CA2309324A1 - Gene that imparts selective proliferation activity - Google Patents

Abstract

Selective amplification of cells is enabled by introducing into cells a gene encoding a fusion protein comprising (a) a mutant estrogen receptor that is unresponsive to a estrogen and that is responsive to a tamoxifen, the derivative thereof, or the metabolite thereof, and (b)_a domain that imparts proliferation activity to the cells upon the association and stimulating the cells with the ligand.

Description

vli,Y-23-00 11:20 +81 298 41 2009 P.04 R-983 Job-004 2000'05/24 WED 00:12 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~004/049 w SPECIFICATION
GENE THAT IMPARTS SELECTIVE PROLIFERATION ACTIVITY
Tec nical Field The present invention relates to the field of genetic engineering, particularly the field of gene therapy.
Background Art Hematopoietic stem cells (HSC), with long-term engraftment and multi-lineage differentiation potential, are ideal targets for gene therapy to treat a number of disorders [Karlsson S., Blood,(1991) 78: 2481-2492; Dunbar CE, and Emmons RVB., Stem Cells,(1994) 12: 563-576]. Retroviral vectors are the most commonly used vehicles for gene transfer into HSC, due to their ability to integrate into the host chromosomes . Although current retroviral vectors have been shown to deliver genetic materials into murine HSC successfully, the transduction efficiency into human HSC is insufficient for clinical applications [Dunbar CE, and Emmons RVB. , Stem Cells, ( 1994 ) 12 s 563-576; Kohn DH, et a1. , Nat.Med.,(1998) 4: 775-780].
A great deal of effort has been made to improve stem cell transduction efficacy, such as developing new vectors, incorporating selectable makers to enrich transduced cells, and utilizing fibronectin to assist viral infection [Naldini L, et al. , Science, ( 1996 ) 272 : 263--267; Pawliuk R, et al. , Blood, ( 1994 ) 84 2868-2877; Persons DA, et al., Blood,(1997) 90: 1777-1786;
Sorrentino BP, et al., Science,(1992) 257: 99-103; Hanenberg H, MRY-23-00 11:20 +81 298 41 2009 P.05 R-983 Job-004 2000'05/24 WED 00:13 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~005/049 et al., Nat.Med.,(1996) 2: 876-882]. We have investigated the feasibility of using fusion genes between cytokine receptors and molecular switches for selective expansion of transduced HSC . We constructed fusion proteins between granulocyte colony-stimulating factor (G-CSF) receptor (GCR, full-length G-CSF
receptor; D GCR, cytokine-binding site was deleted) and the hormone-binding domain (HBD) of estrogen receptor (ER) [zto K, et al. , Blood, ( 1997 ) 90 : 3884-3892 ] . The GCR portion was employed as a growth signal generator, which is physiologically triggered by receptor dirnerization upon G-CSF binding[Avalos BR.,Blood,(1996) 88 : 761-777 ] . The ER-HBD acts as a molecular switch to convert the fusion counterpart to function in an estrogen-dependent manner [Mattioni T, et al. , Methods Cell.Biol. , ( 1994 ) 43 : 335-352 ] . When we introduced the fusion constructs (GCRER and, D GCRER) into interleukin-3 (IL-3)-dependent Ba/F3 cells and murine bone marrow (BM) cells, estrogen supported their growth in a controllable manner [ Ito K, et al. , Blood, ( 1997) 90: 3884-3892 ] .
These results suggested the feasibility of using fusion genes of GCR and ER-HBD, which we designated as ~ selective amplifier genes ' , to expand transduced HSC.
However, there are several concerns about the GCRER/estrogen system for in vivo stem cell expansion. The appreciable levels of estrogen in human plasma, especially in women, may evoke undesired activation of the fusion proteins [UShiroyama T, et al., Acta.Obstet.Gynecol.Scand.,(1989) 68: 139-143; Ushiroyama T, et al., Horm.Res.,(1995) 44: 64-68]. In addition, possible adverse effects of exogenous estrogen, including the stimulatory role on some carcinoma cells from estrogen--dependent tissues, may limit Ml,Y-23-00 11:20 +81 298 41 2009 P.O6 R-983 Job-004 2000'05/24 WED 00:13 FAX +8.1 298 41 2009 SHIMIZU PATENT OFFICE I~006/049 its applicability [Vessey MP., verh.Dtsch.Ges.Path.,(1997) 81:
493-501; Joles JA, et al., J.Am.Soc.Nephrol.,(1997)8:1870-1876].
To overcome these drawbacks, we employed a mutant murine ER with substitution of arginine for glycine-525 (G525R). This mutant receptor is unresponsive to estrogen yet retains an affinity for a synthetic ligand 4-hydroxytamoxifen ( Tm) [ Danielian PS, et al . , Mol.Endocrinol.,(1993) 7: 232-240; Littlewood TD, et al., Nucleic Acids Res.,(1995) 23: 1686-1690]. We constructed cDNAs encoding fusion proteins GCRTmR ( full-length GCR and TmR-HBD) and ~GCRTmR
(lacking the G-CSF-binding site), and examined whether the chimeric genes would confer responsiveness to the appropriate stimulus on Ba/F3 and murine BM cells.
Disclo ure of the Invention The present invention seeks to overcome the problem of poor gene introduction efficiency by selectively amplifying in vivo or ex vivo hematopoietic stem cells into which a gene for treatment has been introduced. The objective of the invention is to provide a fundamental technique for gene therapy targeting hematopoietic stem cells.
In the field of gene therapy today, there are numerous problems to be overcome concerning the efficiency of gene introduction into target cells and the expression efficiency of the introduced gene. It is therefore obvious that establishing a system for selectively amplifying only the target cells containing the introduced gene will produce a major breakthrough.
In particular, if such a system is established for hematopoietic stem cells, which are the origin of many blood cells such as red MAY-23-00 11:20 +81 298 41 2009 P.07 R-983 Job-004 2000'05/24 WED 00:14 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE l~ 007/049 blood cells or white blood cells and which are considered to be the most preferable target cells for gene therapy, it would contribute significantly to the field of gene therapy.
G-CSF, which was traditionally thought to be a cytokine ( a hematopoietic factor) that selectively proliferates neutrophils, can also proliferate hematopoietic stem cells. The G-CSF receptor dimerizes itself when it is activated. A estrogen receptor dimerizes itself upon stimulation with estrogen. Considering these facts, the present inventors have constructed a chimeric gene between the G-CSF receptor gene and the estrogen receptor gene, introducing the chimeric gene into cells, and externally stimulating the cells by estrogen to forcibly dimerize the G-CSF
receptor portion of the chimeric gene praduct(W097/32971).
However, in this system, endogenous estrogen may activate the fusion proteins in vivo, depending on the hormonal states of the cell.
The present inventors replaced estrogen receptor with a mutant receptor(TmR) which specifically binds to 4-hydroxytamoxifen(Tm), to overcome limitations with wild-type estrogen receptor. IL-3-dependent Ba/F3 cells and hematopoietic progenitor cells transducted with the resultant fusion proteins(GCRTmR and d GCRTmR) were examined for ligand-inducible grows.
As a result, GCRTmR- and d GCRTmR-expressing Ba/F3 showed IL-3-independent growth in response to Tm,while the cells were unresponsive to estrogen at concentrations up to 10- 7 -10- g M.
Furthermore, murine bone marrow cells transducted with GCRTmR and d GCRTmR formed colonies in methyl-cellulose medium in response MAY-23-00 11:20 +81 298 41 2009 P.08 R-983 Job-004 2000'05/24 WED 00:14 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~008/049 to Tm, while virtually no colonies appeared with 10-7 M estrogen or without cytokines.
These results suggest that influences of the endogenous estrogen can be almost eliminated by using the GCRTmR/Tm or d GCRTmR/Tm system to expand gene-modified hematopoietic stem/progenitor cells.
Thus, the present invention was completed by developing a new system in which gene-modified hematopoietic stem cells selectively expanded as a result that G-CSF receptor of the chimeric fusion protein is activated by stimulation of exogenous 4-hydroxytamoxifen without the influences of the endogenous estrogen_ The present invention relates to:
1. A fusion protein comprising (a) a mutant estrogen receptor that is unresponsive to a estrogen and that is responsive to a tamoxifen, the derivative thereof, or the metabolite thereof, and ( b ) a domain comprising a cytokine receptor or a part thereof that imparts proliferation activity to a cell upon the association.
2. The fusion protein of (1) , wherein the "domain comprising a cytokine receptor or a part thereof that imparts proliferation activity to a cell upon the association" is derived from a G-CSF receptor or c-mpl.
3. A DNA encoding the fusion protein of (1) .
4. A vector comprising a DNA of (3) .

