise indicated conventional techniques of cell biology, molecular biology, cell culture, immunology and the like which are in the skill of one in the art. These techniques are fully disclosed in the current literature and reference is made specifically to Sambrook, Fritsch and Maniatis eds., "Molecular Cloning A Laboratory Manual, 2°° Ed., Cold Springs Harbor Laboratory Press, 1989); the series Methods of Enzymology (Academic Press, Inc.); and Antibodies: A
Laboratory Manual, Harlow et al.., eds., (1987).
All publications and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications cited herein are hereby incorporated by reference in their entirety.
2D The invention generally described above will be more readily understood by reference to the following examples, which are hereby included merely for the purpose of illustration of certain embodiments of the present invention and are not intended to limit the invention in any way.

WO 00/23567 ' PCT/EP99/07741 EXPERIMENTAL
Ex~~le 1 - human cells:
Cells:
Following informed consent, leukaphersis samples are obtained from normal donors mobilized with ?.5 or 10 ~tg/kg/day of G-CSF for 5-6 days. CD34+ cells are enriched from leukaphersis samples at SyStemi:K using an Isolex 300SA or 300I (Baxter Healthcare Corp., Deerfield, Illinois).
Purification of CD34+Thy-1+ cells from fresh MPB:
To select for CD34+'Thy-1+ cells by flow cytometry, anti-CD34, (PR20, SyStemix Inc., Palo Alto CA) is directly conjugated to CY-5; and anti-human Thy-1 (PR13 SyStemix Inc.) is directly conjugated to phycoerythrin (PE). Purified mouse IgGI (Sigma, St.
Louis, MO) is directly conjugated to PE or Cy5 and used for isotype controls. Isolex-selected MPB CD34+
cells are resuspended at 10' cel)s/mL in the staining buffer (SB) consisting of Iscove's modified Dulbecco's medium (IMDM) without phenol red (JRH Biosciences, Lenexa, KS), 2% fetal bovine serum (FBS) (Gemini Bioproducts, Calabasas, CA), 0.1% heat inactivated human gamma-globulin (Gamimune) (Miles Inc., Elkhart, IN) and 10 mM HEPES (JRH
Biosciences). Cells are stained with anti-CD34-CY5 (5 pg/mL) and anti-Thy-1-PE
(25 It a0 g/mL) or appropriate isotype controls, IgG 1-CY5 and IgG 1-PE, for 30 minutes at 4°C, washed and resuspended in cold SB at 5x106 cells /mL. Propidium iodide (PI) (Boehringer Mannheim Biochemicals, Indianapolis, IN) is added at 1 ~tg/mL to detect nonviable cells.
CD34''Thy-1+ and CD34~"Thy-1- cell populations are sorted on a Becton Dickinson FACStar Plus TM equipped with 5W argon laser (excitation 488nm) (Becton Dickinson, San Jose, :z5 CA). Sort regions for forward versus side scatter, PI, CD34, and Thy-1 are established to select live CD34'Thy-1+ or C'D34~Thy-1' cell populations. Sorted cell populations are reanalyzed to ensure clean separation of cell populations.
Cytokines and Cell Culture:
3o Recombinant human thrombopoietin (TPO) is obtained from R & D Systems, WO 00/23567 ' PCT/EP99/07741 Minneapolis, MN. Recombinant Flt3 ligand (FL) and c-kit ligand (KL) are produced at SyStemix Inc., Interleukin (IL)-6 and leukemia inhibitory factor (LIF) are obtained from Novartis Inc., Basel, Switzerland. Cells are cultured for approximately 112 hours at 2 x 105 cells/mL (CD34+ cells) or 5 x 1.05 cells/mL (CD34+'Thy-1+ cells) in 6 or 24 well flat bottom plates (Corning Costar Corp., Cambridge, MA) at 37 °C in a humidified incubator with approximately 100% saturation.. Cells are cultured in culture medium (CM) [X-Vivo-15 medium, (BioWhittaker, Walke;rsville, MD) containing 1% bovine serum albumin (BSA) (Sigma)], supplemented with thc: cytokines: TPO (50 ng/mL), FL (100 ng/mL), and KL (100 ng/mL). TSA is supplied at '.i nglmL; TPX is supplied at 0.25 nM and 0.5 nM
and chlaunydocin is supplied at 0.25 nM or 0.50 nM. TSA is purchased from Wako Bioproducts, Richmond VA), and stored in absolute ETOH at -20 °C at a concentration of lmg/mL. TPX
and chlaunydocin are provided lby Novartis Pharmaceuticals Corporation, East Hanover, NJ
and stored in DMSO at -20 °C apt a concentration of lmM.
As shown in Figure l,. when CD34+ cells are cultured for 5 days with growth supporting cytokines (TPO, FL and KL) and the HDAC-inhibitors TSA, TPX, or chlamydocin, there is a substantial increase in the number of Thy-1+ cells over cultures which do not contain HDAC-inhibitors. Among the HDAC-inhibitors tested, chlamydocin induced the greatest increase, an average of 7 fold. These results are the average of four experiments, and the error bars show the standard deviation from the mean.
Isolation of cell populations poas culture:
Post culture cells are harvested from tissue culture wells, enumerated, and stained with anti-CD34-Cy5 and anti-Thy-l-PE or the appropriate isotype controls as described above.
Cell populations are isolated with a FACStar Plus flow cytometer as described above for sorting pre-culture cells. Sorted cell populations are reanalyzed to ensure that a majority of events for each population falls. within each sort region, and to ensure that contamination of the Thy-1 populations by Thy-1~ cells is less than 5%.

