WO1995028490A1 - Vecteur d'expression contenant un promoteur non viral - Google Patents

Vecteur d'expression contenant un promoteur non viral Download PDF

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Publication number
WO1995028490A1
WO1995028490A1 PCT/US1995/004583 US9504583W WO9528490A1 WO 1995028490 A1 WO1995028490 A1 WO 1995028490A1 US 9504583 W US9504583 W US 9504583W WO 9528490 A1 WO9528490 A1 WO 9528490A1
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cells
gene
promoter
construct
mammal
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PCT/US1995/004583
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English (en)
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Anlong Xu
Shinichi Kudo
Minoru Fukuda
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Alliance Pharmaceutical Corp.
La Jolla Cancer Research Foundation
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Application filed by Alliance Pharmaceutical Corp., La Jolla Cancer Research Foundation filed Critical Alliance Pharmaceutical Corp.
Priority to AU22473/95A priority Critical patent/AU2247395A/en
Publication of WO1995028490A1 publication Critical patent/WO1995028490A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/108—Plasmid DNA episomal vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the present invention relates to an expression vector containing a non-viral promoter. More specifically, it relates to an expression vector containing the regulatory region of the human leukosialin gene which can drive the highly efficient expression of a heterologous gene in transfected human cell lines.
  • Gene therapy refers to transfer of new genetic material to the cells of an individual with resulting therapeutic benefit to the individual (Morgan and Anderson, (1993) A ⁇ u. Rev. Biochem. , 62:191-217) .
  • ADA adenosine deaminase
  • SCID severe combined immunodeficiency syndrome
  • PEG polyethylene glycol
  • Retroviral vectors encoding human ADA have been used to reconstitute human T-cells from patients affected by ADA " SCID (Ferrari et al. , (1992) Blood,
  • CD18 Leukocyte Adhesion Deficiency
  • Factor IX Factor IX
  • Hypercholesterolemia In addition to the treatment of genetic diseases, gene therapy may be applied to other disorders including AIDS and cardiovascular disease.
  • agents which prevent clot formation such as tissue plasminogen activator, have been introduced into endothelial cells (Dichek et al., (1991) Blood, 77:533-541) .
  • Gene therapy necessarily requires a strong promoter capable of driving expression of a desired heterologous gene in transfected cells.
  • Previously described expression vectors contain the SV40 early or late promoters (Templeton and Eckhart, (1984) Mol. Cell Biol . , 4:817; Sprague et al, (1983) , J " . Virol., 45:773; Lusky and Botchan, (1981) Nature 293:79) , long terminal repeat (LTR) sequences from mouse mammary tumor virus (MMTV) which acts as a promoter when induced by glucocorticoids such as dexamethasone .(Alton and Vapnek, (1979) Nature, 282:864; Lee et al. , (1981) Nature, 294:228; Mulligan and Berg, (1981) Proc. Natl . Acad.
  • LTR long terminal repeat
  • HTLV human T-cell leukemia virus
  • HTLV human T-cell leukemia virus
  • adenoviral vectors More recently, the used of adenoviral vectors has been described (Lemarchand et al., (1992) Proc . Natl . Acad. Sci . U. S.A . , 89:6482-6486) .
  • the expression vectors mentioned above all contain virus- derived promoters and do not exhibit tissue specificity which necessarily limits their usage. For example, these vectors cannot be used for tissue-specific in vivo gene expression (gene therapy) . In addition, there are at least theoretical risks associated with the use of virus-derived sequences in human gene therapy, including potential mutation and subsequent viral replication.
  • Leukosialin also known as CD43, is a highly glycosylated protein expressed on the surface of T-lymphocytes, granulocytes, monocytes, platelets and hematopoietic stem cells
  • Monoclonal antibodies against LS stimulate T-cell proliferation (Mentzer et al. , (1987) J. Exp. Med. , 165:1383) as well as natural killer cell and monocyte activation (Vargas-Cortes et al., (1988) Scand. J. Immunol . , 27:661; Nong et al. , (1.989) J. Exp . Med . , 170:259.)
  • the transcriptional regulatory region of the LS gene contains a guanine-rich element, but not a TATA or CAAT box typical of eukaryotic promoter regions (Kudo and Fukuda, (1991) J. Biol . Che . , 266:8483) .
  • a 14 nucleotide sequence located at position -53 to -40 (40 to 53 nucleotides upstream of the transcription start site) was critical to promoter function based on transient transfection assays (Kudo and Fukuda, ibid.) .
  • One embodiment of the present invention is a DNA construct comprising a heterologous gene, and a leukosialin promoter region operably linked to the heterologous gene in an expression vector.
  • the construct further comprises viral splicing signals and polyadenylation sequences.
  • these viral sequences are derived from SV40.
  • the heterologous gene is adenosine deaminase and the leukosialin promoter region consists essentially of SEQ ID NO: 1.
  • the leukosialin promoter region may be either truncated or elongated.
  • the cells are either T cells or T cell derivatives.
  • the T cells are either Jurkat or CEM cells.