5. A cell carrying the vector of {4) .

6 . Amethod for selectively proliferating the cell of (5) , which comprises exposing the cell of (5) to a tamoxifen~ the derivative thereof, or the metabolite thereof.

- MAY-23-00 11:20 +81 298 41 2009 P.09 R-983 Job-004 2000'05/24 WED 00:15 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE C~ 009/049 7 . A vector comprising a desired exogenous gene and a DNA
encoding a fusion protein comprising (a) a mutant estrogen receptor that is unresponsive to a estrogen and that is responsive to a tamoxifen, the derivative thereof, or the metabolite thereof, and (b) a domain that imparts proliferation activity to a cell upon the association.

8. The vector of (7) , wherein the "domain that imparts proliferation activity to a cell upon the association" is derived from a cytokine receptor.

9 . The vector of ( 8 ) , wherein the cytokine receptor is a G-CSF receptor or c-mpl.
. The vector of (7) , wherein the "ligand-binding domain"
is derived from a steroid hormone receptor.
11 . The vector of (7) , wherein the steroid hormone receptor is an estrogen receptor.
12. A cell carrying the vector of (7) .
13 . Amethod for selectively proliferating the cell of ( 12 ) , which comprises exposing the cell of (12) to a tamoxifen, the derivative thereof, or the metabolite thereof.
14 . A kit comprising ( a ~ the vector of ( 4 ) or ( 7) , and (b) a tamoxifen, the derivative thereof, or the metabolite thereof .
A mutant estrogen receptor used in the present invention that is unresponsive to a estrogen and that is responsive to a tamoxifen, the derivative thereof, or the metabolite thereof is preferably that having substitution of glycine-525 with other amino acid, for example arginine, lysine [Mol. Endocrinol.,(I993) 7 : 232--40 ] .