WO 00/23567 ' PCT/EP99/07741 Cobblestone-area forming cell (CAFC) assay:
Cell populations are plated at limiting dilution (from 100-0.78 cells/well, 24 wells per dilution) on pre-formed murine stromal monolayers, as described in Young et al., Blood 88:1619 ( 1996). The culture medium consists of a mixture of equal portions of RPMI and IMDM medium (JRH BioSciences, Woodland, CA), 1mM sodium pyruvate (JRH
BioSciences), 5 x 10'5 M 2-mercaptoethanol (Sigma, St. Louis, MO), 10% FBS
(Hyclone, Logan, UT), and cytokines, LIF (50 ng/mL) and IL-6 (10 ng/mL). Cultures are fed at weekly intervals by replacing half of ~~the medium, and scored at week 5 for cobblestone area ;t0 formation. CAFC containing wells are scored microscopically, and their frequencies estimated statistically as described below.
A frequency readout is f;enerated from the numbers of positive wells in each row of the CAFC assay limiting dilution series using a SAS statistical analysis program incorporating U5 the maximum likelihood estimate method and the chi-square test of linearity (Biostatistics consulting, Palo Alto, CA). Limiting dilution readouts are considered to fit a linear model if the conservative chi-squared test of linearity was greater than 0.05. The significance of differences between CAFC frequency readouts is determined using Anova (Microsoft), where P less than 0.05 is considered to be significant.
:!o As shown in Figure 2A, the number of Thy-1'' cells and the total CAFC activity among the Thy-1+ fraction of cells increased 2 to 3 fold in cultures containing chlamydocin, versus cultures without chlamydocin. 7.'otal cell numbers are the same in cultures with or without chlamydocin. However, total CAFC activity in the chlamydocin containing cultures is ;!5 increased two-fold. (Figure 2B). The results indicate that stem cell replication or self renewal, measured by Thy-1+ ptuenotype as well as function is increased by exposing cells to HDAC-I during culture.

WO 00/23567 ' PCT/EP99/07741 SCID-hu bone assay:
The SCID-hu bone assay as described in Luens et al., Blood 91:1206 (1998) is utilized. The hosts are C.B. 17 scid/scid mice implanted with human fetal bones (Average gestation age 22 weeks) at 8-10 weeks prior to use in assays. Starting donor cell populations for SCID-hu assays are CD:34 selected as described above. Post-culture donor cell populations are purified by flow cytometry for expression of the Thy-1 antigen as described above. The hosts are whole body irradiated (400 rads), then injected with 5,000 or 15,000 cultured Thy-1+ cells per human fetal bone graft. Eight weeks after the injection, the mice are sacrificed. Bone marrow is harvested from the grafts and stained with anti-W6/32-PE for pan human leukocyte antigen HLA class I major histocompatibility complex detection versus an appropriate FITC-conjugated antibody directed toward the HLA allo-type of the donor cells.
The cells are analyzed on a FACScan (Becton Dickinson Immunocytometry System).
Grafts showing a minimum of 1 % donor cells are scored as positive. The engraftment rate is the ~s number of grafts positive for donor cells out of the number of grafts injected. Table 1 illustrates that stem cell function is maintained on a per cell basis among the Thy-1+ expressing cells from cultures containing chlamydocin.