  • the cells are transfected by cytofection (transfection with vector-cationic lipid complex) , calcium phosphate precipitation, electroporation or DEAE-Dextran.
  • the cells are transfected in vi tro .
  • the cells are transfected in vivo.
  • Still another embodiment of the invention is a method for introducing a gene into the cells of a mammal, comprising isolating cells from the mammal, transfecting the cells with the leukosialinpromoter-containing construct described hereinabove, wherein the construct contains the gene, and reintroducing the cells into the mammal.
  • the mammal lacks the gene.
  • the cells are T-cells, the mammal is human and the gene encodes adenosine deaminase.
  • the invention also provides a DNA expression cassette comprising the leukosialin promoter located upstream of a predetermined restriction site in operable juxtaposition to a heterologous gene inserted at the restriction site, wherein the promoter drives expression of the gene.
  • Yet another embodiment of the present invention is an expression vector comprising a promoter operably linked to a heterologous gene, the improvement comprising the promoter comprising the leukosialin promoter.
  • FIG 1 is a schematic diagram illustrating the construction of the pCAT-KX expression vector containing the human LS promoter. Amp, ampicillin resistance; CAT, chloramphenicol acetyltransferase.
  • Figure 2 shows schematic diagrams of the six expression vectors used in the transient transfections and CAT assays. CMV, cytomegalovirus; MMTV, mouse mammary tumor virus; LTR, long terminal repeat.
  • the present invention discloses a eukaryotic expression vector containing a human leukosialin (LS) promoter operably linked to a heterologous gene.
  • LS human leukosialin
  • the experiments described herein utilize the CAT gene to demonstrate the efficient cell-specific expression of the LS promoter-containing expression vector, any desired gene may be linked to this promoter using methods well known in the art of molecular biology.
  • the expression vector is, in essence, a "cassette”
  • any desired gene can be rapidly incorporated into the SalJ restriction site and operably linked to the LS promoter as described in Example 1.
  • the incorporated gene may be either prokaryotic or eukaryotic, although eukaryotic genes are highly preferred. Efficient expression of a reporter gene is generally accepted to be indicative of the ability of a promoter to drive expression of a heterologous gene.
  • the expression vector also contains a viral origin of replication and polyadenylation signals, preferably from Simian Virus 40 (SV40) , although other viral sequences including those from adenovirus, rous sarcoma virus or any other virus capable of operably providing these components is contemplated.
  • SV40 Simian Virus 40
  • the expression vector may also contain a selectable marker including, but not limited to, ampicillin resistance, streptomycin resistance, tetracycline resistance, neomycin resistance or hygromycin resistance.
  • this expression vector results in high-level expression of a heterologous gene in a number of hematopoietic cell types (Example 3) , it exhibits T-cell specificity compared to other commonly used expression vectors and will be particularly useful in T-cell specific gene delivery and in the treatment of gene deficiencies in T-cells. Since the promoter also drives high-level gene expression in other human cell types (Example 3) , it will also be useful in the treatment of other genetic and non-genetic defects including, but not limited to, cardiovascular disease, cancer and AIDS. The instant invention will be useful in the production of increased amounts of recombinant proteins in mammalian cells, particularly T-cells.
  • the vector may be introduced into T cells or T cell derivatives by a number of transfection techniques well known in the art including calcium phosphate precipitation, lipofection, cytofection, DEAE- dextran, electroporation, antibody conjugation and the like.
  • the cells may be expanded, lysed and the expressed protein isolated by conventional protein purification techniques.
  • T- cells may also be collected from a patient, transfected with the LS promoter-driven expression vector containing a gene of interest, and reintroduced into the patient, resulting in high level expression of a defective gene in vivo .
  • the LS promoter- driven expression vector containing a gene of interest may also be directly injected into a mammal and taken up by cells in vivo.
  • the expression vector may be transfected into any mammalian cell type, although preferred cells are T cells or T cell derivatives expressing LS, since these cells will also contain accessory proteins necessary for the highest expression levels of the heterologous gene linked to the LS promoter. Such accessory proteins include, for example, transcriptional activators and other DNA binding proteins.
  • the amount of LS-containing expression vector used for cell transfection in vi tro is between about 1 ⁇ g and about 100 ⁇ g to transfect between about 5 x 10 5 and 1 x 10 7 cells, depending on the cell type and transfection technique employed. In a particularly preferred embodiment, between about 5 ⁇ g and about 50 ⁇ g is used per 8 x lO 5 - 8 x lO 6 cells.
  • the amounts of vector and cells used for transfection will vary depending on the cell type and transfection method used. However, a person having ordinary .skill in the art can easily optimize the transfection conditions for any given vector and cell type without undue experimentation.
  • the vector may be delivered to mammalian cells in any conventional manner, including cationic lipid mediated transfection, calcium phosphate precipitation, electroporation, microinjection, gold particle bombardment, with viral capsid proteins, or as naked DNAdirectly injected into mammalianmuscle.
  • Example 1 consists of nucleotides -91 to +439, although longer sequences encompassing this region as well as truncated LS promoter regions containing part of this promoter region are also contemplated.