MA'Y-23-00 11:20 +81 298 41 2009 P.10 R-983 Job-004 2000'05/24 WED 00:15 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~010/049 Any cytokine receptor can also be used in the present invention as long as it imparts proliferation activity to a cell upon association. Examples of the cytokine receptor are those belonging to the cytokine receptor family including G-CSF and c-mpl and those belonging to the tyrosine kinase receptor family including c-kit and flk2/flt3.
As the "domain which imparts proliferation activity to a cell" of the fusion protein according to the present invention, it is possible to use a molecule that transmits the intracellular proliferation signal, for example, an entire molecule of a cytokine receptor. It is also possible to use only a domain in the molecule that imparts proliferating activity to a cell . The latter approach is advantageous in proliferating the cell as it is because the domain proliferates the cell into which the fusion protein-coding DNA has been introduced without differentiating it. Furthermore, the vector used in the present invention includes not only a single vector molecule containing the fusion protein-coding DNA and a single vector molecule containing the fusion protein- coding DNA
and the exogenous gene, but also includes a vector system of multiple vector molecules comprising a combination of a vector containing the fusion protein-coding DNA and a vector containing the exogenous gene, for example, a binary vector system. Such a vector system of multiple vector molecules is usually introduced into a cell by co-transformation.
When a DNA encoding the fusion protein and an exogenous gene are inserted into the same vector, they may be made .into a dicistronic form containing an internal ribosome entry site (zRES) { published PCT Application in Japan No . Hei 6-509713 ) . For example, MA'Y-23-00 11:20 +81 298 41 2009 P.11 R-983 Job-004 2000'05/24 WED 00:16 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~011/049 it is possible to use a vector having a structure containing, from 5' to 3' , a promoter, an exogenous gene, IRES, and a DNA encoding the fusion protein or a vector having a structure containing, from 5'to 3' , a promoter, a DNA encoding the fusion protein, IRES, and an exogenous gene . The former type is generally used to allow most of the cells expressing the fusion protein gene to express the exogenous gene.
Moreover, in the present invention, the cell into which the vector is introduced includes hematopoietic stem cells, lymphatic cells, and cells other than these blood cells. In particular, hematopoietic stem cells that can self-proliferate are preferable in the present invention. Although the exogenous gene to be introduced into the cell in the present invention is not particularly limited, a normal gene corresponding to a defective gene is generally used in the field of gene therapy.
~~,y~ef Description of the Drawings Figure 1. Structures of the molecules involved in this study.
(A) the murine G-CSF receptor (GCR) , (B) a GCR derivative deleting the G-CSF-binding site (D GCR), (C) a mutant estrogen receptor specific for 4-hydroxytamoxifen (TmR), and (D, E) the fusion proteins between GCR and TmR ( GCRTmR ) or D GCR and TmR ( D GCRTmR ) .
Extracellular, extracellular region; G, G-CSF-binding site (amino acids 5-195); TM, transmembrane domain; Cytoplasmic, cytoplasmic portion; TA, transactivation domain; DNA, DNA-binding domain; HBD, hormone-binding domain; G525R, arginine substitution for glycine-525 in mouse estrogen receptor.
Figure 2. Retroviral vectors used in this study. (A) MAY-23-00 11:20 +81 298 41 2009 P.12 R-983 Job-004 2000'05/24 WED 00:16 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE 1012/049 MSCV/GCRTmR-IRES-CDBa. (B) MSCV/ D GCRTmR-IRES-CDBa. (C) MSCV/
GCRTmR-IRES-EGFP. (D) MSCV/IRES-EGFP. LTR, long terminal repeat;
GCRTmR, cDNA for GCRTmR; D GCRTmR, cDNA for D GCRTmR; IRES, encephalomyocarditis virus-derived internal ribosome entry site;
CDBa, cDNA for murine CDBa.
Figure 3. Western blot analysis of BaF/GCRTmR and BaF/0 GCRTmR cells. Lysates of control Ba/F3 (C; lane 1), BaF/GCRTmR
clones (lanes 2-4) and BaF/ D GCRTmR clones (lanes 5-7) were electrophoresed and transferred onto PVDF membranes.The membranes were hybridized with an anti-GCR (top) or an anti-ER (bottom) , antibody, and GCRTmR fusion protein (ca. 140 kDa) and D GCRTmR
fusion protein (ca.120 kDa) were visualized with an ECL kit (Amersham).
Figure 4. Growth curves of parental Ba/F3 cells (A), BaF /GCRTmR c lori a 1 ( 8 ) , c lone 2 ( C ) , c lone 3 ( D ) , BaF / D
GCRTmR c Zone 1 ( E ) , clone 2 ( F ) and clone 3 ( G ) . Cells were incubated with IL-3 (closed squares), G-CSF (closed circles), Tm (closed triangles), Ez (closed diamonds) or no stimulator (open squares). The graphs represent cumulative A49o-As~o values of XTT assay (means~SD of triplicate determinants).
Figure 5. Long-term culture of GCRTmR-t3ransduced Ba/F3 cells . BaF/GCRTmR clone 1 ( squares ) , clone 2 ( circles ) and clone 3 ( triangles ) were incubated with Tm ( closed symbols ) for 36 days .
Aliquots of the cells were periodically deprived of Tm and evaluated for viability (open symbols). The growth curves represent cumulative A490-A620 values of XTT assay (means~SD of triplicate determinants).
Figure 6. Dose-dependence assay of GCRTmR-transduced Ba/F3 . MFY-23-00 11:20 +81 298 41 2009 P.13 R-983 Job-004 200005/24 WED 00:17 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~013/049 cells . BaF/GCRTmR clone 1 ( squares ) , clone 2 { circles ) and clone 3 ( triangles ) were incubated with various concentrations of E2 (A) or Tm (B). XTT assay was performed on day zero and day four, and graphs represent the ratios of day four A,9o-A6zo to day zero A49o-Abzo (means ~ SD of triplicate determinants).
Figure 7 . Structures of the plasmid used in this study. These vectors express the fusion proteins between (A) pGCR and ER, (B) Mpl and ER, (C) D GCR-Mpl and ER, (D) D GCR-Mpl and TmR.
Figure 8. Cell proliferation assay of Ba/F3 cells transduced with the chimeric genes, O GCR-ER(left), Mpl-ER(right).
Figure 9. Cell proliferation assay of Ba/F3 cells transduced D GCR-Mpl-ER.
Figure 10. Proliferation of Ba/F3 cells expressing p GCR-Mpl-TmR. (Left)Growth curves of parental Ba/F3 cells expressing ~ GCR-Mpl-TmR. (Right)Long-term culture of Ba/F3 cells transduced with D GCR-Mpl-TmR. A representative clone of BaF/,L~
GCRmpITmR was cultured in the presence of IO-'M Tm, and was split every three to four days . 27 days later, the cells were washed and further cultured in the presence or absense of Tm. MTS assays were performed as described below.
Figure 11. Expression of CD34 in the bone marrow cells into which a OGCR-Mpl-TmR-IRES-EGFP" has been introduced, detected by f low cytometry.
Best Mode for Implemen inQ the Invention Example 1 Examination for ligand-inducible growth using the selective amplifier gene encoding the fusion proteins, GCRTmR and D GCRTmR .