Thy-1+ cells Dose Engraftment Rate from cultures (-) CHL 5,000 1/3 15,000 4/4 (+) CHL 5,000 2/3 15,000 3/3 CHL, is chlamydocin Dose = cultured Thy-li~ cells per human fetal bone graft.
Engraftment Rate = the number of positive grafts for donor cells out of the number of grafts injected.
:25 WO 00/23567 ' PCT/EP99/07741 NOD SCID repopulation assay:
Trafficking to and enl;raftment in mouse bone marrow by human hematopoietic cells injected IV is measured by tlhe NOD SCID repopulation assay. Six to ten week old NOD
SCID mice (Jackson derived" and bred at SyStemix) are irradiated with 350 rads. Human MPB cells are injected into the tail vein or orbital sinus. Thy-1+ cells are not repurifed after culture for the present engraftment assays; instead, equal cell numbers from the whole culture are injected. Six weeks later l:he mice are sacrificed, and marrow cells from the long bones of the hind limbs are recovered. Cells are fluorescently labeled with anti-CD45-APC which detects human cells and analyzed on a FACS Calibur~. Results are illustrated in Figure 6.
When cell dose is low and chl~amydocin is supplied in a 24 hour incubation, the engraftment is improved.
Division tracking dye labeling:
Isolex selected CD34+ cells are labeled with carboxyfluorescein-diacetate succinimidylester {CFSE) dye (Molecular Probes Inc., Eugene, OR) at a cell concentration of 3-5 x 106 /mL and a dye concentration of 1.25 p.M in IMDM without phenol red in the dark, at room temperature, for 10 minutes. The labeling is stopped by the addition of 1l5 volume of FBS and 10 fold volume of cold CM. Cells are washed and resuspended in CM. An aliquot of dye labeled cells is fixed with 1 % paraformaldehyde and stored at 4 °C to be used as a marker for the fluorescence intensity of undivided cells post-culture.
Analysis is performed on a FACSCalibur (Becton Dickinson, Immunocytometry System).
As shown in Figure 3, Thy-1 expression in culture after 5 days in the presence of chlamydocin is retained after at least 4 cell divisions, whereas it is more rapidly lost with cell division in cultures without chl,amydocin.
Analysis of cell phenotype post-culture:
Expression of CD34 and Thy-1 antigens is evaluated using anti-CD34-CYS and anti-Thy-1-PE as described above. FITC-conjugated monoclonal antibodies to human CD2, CD 14, CD 15, CD33, and CD 19 (Becton Dickinson, San Jose CA) are used to evaluate WO 00/23567 ~ PCT/EP99/07741 expression of lineage antigens. Analysis is performed on a FACSCalibur.
Using the above described methods, it is determined that the expression of the surface lineage antigens CD2, CD 14, ~md CD 19 is negligible both before and after 5 day culture with or without HDAC-inhibitors. 'The expression of CD 15 and CD33 was increased post-culture, but on a similar proportion of cells in cultures with or without HDAC-inhibitors. Figure 4 shows that the proportion of Thy-1+ cells that express CD15 after 5 days of culture is approximately 30%, with or without HDAC-inhibitors. These results indicate that although expression of primitive antigens such as CD34 and Thy-1 is increased on cells in cultures containing HDAC-inhibitors, the expression of other surface antigens, such as those associated with differentiation, is not.
Example 2 - murine cells:
i5 Isolation of the murine stem cells:
Bone marrow is harvested from 4 week old BA.l mice (C57BIJKa.AKR/Jsys) and enriched in Sca-1 expressing cells using a biotin-conjugated anti Sca-1 antibody (Pharmingen) and positive selection using magnetic columns (Miltenyi Biotec Inc.). The Sca-1 enriched cells are further labeled with c-Kit-APC, Thyl.l-FITC, streptavidin-Texas Red and a panel of PE
2o conjugated-lineage (Lip) antibodies directed against Mac-1, Gr-1, B220, CD2, CD4, CDS, CD8 (all antibodies purchased from Pharmingen). Propidium iodide (PI) is added at 1 wg/ml to exclude non-viable cells. The c;-Kit''Thy-1'~,in'°~-Sca-1'' subset, which is highly enriched for stem cell activity (Morrison a.nd Weissman, Immunity, 1:b61-673, (1994)) is isolated by FACS on a Vantage sorter (Bec;kton Dickinson, San Jose, CA).
Culture conditions:
The sorted cells are seeded in liquid culture at a density of 5x104 cellslml in Xvivol5 media (BioWhittaker) in the presence of murine interleukin (mIL)-3 (10 ng/ml), mIL-6 (10 nglml) and murine Stem Cell i:actor (mSCF) ( 100 nglml) (purchased from R&D
Systems).
3o TSA (Wako Bioproducts, Richmond VA) or chlamydocin (Novartis Pharmaceuticals) are added to the culture at concentrations ranging from 0.5 to 10 ng/ml and 0.25 to 2 nM, WO 00/23567 ~ PCT/EP99/07741 respectively. The cells are cultured 4 days in a 37°C incubator with 5%
COz in mufti-well flat bottom tissue culture plates (either 9b well, 48 or 24 well plates depending on the initial number of cells seeded).
Analysis of cells post culture:
After 4 days of culture, the cells are harvested and an aliquot is stained with Trypan Blue to determine the number of viable and non-viable cells using a Neubauer hemacytometer.
At least 200 total cells are scored. A portion of the cultured cells (equivalent to at least 40 000 viable cells) ares then stained as described above. The profile of c-Kit , Thy-1, Lin and 1o Sca-1+ expression is then deternnined by FACS using a Vantage sorter. The limits of the gates used to determine the percentage of cells that fall into the c-Kit+'Thy-1'"Lin'°'-Sca-1+ subset is defined as the frontiers separating the bimodal population for each stain observed separately.
These limits are set with cells treated with mIL-3, mIL-6 and mSCF alone. The number of c-Kiti'Thy-1+Lin'°'-Sca-l; cells preaent in the culture is calculated by multiplying the proportion of cells in the c-Kit'"Thy-1+L,in'°'~Sca-1+ subset by the total number of viable cells present in the culture.
Results from a representative experiment are summarized in Table 2 and illustrated in Figures SA, SB and SC.