MAY-23-00 11:20 +81 298 41 2009 P.14 R-983 Jab-004 20,00 05/24 WED 00:17 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE ~ 014/049 ( 1 ) Construction and expression of GCRTmR and ~GCRTmR chimera.
A murine IL-3 expression plasmid pBMG-hph-IL3 was constructed for high-titer production of the cytokine by cultured cells . This plasmid contains a Rous sarcoma virus promoter-driven IL-3 cDNA, and a hygromycin B-resistance gene (from pY3 plasmid) in the pBMGNeo backbone [Gorman CM, et al., Proc.Natl.Acad.Sci.USA.,(1982) 79: 6777-6781; Fung MC, et al., Nature,(1984) 307: 233-237; Blochlinger K, and Diggeimann H., Mol_Cell.Biol.,(1984) 4:2929-2931;Karasuyama H, and Melchers F., Eur.J.Immunol.,(1988) 18: 97-104].
The structures of GCR, D GCR, ~m-responsive mutant estrogen .
receptor (TmR), and the newly constructed selective amplifier gene products (GCRTmR and , ~GCRTmR) are shown in Figure 1. GCRTmR was a fusion construct between the full-length mouse GCR and the G52SR
mutant murine ER, which was expected to bind G-CSF and Tm to transmit growth signals. As a derivative, D GCRTmR was constructed by deleting the G-CSF-binding domain (amino acids 5-195)from GCRTmR.
We cloned the GCRTmR and ~GCRTmR eDNAs to make bicistronic retrovirus vectors which also harbored the murine CDBa gene as a second cistron (MSCV/GCRTmR-IRES-CDBa and MSCV/ D GCRTmR-IRES-CDBa; Figure 2).
To transduce the modified selective amplifier genes into hematopoietic cells, bicistronic expression vectors were constructed as follows. The murine phosphoglycerate kinase promoter-neomycin phosphotransferase gene cassette (EcoR I-Sal I) in MSCV2.2 retrovirus (a gift from Dr. R. G_ Hawley, University of Toronto, Canada ) was replaced with the murine CDBa cDNA under control of the encephalomyocarditis virus (EMCV)-derived internal MAY-23-00 11:20 +81 298 41 2009 P.15 R-983 Job-004 2000 05/24 WED 00:18 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~015/049 ribosome entry site sequence (IRES; nucleotides 259-B33 of EMCV-R
genome) to construct MSCV/IRES-CDBa [Hawley RG, et al., Gene Ther.,(1994) 1: 136-138; Nakauchi H, et al., Proc.Natl.ACad.Sci.USA., (1985) 82: 5126-5130, Duke GM, et al., J.Virol. , ( 1992 ) 66 : 1602-1609 ] . The murine cDNAs encoding GCR and ~GCR were obtained as BamH I-Pme I fragments from pMX/ GCRER and pMX/ D GCRER respectively [Ito K, et al., Blood,(1997) 90:
3884--3892 ] . TmR cDNA was derived from pHS + ERTM ( a gift from Drs .
T. D. Littlewood and G. I. Evan, Imperial Cancer Research Fund, London, UK) [Danielian PS, et al., Mol.Endocrinol.,(1993) 7:
232-240; Littlewood TD, et al., Nucleic Acids Res.,(1995) 23:
1686-1690], by polymerase chain reaction (PCR) with primer A
(5'-TAC GTT TAA ACG ATC CGG GCA CTT CAG GAG-3'; SEQ ID NO:l) and primer B (5'-CTG TCG ACA CTA GTA GGA GCT CTC AGA TCG-3'; SEQ ID
NO: 2 ) , creating a Pme I site in the 5 ~ -end and a Sal T site in the 3'-end [Littlewood TD, et al., Nucleic Acids Res.,(1995) 23:
1686-1690; Kodiara H, et al., Jpn.J.Cancer Res.,(1998) 89:
741-747]. Along with TmR cDNA, the GCR or D GCR was cloned respectively into Bgl II-Xho I site of MSCV/IRES-CD8a by trimolecular ligation, and the resultant vectors were designated as MSCV/GCRTmR-IRES-CDBa and MSCV/p GCRTmR-IRES-CDBa.
MSCV/GCRTmR-IRES-EGFP was constructed to transduce primary murine hematopoietic cells by replacing the murine CDSa cDNA in MSCV/GCRTmR-IRES-CDBa with a fragment encoding the enhanced green fluorescent protein (EGFP; derived from pEGFP-1, Clontech, Paio Alto, CA) . IRES-EGFP without a selective amplifier gene was cloned into MSCV2.2 backbone as a control (MSCV/IRES-EGFP).
To prepare GCRTmR and ~ GCRTmR gene-containing retroviruses, MAY-23-00 11:20 +81 298 41 2009 P.16 R-983 Job-004 200005/24 WED 00:18 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE C~O16/049 r BOSC23 cells were lipofected with MS CV/ GCRTmR-IRES-CDBa or MSCV/
p GCRTmR-IRES-CDBa, and the supernatants were harvested. The viral supernatants were used to infect IL-3-dependent Ba/F3 cells according to a fibronectin-assisted infection procedure [Hanenberg H, et al., Nat.Med.,(1996) 2: 876-882].
Ba/F3 cells (Riken Gene Bank RCB0805, Tsukuba, Japan) were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Bioserum, Victoria, Australia), 100 units/ml(u/ml) penicillin-100,icg/ml streptomycin (Irvine Scientific, Santa Ana, CA), and 0.5% conditioned medium of C3HlpTl/2 cells (Riken Gene Bank RCB0247) transfected with pBMG-hph-IL3 as a murine IL-3 source.
Titration studies with mouse BM progenitors revealed that this dose of the conditioned medium had an equivalent titer to 100 U/ml IL-3.
BOSC23 (American Type Culture Collection [ATCC] CRL-11554, Mantissas, vA) and GP+ E86 (kindly provided by Dr. A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA) ecotropic packaging cells were maintained in Dulbecco's modified Eagle medium (Life Technologies) containing 10% FBS.
All retroviral transduction experiments were performed in P2 facilities, according to the institutional recombinant DNA
biosafety guidelines. BOSC23 cells were transfected with MSCV/GCRTmR-IRES-CD8a or MSCV/ D GCRTmR-IRES-CDBa using Lipvfectamine (Life Technologies) and the viral supernatants were harvested on day two post-lipofection. Fibronectin-assisted transduction of Ba/F3 cells was carried out on 6-well plates precoated with RetroNectin (Takara Shuzo, Otsu, Japan) according MAY-23-00 11:20 +81 298 41 2009 P.17 R-983 Job-004 2000 05/24 WED 00:19 FAX +B1 298 41 2009 SHIMIZU PATENT OFFICE 0 017/049 to a standard procedure [Hanenberg H, et al. , Nat.Med. , ( 1996 ) 2 876-882 ] . After retroviral infection, the transduced Ba/F3 cells were selected with a Magnetic Cell Sorting ( MACS ) system ( Miltenyi Biotec, Bergisch Gladbach, Germany). Aliquots of 1 x 10' Ba/F3 cells were incubated with anti-CDBa antibody-conjugated microbeads, and CDBa-positive cells were recovered according to the manufacturer ~ s protocol. The selected cells were analyzed for CDea expression by fluorescence-activated cell sorting(FACS) with a fluorescein isothiocyanate (FITC)-labeled anti-marine CDBa antibody (Pharmingen, San Diego, CA) and a FACScan (Becton Dickinson, Palo Alto, CA). Successfully transduced Ba/F3 cells (BaF/GCRTmR and BaF/~ GCRTmR) were cloned by limiting dilution, with an initial incubation with IL-3 for six days followed by expansion with 10-' M Tm (Sigma, St. Louis, MO).
After transduction, CDBa expression in an aliquot of Ba/F3 cells was analyzed by FRCS, and the transduction efficiency was estimated to be between 38 % and 54% by counting CDBa-positive cells .
The remainder of the cells were subjected to MACS selection, and nearly 100% of the recovered Ba/F3 were CDBa-positive.
Subsequently, the selected Ba/F3 cells were cloned by limiting dilution; 11 out of 52 isolates of HaF/GCRTmR and nine out of 20 isolates of BaF/D GCRTmR showed Tm-responsive growth. Three of these isolates expressing GCRTmR or ~GCRTmR were randomly chosen for further characterization, and FAGS confirmed CD8a expression in these clones.
Expression of GCRTmR and D GCRTmR in the selected Ba/F3 clones were determined by a western blot as shown in Figure 3.
Aliquots of 1 x 10' parental and transduced Ba/F3 cells were MAY-23-00 11:20 +81 298 41 2009 P.18 R-983 Job-004 200005/24 WED 00:19 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~018/049 lysed with NP-40 lysis buffer ( 1$ NP-40, 150 mM NaCl, 50 mM Tris-HCl [pH 7.4~, 500 U/ml aprotinin, and I mM phenylmethylsulfonyl fluoride). Protein concentrations of the lysates were determined by BCA Protein Assay (Pierce, Rockford, IL) . Protein samples ( 10 ,u g/lane) were electrophoresed on 7.5$ sodium dodecyl sulfate-polyacrylamide gels and electroblotted onto Immobilon-1~
polyvinylidene fluoride (PVDF) membranes (Millipore, Yonezawa, Japan). After blocking with 4~ bovine serum albumin (Boehringer Mannheim) , the membranes were incubated with an anti-GCR antibody (M-20; Santa Cruz Biotechnology, Santa Cruz, CA) or an anti-ER
antibody (MC-20; Santa Cruz Biotechnology). The fusion proteins were visualized by an ECL system (Amersham, Little, Chalfont, UK) .
Probing with either an anti-GCR or an anti-ER antibody revealed a peptide of 140 kDa ( lanes 2-4 ) in the GCRTmR-transduced clones, and a 120 kDa protein ( lanes 5-7 ) in the p GCRTmR-transduced clones, with the same apparent molecular weights for the fusion proteins GCRER and p GCRER described previously [ Ito K, et al., Blood,(1997) 90: 3884-3892 ] .
( 2 ) Proliferation of Ba/F3 expressing GCRTmR or ~GCRTmR.
Representative BaF/GCRTmR and BaF/ D GCRTmR clones were stimulated by IL-3, 10-9 M human G-CSF (rhG-CSF; provided by Chugai Pharmaceuticals , Tokyo, Japan ) , 10-' M Tm or 10-' M ~3 -estradiol ( Ea ;
Sigma) . Cultures containing IL-3 or G-CSF were split every three to four days, while Tm-containing cultures were diluted every four days. Cell proliferation assay was performed periodically in 96-well microtiter plates by the 2,3-bis (2-methoxy-4-vitro-5-sulfophenyl)-5- [(phenylamino)-carbonyl] -2H-tetrazolium ...,.._..,~-.-~_ ____-__~ ~_... .. ___.~..___ . .

MAY-23-00 11:20 +81 298 41 2009 P.19 R-983 Job-004 2000 05/24 WED 00:20 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~019/049 hydroxide (XTT) method using Cell Proliferation Kit zz (Boehringer Mannheim, Mannheim, Germany) (Scudiero DA, et al., Cancer Res.,(1988) 48: 4827-4833]. For hormone dose-response analysis, transduced Ba/F3 cells were stimulated by various concentrations of Tm or E~, and XTT assay was performed on day zero and day four.
The final concentration of ethanol used to dilute Tm and »z was 0.1$ in all the culture conditions.
Parental Ba/F3 cells were dependent on IL-3, and all cells died upon its withdrawal. G-CSF, Tm, and EZ did not support untransduced Ba/F3; switching from IL-3 to any of these stimuli resulted in rapid and extensive apoptosis (Figure 4A). GCRTmR-transduced Ba/F3 cells proliferated not only with IL-3, but also grew continuously with either 10-9 M G-CSF or 10-' M Tm. The growth rates of the GCRTmR-transduced clones were almost identical regardless of whether the cells were incubated with IL-3 or G-CSF, but their responses to Tm varied as shown in Figure 4B-D. Among three clones examined, clone 3 showed the greatest growth rate with Tm which was comparable to those with IL-3 and G-CSF. Clone 2 showed the least response and clone 1 showed an intermediate response to Tm. The amount of GCRTmR protein in clone 2 seemed a little less than the other two clones (Figure 3), which may account for the clonal variation in sensitivity to Tm. In contrast to Tm stimulation, 10-' M EZ had no effect on BaF/GCRTmR clones. In our previous study, Ez at this dose was optimal to support Ba/F3 cells expressing GCRER or D GCRER [Ito K, et al., Blood,(1997) 90:
3884-3892]. Thus, these observations suggested that GCRTmR was selectively activated by Tm while it was inert to Ez. Meanwhile, the BaF/~GCRTmR clones did not respond to G-CSF but proliferated MAY-23-00 11:20 +B1 298 41 2009 P.20 R-983 Job-004 20,00 '05/24 WED 00:20 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE CdJ020/049 in response to Tm (Figure 4E-G). As was seen in the GCRTmR-transduced Ba/F3, variable sensitivity to Tm was observed among BaF/ ~GCRTmR clones, which may also depend an ~GCRTmR expression level.
When the BaF/GCRTmR clones were subjected to long-term culture Without IL-3, Tm alone could support the cells for at least 36 days, and the cells stopped growing and died within 24 hours upon removal of Tm from the media (Figure 5 ) . Thus, BaF/GCRTmR cells maintained a Tm-dependence throughout the culture period, and the on/off switching of the growth signal via GCRTmR was effectively controlled by Tm.
(3) Hormone dose-dependence of GCRTmR-mediated growth.
To determine the specificity of GCRTmR for Tm against EZ, growth rates of BaF/GCRTmR clones were examined at various concentrations of Tm and E2 ( Figure 6 ) . BaF/GCRTmR clone 3 partially responded to 10-° M Tm, and all the three clones grew well with Tm at 10-' - 10'6 M. In contrast, these clones were refractory to estrogen; no growth was observed with up to 10-' M EZ . Clone 3 showed a limited response to IO-6 M Ez, while the other clones were inert with this dose of EZ. Thus, GCRTmR-expressing cells appeared to be at least 100-fold more sensitive to Tm against E2. Since E, induced proliferation of GCRER-expressing Ba/F3 at 10-1° M or greater concentrations[Ito K,et al.,Blood,(1997)90:3884-3892], GCRTmR-expressing Ba/F3 cells were at least 1000-fold more resistant to Ea than GCRER-expressing cells. When Ez yr Tm was added to the parental and transduced Ba/F3 cultures with IL-3, neither reagent showed toxic effects at concentrations up to 10'6 M. Taken MAf-23-00 11:20 +81 298 41 2009 P.21 R-983 Job-OD4 20p0 05/24 WED 00:21 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE l~ 021/049 together, the optimal concentration of Tm to stimulate BaF/GCRTmR
appeared to be 10-' - 10-6 M.
( 4 ) Clonogenic progenitor assay of transduced murine bone marrow cells.
BM cells from 5-FU-treated mice were transduced respectively with retrovirus vectors containing the genes for EGFP, GCRER, GCRTmR and O GCRTmR.
GCRTmR, D GCRTmR and EGFP retroviral supernatants were prepared by transfecting BOSC23 cells as described above.
GCRER-viral supernatant was obtained from a selected GP + E86/GCRER
producer clone [Ito K, et al., Blood,(1997) 90: 3884-3892].
Six-week-old C57HL/6 mice (purchased from Clea, Tokyo, Japan) were injected intraperitoneally with 150 mg/kg 5-fluorouracil (5-FU;
F. Hoffmann-La Roche, Basel, Switzerland), and BM cells were flushed from femora and tibiae two days later. The cells were prestimulated with 100 ng/ml recombinant rat stem cell factor ( rrSCF; kindly provided by Amgen, Thousand Oaks, CA) and 100 U/ml recombinant human IL-6 (rhlL-6; kindly provided by Ajinomoto, Yokohama, Japan ) in a -MEM containing 2 0$ FBS at a starting density of 2 x 106 cells/ml. After 48 hours of prestimulation, BM cells were incubated (5 x 105 cells/ml) in the viral supernatants on Retro-Nectin-coated plates in the presence of 100 ng/ml rrSCF and 100 U/ml rhlL-6 for three days, with the vector-containing media changed five times. Mock transduction was similarly performed without using a viral supernatant.
The transduced and untransduced BM cells were harvested, and plated onto Petri dishes at 1 x 105 cells/dish with 1 ml StemPro ' MAY-23-00 11:20 +81 298 41 2009 P.22 8-983 Job-004 200 05/24 WED 00:22 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~ 022/049 Y