HDAC-I added Viable: cell % of non viable % of KTLS Fold expansion (x1173) cells of KTLS cells None 1 9:50 16% 8% 3.1 TSA 610 6% 58% 7.2 (3ng/ml) Chlamydocin 450 3% 57% 5.0 (1nM) KTLS is c-Kit'Thy-1'Lin'°''Sca-1+

WO 00/23567 ~ PCT/EP99/07741 In this experiment SOxlO'' sorted c-Kit;'Thy-1'°Lin'd'Sca-1+(KTLS) cells are cultured in lml of X vivol5 media supplemf;nted with mIL-3, mIL-6 and mSCF in a 24 well-plate. When indicated TSA (3ng/ml) or chlamydocin (1nM) is added to the culture. After 4 days, the cells are collected for cell count and FACS analysis. The number of viable cells recovered after 4 days of culture is diminished 2 to 4 fold by the addition of HDAC-I to the growth-media.
Compared to cells grown in the absence of HDAC-I, the vast majority of cells treated with TSA or chlamydocin remains Lizi negative (93 or 88% versus 55%) and expresses high levels of Thy-1 (95-96% versus 30%) and Sca-1 (94 or 88% versus 46%). A higher percentage of i.0 cells grown with either HDAC-I also remain positive for c-Kit compared to cells cultured without HDAC-I (67 and 73% versus 51%). While only 8% of the cultured cells retained the stem cell phenotype (c-Kit'Thy-1'"Lin'°'-Sca-1+) when grown in the absence of HDAC-I, these cells represent 58 and 57% of thn cells cultured with TSA or chlamydocin, respectively. Over the 4 day-culture period, the c~-Kit~T'hy-1'°Lin~'~-Sca-1+cells grown without the addition of HDAC-I underwent a 3.1-fold expansion while those cultured with TSA or chlamydocin underwent a 7.2- or 5.0-fold expansion, respectively.
Figures SA, SB and SC illustrate the profile of Lin, Thyl, cKit and Sca-1 which are analyzed after 4 days of culture in growth media alone (A) or media supplemented with 3 2,0 ng/ml of TSA (B) or 1nM of chlamydocin (C). The limits of the regions used to calculate the percentage of cells that fall into each gate are depicted on the dot plots.
The nonviable cells are excluded from the analysis.
Example 3 - RetroNectin~ Transduction:
2.5 Retroviral Infection:
Following informed consent, as described above in example 1, the CD34'' of CD34'Thy-1+ enriched cells are cultured at 106 cells per ml in 5 mL cultures in X-Vivo-15 medium (BioWhittaker, Walkerswille, MD) for 48 hours at 37°C in 5% CO2.
The cultures are 30 supplemented with the cytolcines: TPO ( 100 ng/mL), (R&D Systems, Minneapolis, MN); KL