medium (Life Technologies) containing 10-9 M rhG-CSF, 10-' M Tm, 10-' M E2, or without stimulator . After ten days of incubation at 37°C in a humidified atmosphere of 5% COz in air, colonies were scored. EGFP expression was examined With an inverted fluorescence microscope (IX70; Olympus, Tokyo, Japan).
Table 1 summarizes the clonogenic progenitor assay.
Table 1 Number of coloniesa Transgene Tm EZ None G-CSF

GCRER 68.0'12.2 100.17.2 19.36.1 184.7111.5 GCRTmR 20.2 1.9 0 0 182.7 15.3 .AGCRTmR 52.0 15.5 0.7 0.9 0.3 0.5 136.5 11.2 EGFPb 0 0 0 108.7 2.1 None 0 0 0 194.0 13.0 'Each value represents mean ~ SD of hexaplicate determinants.
bEGFP: enhanced green fluorescent protein.
When stimulated by G-CSF, 100-200 colonies were observed in every culture dish, no matter what supernatant was used during transduction. Untransduced or EGFP-transduced BM cells yielded no colony with Ez or Tm alone. GCRER-transduced BM cells gave rise to about 100 colonies in response to Ez, 60-80 colonies with Tm, and even about 20 colonies were formed without any stimulator.
Comparable background colonies were observed in our previous study [ Ito K, et al. , Blood, ( 1997 ) 90 : 3884-3892 ] , implying nonspecific activation of GCRER by some estrogen-like substances in the media .
In contrast, GCRTmR- and ~GCRTmR-transduced BM showed very strict . MA'i'-23-00 11:20 +81 298 41 2009 P.23 R-983 Job-004 2000 05/24 WED 00:22 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~ 023/049 responses. GCRTmR-transduced BM gave rise to about 20 colonies out of 1 x 105 cells in the presence of 10-' M Tm, but absolutely no colonies were formed with 10-' M Ez or without a stimulator. D
GCRTmR-transduced BM yielded about 50 colonies with Tm, while minimum number of colonies (less than one out of 1 x 105 cells) were formed in the dishes With E2 or no stimulator. These results clearly demonstrated that TmR-containing chimeric receptors (GCRTmR and ~GCRTmR) transmitted growth signals very specifically in response to Tm, with negligible activation by EZ in our culture setting. Morphologically, the Tm-induced colonies were mostly myeloid and mixed colonies, including a few erythroid ones; this result paralleled our previous finding on EZ-induced colonies derived from GCRER- and D GCRER-transduced BM [Ito K, et al., Blood,(1997) 90: 3884-3892].
When BM cells were infected by MSCV/GCRTmR-IRES-EGFP
retrovirus and subjected to progenitor assay, a total of 121 colonies were formed out of 6 x 105 cells in response to 10-' M Tm.
Most of the Tm-.respons~.ve colonies fluoresced when observed with an inverted fluorescence microscope, indicating that the transduced progenitors expressed both GcRTmR and EGFP trans-genes during colony formation.
Example 2 Examination for ligand-inducible growth using the selective amplifier gene encoding the fusion proteins, D GCR-Mpl-ER and L~GCR-Mpl-TmR.
( 1 ) plasmid construction All enzymes used were purchased from New England Biolabs Inc (Beverly, MA). A mammalian expression vector pCMX-MfasER (kindly MAf-23-00 11:20 +81 298 41 2009 P.24 R-983 Job-004 20Q0 05/24 WED 00:22 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~024/049 provided by Dr A. Kakizuka, Kyoto University, Kyoto, Japan) , which contains the sequence encoding the HBD of rat estrogen receptor (ER) was digested with BamHI and EcoRI. The BamHI-EcoRI fragment containing the ER-HBD was separated by agarose gel electrophoresis and electroelution. This fragment was subcloned into BamHI-EcoRI
site of the plasmid pBluescript (TOYOBO, Japan) by ligation.
Further, tamoxifen receptor (TmR) cDNA was derived from a retroviral vector MSCV-DGCRTmR-IRES-EGFP ( kindly provided by Dr R. Xu, Jichi Medical School, Tochigi, Japan) which contains the sequence encoding the HBD of mouse TmR, by polymerise chain reaction (PCR) with primer A (5'-CTGGATCCGGGCACTTCAGGAGAC-3'; SEQ
ID N0:3, creating a BamHI site) and primer B (5'-CTGTCGACCACTAGTAGGAGCTCTCA-3'; SEQ ID N0:4, creating a Salt site).
This cDNA was subcloned into BamHI-Sall site of the pBluescript by ligation. On the other hand, a mammalian-expression vector pcDNA3 . 1-a-mpl ( kindly provided by Dr M. Takatoku, Jichi Medical School, Tochigi, Japan) which contains the cDNA for human c-mpl between the EcoRI and Xbal sites was digested with EcoRI and SacI .
The EcoRI-SacI fragment containing most of the extracellular domain of c-mpl was separated by agarose gel electrophoresis and electroelution. Further, the rest c-mpl cDNA between SacI site and the c-terminal cytoplasmic domain was constructed by PCR using the pcDNA-c-mpl as a template with primer C (5'-CCCACCTACCAAGGTCCCTGG-3'; SEQ ID N0:5) and primer D (5'-CGGGATCCAGAGGCTGCTGCCAATAG-3' ; SEQ ID N0:6, creating a BamHI
site). Then, the murine phosphoglycerate kinase (pgk) promoter-neomycin phosphotransferase gene (neo) cassette (EcoRI-BamHI) in MSCV2.2 retrovirus (a gift from Dr_ R. G. Hawley, ~~rwmw.o~. -.......,..... ......_.... .~.__. ........._ MAY-23-00 11:20 +81 298 41 2009 P.25 R-983 Job-004 20p0 05/24 WED 00:23 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~1025/049 r University of Toronto, Canada) was replaced with the EcoRI-SacI
fragment of c-mpl and the Sacl-BamHI fragment of c-mpl by trimolecular ligation to construct MSCV-mpl. The pBluescript-ER
and the pBluescript-TmR were digested with BamHI and Sall, and the ER and TmR fragments were separated by agarose gel electrophoresis and electroelution. These fragments were cloned into BamHI-Sall site of MSCV-mpl by ligation . The resultant vectors were designated as MSCV-mpl-ER or MSCV-mpl-TmR.
MS CV-~GCRmpl-ER and MS CV-~GCRmpl-TmR were constructed as follows. MSCV-pGCR-ER (kindly provided by Dr. KM Matsuda, Jichi Medical School, Tochigi, Japan), in which the GCR binding domain was deleted, was digested with HindIII and KpnI, and the HindIII-KpnI fragment containing a part of the ~GCR was separated by agarose gel electrophoresis and electroelution. The OGCR cDNA
between KpnI site and the transmembrane region was constructed by PCR using the MSCV- D GCR-ER as a template with primer E (5'-GAGTGGGTACCTGAGGCCCCTAGG-3'; SEQ ID N0:7) and primer F (5'-AACTCGAGGCAGCAGAGCCAGGTCAC-3';SEQID NO:B,creating a Xhol site).
on the other hand, the cDNA containing the cytoplasmic region of c-mpl was constructed by PCR using the pcDNA-c-mpl as a template with primer G (5'-AACTCGAGAGGTGGCAGTTTCCTGCA-3'; SEQ ID N0:9, creating a Xhol site) and primer D. The extracellular region of D GCR and the cytoplasmic region of c-mpl were cloned into HindIII-BamHI site of pEGFP-Nl (Clontech, Palo Alto, CA, USA) by ligation ( pEGFP-~ GCRmpl ) . Then, pgk and neo cassette ( BglI I-Sall ) in MSCV2.2 were replaced with the BglII-BamHI fragment containing the ~GCRmpI and the BamHI-SalI fragment containing the ER or the TmR. The resultant constructs were designated as MSCV-~GCRmpl-ER

MAY-23-00 11:20 +81 298 41 2009 P.26 R-983 Job-004 20.00 05/24 WED 00:24 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE C~02C/049 or MSCV- ~ GCRmpl-TmR.
MSCV- D GCRmpl-TmR-IRES-EGFP was constructed as follows.
MSCV-~GCR-TmR-IRES-EGFP was digested with HpaI and Clal, and the HpaI-ClaI fragment containing IRES (internal ribosome entry site)-EGFP was separated by agarose gel electrophoresis and electroelution. Then, pgk and neo cassette (BglIi-Sall) in MSCV2.2 was replaced with this fragment. The resultant constructs MSCV-IRES-EGFP was digested with Xhol and Clal, and the XhoI-ClaI
fragment containing IRES-EGFP was separated by agarose gel electrophoresis and electroelution. This fragment was cloned into SalI-ClaI site of MSCV-0 GCRmpl-TmR by ligation, and the resultant constructs was designated as MS CV-~GCR-TmR-IRES-EGFP. All these constructs were confirmed by sequence analysis.
( 2 ) Cell proliferation assay Since TPO is known to stimulate the growth of not only the megakaryocyte lineage but also primitive hematopoietic cells, the intracellular signals from Mpl may be appropriate for selective amplification of transduced HSC.
Structures of the vectors used in this study were schematically shown in Figure 7 . BosC23 cells were transfected with MSCV/ D GCR-ER or MSCV/Mpl-ER or MSCV/ O GCR-Mpl-ER or MSCV/ D
GCR-Mpl-TmR-IRES-EGFP using Transfection MBS Mammalian Transfection Kit (Stratagene) and the viral supernatants were harvested on day two post-transfection. Fibronectin-assisted transduction of IL3-dependent Ba/F3 cells was carried out on 6-well plates precoated with CH296 fibronectin fragment (Retronectin;
Takara Shuzo) according to a standard procedure. After retroviral - MAC'-23-00 11:20 +81 298 41 2009 P.27 R-983 Job-004 2Q00 ,05/24 WED 00:24 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~027/049 , infection, D GCR-Mpl-TmR-IRES-EGFP transduced Ba/F3 cells (BaF/
p GCR-Mpl-TmR) were selected with EPICS Elite ESP Cell Sorter.
GFP-positive cells were removed and were cloned by limiting dilution with 10-'M 4-hydroxytamoxifen (Tm).
A quantity of 4x103 untransduced or transduced Ba/F3 cells in 100,u1 was cultured in the presence or absence of lng/m1 rmIL-3, 100ng/ml recombinant human thrombopoietin (rhTPO), or 10-'M ,(3-estradiol (E2; Sigma), or 10-'M Tm in 96-well microtiter plates.
Cell proliferation assay was periodically performed using CellTier 96 Aqueous One Solution Cell Proliferation Assay {3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS); Promega, Madison, WIC
essentially according to the manufacturer's instruction. In brief, 20,cc1 per well of MTS-labeling mixture was added. After the incubation at 37°C for 2 hours, the spectrophvtometeical absorbance was measured at the wave length of 490 nm and 650 nm.
Mpl-mediated signals have been employed in selective amplifier genes. When Mpl-ER chimera was expressed in the interleukin-3 (IL-3)-dependent Ba/F3cells, the cells acquired the ability to proliferate in response to both estrogen and TPO in the absence of IL-3 (Figure 8).
However, attempts to minimize the effects of serum TPO by deleting the extracellular domains of Mpl resulted in a total loss of the response to estrogen as well as TPO.
To solve this issue, the extracellular portion of Mpl-ER was replaced with that of D GCR-Mpl-ER. Transduction of Ba/F3 cells with the D GCR-Mpl-ER gene conferred an estrogen-dependent growth ability on Ba/F3, while the cells were unresponsive to G-CSF or .""~"_ _.____... __ _..._ ,MAC'-23-00 11:20 +81 298 41 2009 P.28 R-983 Job-004 2Q00 05/24 WED 00:25 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE f~028/049 s TPO (Figure 9).
A comparative study for the stimulatory effect of estrogen suggested that the Ba/F3 cells expressing D GCR-Mpl-ER
proliferated better than the cells expressing D GCR-ER or Mpl-ER.
To eliminate the effects of endogenous E2, ER was replaced with a mutant receptor (TmR) which specifically binds to Tm. D
GCR-Mpl-TmR-expressing Ba/F3 cells showed Tm-dependent growth, while the cells were unresponsive to E2 (Figure 10).
(3) Transduction of murine bone marrow (BM) cells D GCR-ER, mpl-ER and D GCRmpI-ER retroviral supernatants were prepared by transfecting BOSC 23 cells as described above.
Six-week-old C57BL/6 mice (purchased from Clea, Tokyo, Japan) were injected intraperitoneally with 150 mg/kg 5-fluorouracil (5-FU;
F. Hoffmann-La Roche, Basel, Switzerland), and 2 days later, BM
cells were flushed from femora with IMDM containing 5$ FBS. The BM cells were collected by density centrifugation using Lympholyte-M (Cedarlane). Approximately 5x146/ml HM cells were prestimulated with 100ng/ml of recombinant murine SCF (rmSCF;
Pepro Tech Inc, London, England) and 100 U/ml of recombinant human IL-6 (rmlL-6; kindly provided by Ajinomoto, Yokohama, Japan) in IMDM (GIBCO-BRL) containing 20$ FBS at 37°C for 48 hours in a humidified atmosphere of 5~ C02 in air. Subsequently, the cells were resuspended in 2ml of viral supernatant containing rmSCF and rhlL-6 at a concentration of 5xlOs cells/ml, transferred to the 6-well plates precoated with 20,CLg/cma of CH296, and then incubated at 37°C in a humidified atmosphere of 5% COZ in air. for 72 hours .