WO 00/23567 ~ PCT/EP99/0774t ( 100 ng/mL), and FL ( 100 ng~mL), SyStemix, Palo Alto CA. The histone deacetylase inhibitor, chlamydocin is supplied to the cultures at 0.50 nM per mL for 72 hours.
Chlamydocin is provided by Novartis Pharmaceuticals Corporation, East Hanover, NJ and is stored in DMSO at -20 °C at a concentration of lmM.
Non-tissue culture-treated plates (Falcon, Lincoln Park, NJ) are coated at 2 ~g per cm2 of fibronectin fragment CH-296 (FN) (BioWhittaker, Walkersville, MD) and incubated for 2 hours at 37°C. Plates are blocked with 2% human serum albumin in phosphate buffered solution (HSAIPBS) for 30 minutes. This is followed by washing with 2.5% Hepes buffer and Hanks Balanced Salt Solution (HBSS).
After 48 hours, cultured cells are centrifuged for 10 minutes at 1200 rpm at 4 °C and resuspended in the same medium as described above. The cells are added to FN-coated plates containing an equal volume of rexroviral supernatant for 20 hour culture at 37°C in 5% C02 without polybrene or protamine sulphate. (Lishan SU et al., 1997, Hematopoietic Stem Cells-Based Gene Therapy for Acquired Immunodeficiency Syndrome: Efficient Transduction and Expansion of RevMlO in Myeloid Cells In Vivo and In Vitro. Blood:Apr. 2283 -2290).
The Moloney murine leukemia virus (MoMLV) vector is prepared from a ProPAk (PP-A.6) packaging cell line (Forestell, et al., 1997, Novel Retroviral Packaging Cell Lines:
Complementary Tropisms and Improved Vector Production For Efficient Gene Transfer, Gene Therapy, 4:600-610). The vector comprises - LTR-NGFR-SV40Neo-LTR- wherein LTR is the viral long terminal repeat; NGFR is the truncated human nerve growth factor receptor, SV40 is the SV40 promoter, and Neo encodes 6418 resistance. (Miller, et al., 1989, Improved Retroviral Vectors for Gene Transfer and Expression. BioTechniques.
7:980 -990).
Cells are removed from the plates by gentle pipetting and centrifuged (as described previously). Cell pellets are resus~pended in X-vivo 15 medium plus 1% BSA.
Viable cells are counted by trypan blue exclusion. After diluting, at a 1:10 ratio of cells to trypan blue, cells are placed in suspension in a hemacytometer. Dead cells are then determined by their blue WO 00/23567 ' PCT/EP99/07741 color. These methods are known t:o those skilled in the art.
FACS analysis of gene expression on CD34+, Thy-1 + and total cells:
After transduction, cells are analyzed by FACS as described below. A subset of the ~~ cells as described above are ;placed in X-vivo 15 culture medium containing GM-CSF, l Ong/ml; EPO, 2 U; IL-3, 10 ng/ml; IL-6, 10 ng/ml; LIF, 100 ng/ml; and ng/ml. Cultures (1.0 ml) are performed in 24-well tissue culture plates (Falcon). At 72 hours, cultured cells are harvested and stained with anti-CD34-APC (Becton Dickinson, San Jose, CA), anti-Thy-1-PE (SyStemix, Palo Alto CA), and anti-NGFR-FITC (Boehringer Mannheim, Indianapolis, IN) or appropriata~ isotype controls (Becton Dickinson, San Jose, CA).
Fluorescence is analyzed on a FAGS Calibur (Becton Dickinson) using standard techniques.
The anti-NGFR-FITC is conjugated at SyStemix. Results summarized in Table 3 demonstrate the application of chlamydocin increased not only the number of cells expressing Thy-1 antigen but also the number of Th;y~ NGFR+ cells.
15~

NGFR Transgene Expression in Primitive Thy-1' MPB Cells After 2G Short-term Culture (3 Days) With TPO, FL, and KL and with or without Chlamydocin %NGFR+ CELL#X
OF THY-1+ 10E5 OF

THY-1+NGFR' AVE SD AVE SD

(-) 11.60 3.9 0.69 0.4 CHL

(+) 11.:?0 3.7 1.68 0.8 CHL

CHL 12.'.33 5.9 1.56 0.6 +

BSA

Fold 2.44 Increase (+) CHL

Footnote to Table 3.