~ MAY-23-00 11:20 +B1 Z98 41 2009 P.29 R-983 Job-004 2000\05/24 WED 00:25 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE ~ 029/049 ..

During this transduction period, viral supernatant was changed five times. Mock transduction was similarly performed without using a viral supernatant.
(4) In vitro clonogenic progenitor assay The transduced and untransduced BM cells were harvested, and 1x10' cells each were plated in 35-mm dish with 1 ml stemPro medium (GIBCO-BRL) in the presence or absence of 100ng/ml of rhTPO or 10-' M of E2 . In some experiments, 100 ng/ml of rmSCF, 100 U/ml of rhIL-6, 100 ng/ml of rhTPO, and 2 U/ml of recombinant human erythropoietin ( rhEpo; Chugai Pharmaceutical ) were added to the cultures . After days of incubation at 37°C in a humidified atmospherE of 5$ Co2 in air, colonies were scored using an inverted microscope.
( 4 )Isolation of cynomolgus CD34-selected bone marrow cells ( BMCs ) Clinically healthy adult cynomolgus monkeys born and reared in Tsukuba Primate Center for Medical Science, National Institute of Health, were used for experiments. BMCs were harvested from femora and suspended in lysis buffer to dissolve red blood cells.
Immunomagnetic selection of the CD34+ cells was accomplished using Dynabeads system (Dynal AS, Oslo, Norway) according to the manufacturer's instruction.
(5) Transduction of CD34-selected BMCs 293T cells were co-transfected with MSCV- p GCRmp1-TmR-IRES-EGFP and pCL-Ampho using Transfecti~on MHS Mammalian Transfection Kit (Stratagene, La Jolla, CA) and the viral supernatants were harvested on day two and three post-transfection .

~ MA'Y-23-00 11:20 +81 298 41 2009 P.30 R-983 Job-004 2000,05/24 WED 00:26 FAX +81 298 41 2009 SHIMIZU PATENT OFFICE 1030/049 ~ ~ ..

Cynomolgus CD34-selected cells were placed 3.n 6-well plates precoated with 20,ccg/cmZ of CH296 and were cultured for 24 hours at 37°C with 5$ COz in Iscove~s Dulbecco~s medium (IMDM) supplemented with 10$ FBS, 50 ng/ml rhIL6, 100ng/ml rhSCF, 100 ng/ml rhFlt-3 ligand (Research Diagnostic Inc) and 100ng rhTPO.
Subsequently, the cells were resuspended in lml of viral supernatant containing all cytokines described above at a concentration of 1x105cells/ml. During this transduction period, viral supernatant was changed six times. Mock transduction was similarly performed without using a viral supernatant.
(6) Suspension culture and FACE analysis After retroviral transduction, CD34-selected BMC were washed and cultured in IMDM supplemented with 10$ FBS, 50 U/ml penicillin-50 ug/ml streptomycin, containing lOOng/ml hrFlt-3 ligand, 10-'M Tm or without stimulator. On the day 34, aliquots of cells were removed from the suspension culture and tested by flow cytometry.
When cynomolgus monkey CD34-selected bone marrow cells transduced with p GCR-Mpl-TmR-EGFP were cultured in the presence of Tm, the ratio of GFP of the cells increased about 3-fold relative to the cells cultured in the absence of Tm (Figure 11).
industrial Apglicabilitv The present invention has made it possible to selectively amplify a cell into which an exogenous gene has been introduced, in response to an external stimulus, thereby enabling effective gene therapy even when the introduction efficiency of the gene into ' MAY-23-00 11:20 +81 298 41 2009 P.31 R-983 Job-004 20.00,05/24 WED 00:26 FAX +81 298 41 2009 SNIMIZU PATENT OFFICE f~031/049 the target cells is low. Furthermore, since the system for selectively amplifying cells of the present invention can be applied to various blood cells, the range of cells targeted in gene therapy has been widened. Therefore, the present invention provides an important basic technology, particularly in the field of gene therapy.

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(i) APPLICANT:DNAVEC RESEARCH INC.
(ii) TITLE OF INVENTION: GENE THAT IMPARTS SELECTIVE PROLIFERATIVE
ACTIVITY
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Claims (14)

1. A fusion protein comprising (a) a mutant estrogen receptor that is unresponsive to a estrogen and that is responsive to a tamoxifen, the derivative thereof, or the metabolite thereof, and (b) a domain comprising a cytokine receptor or a part thereof that imparts proliferation activity to a cell upon the association.

2. The fusion protein of Claim 1, wherein the "domain comprising a cytokine receptor or a part thereof that imparts proliferation activity to a cell upon the association" is derived from a G-CSF receptor or c-mpl.

3. A DNA encoding the fusion protein of Claim 1.

4. A vector comprising a DNA of Claim 3.

5. A cell carrying the vector of Claim 4.

6. A method for selectively proliferating the cell of Claim 5, which comprises exposing the cell of Claim 5 to a tamoxifen, the derivative thereof, or the metabolite thereof.

7. A vector comprising a desired exogenous gene and a DNA
encoding a fusion protein comprising (a) a mutant estrogen receptor that is unresponsive to a estrogen and that is responsive to a tamoxifen, the derivative thereof, or the metabolite thereof, and (b) a domain that imparts proliferation activity to a cell upon the association.

8 . The vector of Claim 7, wherein the "domain that imparts proliferation activity to a cell upon the association" is derived from a cytokine receptor.

9. The vector of Claim 8, wherein the cytokine receptor is a G-CSF receptor or c-mpl.

10. The vector of Claim 7, wherein the "ligand-binding domain" is derived from a steroid hormone receptor.

11. The vector of Claim 7, wherein the steroid hormone receptor is an estrogen receptor.

12. A cell carrying the vector of claim 7.

13. A method for selectively proliferating the cell of Claim 12, which comprises exposing the cell of Claim 12 to a tamoxifen, the derivative thereof, or the metabolite thereof.

14. A kit comprising (a) the vector of Claim 4 or Claim 7, and (b) a tamoxifen, the derivative thereof, or the metabolite thereof.