WO 00/23567 ~ PCT/EP99/07741 For (+) and (-) CHL, each value is the average (AVE) of 5 experiments.
For CHL + BSA each value is the average of 3 experiments.
SD = standard deviation.
CHL is Chlamydocin supplied at 0.5 nM/mL.
BSA is Borine Serum Albumin and is supplied at 1.0 %n.
Fold Increase = (+) average CHI. / (-) average CHL
In Vitro Assay for Transduction .of Primitive PHP:
1 o After approximately 72 hours of transduction, the cells are counted and twenty thousand cells per culture condition (+ or - chlamydocin) are plated on top of SyS 1 marine stromal cells (Young et al., Blood, vol. 87: 545 1996) in 2 wells in 24-well plates (Corning Science Products, Acton MA) for 5-week culture in the presence of exogenous human IL-6 (20 ng/ml) and LIF ( 100 ng/mi). Plates are fed weekly by exchange of half the medium volume. The medium includes a mixture of 50/50 RPMI/IMDM plus 10% fetal calf serum.
After 5 weeks in stromal culture, there is no significant difference between %
NGFR in CD34+
cells for cultures with or without chlamydocin. (Data not shown) FACS Analysis of Gene Expression on CD34+ and total cells cultured on SYSI
Stroma:
Cells from above are harvested and filtered through 70mm cell strainers (Falcon) to remove stromal cell clumps. Viable hematopoietic cells are counted, and the cells cultured for 3 days in X Vivo 15 medium containing GM-CSF, 10 nglml; EPO, 2U; IL-3, 10 ng/ml; IL-6, 10 ng/ml; LIF, 100 ng/ml; and K:L, 100 ng/ml. Cells are stained with anti-NGFR-FITC and anti-CD34-APC (Becton Dickinson). Cells are then analyzed on a FACS Calibur.
Further LTC-CFCAssays for SV40Neo on LTC-CFC can be performed' Triplicates of 40,000 celas/ml from C are placed into methylcellulose colony assays (MethoCult, StemCell Technologies, Vancouver, Canada) with GM-CSF, 10 ng/ml;
EPO, 2U;
IL-3, 10 ng/ml; IL-6, 10 ng/ml; and KL, 100 ng/ml. Hematopoietic colonies are scored after 12 - 14 days according to standard criteria known in the art.
Individual colonies (64) are placed into 50 ltl of lysis buffer as described below.
Lysates are incubated overnight at 37 °C and heat inactivated at 95°C for 15 minutes, before storage at -20°C. PCR assays ~~re performed to test transgene marking with SV40Neo. B-WO UO/23567 ' PCT/EP99/07741 globin is used as a positive control for the presence of DNA. PCR lysis buffer solutions, A and B, are mixed at a ratio of 1:1 with 144 mg Proteinase K. Lysis buffer solution A includes 100 mM KCL, 10 mM Tris HCL, and 2.5 mM MgCl2. Lysis buffer solution B includes 10 mM
Tris HCL, 2.5 mM MgCl2,1 % 'rween, and 1 % NP40. The final concentration obtained is 100 mg/ml Proteinase K. For PCR analysis, 10 p.l ( 1000 cells) and 5 p.l (500 cells) aliquots are dispensed into thermocycle plates. Specific known primers and probes are used for PCR:
PCR is carried out in 30 ~1 volumes, containing 10 mM Tris-HCL (pH 8.3), 1.5 mM
MgCl2, 50 mM KCL, 0.1666 m~Ivl each of dATP, dCTP, dGTP, dTTP, 0.833 ~tM of SvNeo to forward primer 1 and SvNeo rE:verse primer 2, 0.227 p.M B-globin forward primer 1 and B-globin reverse primer 2, and 1. unit of Taq DNA polymerase. The PCR program can be preformed on a Perkin-Elmer 9600 thermocycler: one cycle of 95 °C for 5 minutes and 40 cycles of 95 °C for 30 seconds, 62 °C for 30 seconds and 72 °C for 1 minute, followed by one cycle of 72 °C for 10 minutes. PCR products are analyzed by gel electrophoresis on 3 i5 agarose gels. These techniques .are well known in the art.
Example 4 - Spinoculation Trar~ a t' Hematopoietic stem cells are purified by selection of CD34+ cells by an immune 2o affinity system from G-CSF mobilized peripheral blood obtained from healthy volunteers using standard practices and also as described in Example 1 above.
The purified CD34+ cells (3 x 106) are cultured in 7m1 Teflon cell culture bags (American Fluoroseal, Inc., Ga.itherburg, MD) for 48 hours in 1.5 ml serum-free X-vivo 15 25 medium. The size of the Teflon bag used in an experiment is dependent on the number of cells to be cultured and subsequently transduced. The medium is supplemented with hematopoietic cell growth factors including '1,"PO, 200 ng/mL; FL, 200 ng/mL; IL-3, 40 ng/mL; IL-6, 40 ng/mL; and LIF, 200 ng/mL in the presence or absence of the histone deacetylase inhibitor, chlamydocin is supplied at 0.50 nM per mL.

WO 00/23567 ~ PCT/EP99/07741 After 48 hours an equal volume ( 1.5 ml) of retroviral supernatant is added to the cultured cells. The retroviral vector and supernatant used in this example is described above in example 3. The cells are centrifuged at 3400 rpm (Sorval RD6000) oriented with the flat side of the bag placed at the bottom of the centrifuge bucket for 4 hours. The cells are resuspended by gentle pumping of the bag, and cells are returned to the incubator. After 20 hours at 37 °C in 5% CO2, the cells are harvested from the bag, and analyzed for stem cell content (CD34+ and CD34''Thy-1'") as described above in Example 3.
to TA_ BLE 4 Cell Recovery Post Transduction Percent Cell Recovery Without With Chlamydocin Chlamydocin Mock: Transduced Mock Transduced Range 83.9 - 115.760.0 - 158.683.2 - 144.381.4 - 151.1 Mean 104.5 95.9 118.5 113.

StDev 12.0 37.0 25.60 25.

n= 6 6 3 6 The data shown in Table 4 indicates that cell recovery after the transduction process is not effected by chlamydocin.
:ZO TABLE 5 The Effect of Chlamydocin on Stem Cell (CD34' Thy-1') Content Post Transduction at Day 3 WO 00/23567 ~ PCT/EP99/07741 % CD34' Thy-1' Starting Post Transduction (Input) (-) Chlamydocin(+) Chlamydocin Range 40.8 - 24.9 - 44.6 49 - 79.8 G9.8 Mean 54.7 38.6 67.1 StDev 9.4 7.6 11.5 The mean represents the average of six experiments.

WO 00/23567 ~ PCT/EP99/07741 The data provided in Tables 4 and 5 are used to calculate the absolute number of HSC
{CD34+ Thy-1+) and the results are illustrated in Table 6. Yields are per 1 x 106 cells at the start of transduction. The results suggest that there are approximately 2 fold more HSCs in the populations transduced with chlamydocin as compared to cells transduced without the chlamydocin. Cells transduc~ed in the presence or absence of chlamydocin showed equivalent content of CD34+ cells {99 -+-/-1.4%) after the 3 day transduction process.
TABLE
Calculation of Chiamydocin Effect on Stem Cell 1o Content Post Transduction # of CD34'Thy-1+ % of input cells Input (starting)547000 100 (-) Chlamydocin370000 G7.6 (+) Chlamydocin758000 138.6 Transgene Expression on HSC
% Cells Expressing NGFR

Total CD34+ CD34+Thy-1+
Cells (-) {+) {-) {+) {-) (+) CHLAM CHLAM CHLAM CHLAM CHLAM CHLAM

Range 8.7-19.37.4-28.4 8.8-19.8 7.6-29.4 5.0-15.15.0-27.2 Mean 13.1 18.1 13. S 18.7 9.1 15.9 StDev 3.9 7 4 7.3 4 7.9 WO 00/23567 ~ PCT/EP99/07741 TA
Relative Effect of Chlamydocin on Thy Content and Thy' Cells That Express Transgene on Day 3 As Compared to No Chlamydocin Value Relative to (-) Chlamydocin % CD34'Thy-1+ % Thy-1+ expressing NGFR

Range 1.63 - 1.96 0.93 - 2.43 Mean 1.75 1.71 StDev 0.12 0.45 Footnote to Table 8: 1.75 x 1.71 = approximately 3 fold increase in the # of Thy+ c;ells that express the transgene in the infusion product.
The mean represents six experiments.
Example 5 - Dose Response F~~r Chlamydocin:
CD34+ selected cells are transduced by spinoculation with the MoMLV vector as described in Example 3. The results shown in Table 9 illustrate chlamydocin operates in a dose dependent manner in increasing the stem cell content (CD34''Thy-1'") of the ex vivo transduced CD34+ cells derived from mobilized peripheral blood. The data indicates that the optimal concentration is between O.SnM and l.OnM for cells incubated for three days.
2o Additionally, the results illustrate there is an increase in the stem cell content relative to the starting CD34+ population in the presence of 0.5 nM Chlamydocin.

WO 00/23567 ~ PCT/EP99/07741 T. ABLE 9 Post Transduction Stem Cell Content CHL Concentration CD34'Thy-1' Cells (nM ) lo 0.0 16.2 O.:l_ 21.3 0.25 30.1 0.'i 54.0 1.0 48.8 Footnote to Table 9. The results are the averages of two experiments.
The CD43+'Thy-1~' cell content at the start of culture (Day 0) was 37 %.

Claims (34)

We claim:

1. A method of promoting self renewal division of hematopoietic stem cells comprising:
a) obtaining a population of hematopoietic cells from a source of hematopoietic cells wherein the obtained hematopoietic cells include a subpopulation of hematopoietic stem cells;
b) culturing the hematolpoietic cells under growth supporting conditions;
c) exposing the cultured cells to an effective amount of a histone deacetylase inhibitor wherein self-renewal divisions of the stem cells is promoted; and d) obtaining a composition of the self renewed hematopoietic stem cells.

2. The method according to claim 1 further comprising obtaining a population of enriched hematopoietic stern cells prior to step b).

3. The method according to either claim 1 or 2 further comprising expanding the composition of self renewed cells.

4. The method according to any one of claims 1 to 3, further comprising selecting the subpopulation of hematopoietic stem cells from the cultured cells.

5. The method according; to any one of the preceding claims, further comprising genetically modifying the hematopoietic stem cells by contacting a gene delivery vehicle comprising a polynucleotide with the hematopoietic stem cells exposed to the histone deacetylase inhibitor.

6. The method according; to any one of the preceding claims, wherein the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, trapoxin, chlamydocin, sodium butyrate, and dimethyl sulfoxide.

7. The composition of self renewed cells obtainable by the method according to claim 1.

8. A culture comprising:
a) isolated mammalian hematopoietic cells which include a subpopulation of engrafting cells capable of self renewal;
b) an effective amount of one or more histone deacetylase inhibitors; and c) effective amounts of growth and self renewal supporting cytokines, wherein said effective amount of histone deacetylase inhibitor promotes self renewal division of the engrafting cells in the culture.

9. The culture of claim 8 wherein the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, trapoxin, chlamydocin, sodium butyrate, and dimethyl sulfoxide.

10. A method of promoting self renewal division of stem cells, comprising treating a population of stem cells with an effective amount of one or more histone deacetylase inhibitors, and allowing the stem cells to self renew.

11. The method according to claim 10 wherein the histone deacetylase inhibitor is selected from the group consisting of trichostatin A, trapoxin, chlamydocin, sodium butyrate, and dimethyl sulfoxide.

12. The method according to either claim 10 or 11, wherein said stem cells are enriched hematopoietic stem cells characterised as CD3+Thy-1+.

13. A method of generating transduced stem cells of a mammal comprising:
(m) treating stem cells in a culture with an effective amount of a histone deacetylase inhibitor;

(n) introducing a heterologous gene into the cultured stem cells using retroviral mediated transfer; and (o) allowing transduction of the stem cells wherein the number of transduced stem cells is increased over the number of transduced stem cells exposed to substantially the same conditions but in the absence of treatment with the histone deacetylase inhibitor.

14. The method according to claim 13 wherein the stem cells are hematopoietic stem cells.

15. The method according to either claim 13 or 14, further comprising separating a population of the transduced stem cells.

16. The method according to any one of claims 13, 14 or 15 further comprising administering an effective amount of a population of the transduced stem cells to a mammalian subject.

17. The method according to claim 16 wherein the stem cells are allogeneic to the subject.

18. The method according to claim 16 wherein the stem cells are autologous to the subject.

19. A method of genetically modifying stem cells comprising:
(s) contacting a. gene delivery vehicle comprising a polynucleotide with a population of stem cells cultured in the presence of an effective amount of a histone deacetylase inhibitor; and (t) obtaining genetically modified stem cells.

20. The method according to claim 19 wherein the gene delivery vehicle is selected from the group consisting of retroviral vectors, DNA viral vectors, naked DNA and a liposome delivery vehicle.

21. The method according to claim 19 wherein the polynucleotide encodes a therapeutic gene.

22. The method according to any one of claims 19, 20 or 21 further comprising expanding a population of the genetically modified stem cells.

23. The method according to claim 19, 20 or 21 further comprising isolating a population of the genetically modified stem cells.

24. The method according to claim 19, 20 or 21 further comprising administering an effective amount of the population of genetically modified stem cells to a subject.

25. A method of restoring hematopoietic capability in a subject comprising:
(a) contacting a population of HSCs with an effective amount of a histone deacetylase inhibitor; (b) transducing the HSCs by exposing the HSCs to a vector including a nucleic acid sequence encoding a therapeutic gene; and (c) administering an effective amount of a population of the trinsduced hematopoietic stem cells to a subject wherein hematopoietic capability is restored.

26. A method of improving engraftment of genetically modified mammalian stem cells comprising:
(z) exposing stem cells in a culture to an effective amount of a histone deacetylase inhibitor;
(aa) introducing a therapeutic gene into the cultured stem cells;
(bb) generating an increase in the number of genetically modified stem cells over that in the absence of exposure to a histone deacetylase inhibitor; and (cc) administering an effective amount of a population of the modified cells to a subject.

27. The method according to claim 2b wherein the cells are hematopoietic stem cells.

28. The method according to claim 27 wherein the stem cells administered to the subject are allogeneic.

29. The method according to claim 27 wherein the stem cells administered to the subject are autologous.

30. A method of transducing stem cells comprising (dd) exposing a population of stem cells to an effective amount of a histone deacetylase inhibitor;
(ee) transducing the stem cells with a heterologous gene; and (ff) obtaining transduced stem cells wherein the number of transduced stem cells is increased over the number of transduced stem cells grown under essentially the same conditions but in the absence of exposure to an effective amount of the histone deacetylase inhibitor.

31. The method according to claim 30 wherein the cells are transduced in a culture which supports stem cell viability and growth.

32. The method according to claim 30 or 31 wherein the cells are exposed to the histone deacetylase inhibitor in vivo.

33. The method according to claim 31 wherein the culture includes a factor selected from the group consisting of IL-3, IL-6, soluble IL-6 receptor, LIF, TPO, KL, and FL, alone or in any combination thereof.

34. A culture comprising:
(hh) a population of stem cells;
(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 gene delivery vehicle including a polynucleotide sequence encoding a marker gene or a therapeutic gene.