  • Other LS promoter regions capable of supporting high levels of heterologous gene expression can be determined by one having ordinary skill in the art by routine experimentation using restriction digestion of either the entire LS gene or the -91 to +439 region and are within the scope of the present invention.
  • mutations of any of these LS promoter sequences such as those produced by site-directed mutagenesis, are also contemplated.
  • Example 1 Construction of an expression vector containing the LS promoter
  • the construction of the pCAT-KX expression vector is summarized in Figure 1.
  • the procedure involves techniques and methods well known in the art of molecular biology, such as those found in Sambrook et al. (Molecular Cloning, a Laboratory Manual , 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989)
  • the plasmid pCAT-Basic (Promega, Madison, I) was digested with SalJ and BamHI and ligated to the SV40 DNA fragment containing the splicing signal and polyadenylation site isolated from pMSG-CAT (Pharmacia, Piscataway, NJ) .
  • the resulting plasmid was called L9-2.
  • the LS promoter comprising nucleotides -91 to +439 (Kudo and Fukuda, 1991; SEQ ID NO:l) was generated by polymerase chain reaction amplification using primers ST- 1 ( 5 ' - TTTAAGCTTTGGGAGCAGGCGGTGGGCAGGATG-3' ; SEQ ID NO:2) and ANM (5'- TTTGTCGACGGCAGCCGGAGAAGCAGAACACGC-3' ; SEQ ID NO:3) to which Hindlll and SalJ sites, respectively, were added.
  • One hundred ⁇ g of the leukosialin genomic clone was used as template.
  • the reaction was performed in a total volume of 50 ⁇ l 10 mM Tris-HCl, pH 8.3, 50 mM KC1, 1,5 mM MgCl 2 , 0.01% (w/v) gelatin, 200 ⁇ M dNTPs, 1 ⁇ M primer and 1 unit Tag DNA polymerase.
  • the amplification conditions were 94°C for 2 min., 55°C for 2 min. and 72°C for 3 min.
  • the amplification product was digested with SalJ and Hindlll and subcloned into the corresponding sites of plasmid L9-2 to form plasmid pKX.
  • the plasmid pCAT-KX was constructed by inserting the cDNA encoding the CAT gene (Pharmacia) into pKX at the SalJ sire. Verification of the correct insertion of the various DNA ' fragments into the expression vector was determined by sequencing across each joined DNA region. pCAT-KX and other commonly used expression vectors were transfected into different eukaryotic cell types as described in the following example.
  • Example 2
  • the following cell lines were used for transfection: Human leukemic Jurkat T cells, T lymphoblastoid CEM cells, human chronic myelogenous leukemia K562 cells, Raji lymphoma cells, human epithelioid carcinoma HeLa cells, and CHO cells. These cell lines are commercially available from the American Type
  • TIB 152 ATCC CCL 119, ATCC CCL 243, ATCC CCL 86, ATCC CCL 2.2 and ATCC CRL 9096, respectively.
  • Jurkat, CEM, Raji and K562 cells were cultured in RPMI 1640 medium containing 20% fetal calf serum (FCS) , 2 mM glutamine, 500 units/ml penicillin and 100 ⁇ g/ml streptomycin.
  • FCS fetal calf serum
  • HeLa and CHO cells were cultured in ⁇ -MEN medium (Gibco BRL, Gaithersburg, MD) containing 10% FCS, 2 mM glutamine, 500 units/ml penicillin and 100 ⁇ g/ml streptomycin.
  • the expression vectors used were pCAT-KX, pMSG-CAT driven by the MMTV LTR promoter (Pharmacia) , pcDL-SR ⁇ -CAT containing the SV 40 ori and HTLV-1 LTR (Takebe et al. , (1988) ⁇ ol. Cell Biol .
  • pcDNAI- CAT containing the CMV promoter (Invitrogen, San Diego, CA)
  • pCAT-Control containing the SV40 promoter and enhancer regions
  • pCAT-Basic Promega
  • pSVOKS-CAT which do not contain a promoter region as a negative control.
  • pSVOKS-CAT was constructed by inserting the CAT gene into pBluescript
  • CHO and HeLa cells (1 x 10 6 cells/dish) were transfected with 20 ⁇ g vector DNA using the LIPOFECTINTM reagent (a cationic lipid or cytofectin) (Gibco BRL) according to the manufacturer's instructions. Briefly, plasmid DNA was complexed with LIPOFECTIN in serum-free medium. The DNA-LIPOFECTIN complex was then added to cells. After a 4 hour incubation, fresh CU-MEN medium containing 10% serum was added. Cells were incubated at 37°C for 2 days to allow transient expression of the transfected CAT gene.
  • LIPOFECTINTM reagent a cationic lipid or cytofectin
  • Jurkat and K562 cells were transfected using the Cell PhectTM calcium phosphate transfection kit (Pharmacia) .
  • Ten ⁇ g vector DNA was combined with calcium phosphate in the cell dishes (5 x 10 6 cells/dish) as per the manufacturer's protocol, and the precipitated DNA incubated with the cells overnight.
  • the precipitated medium was replaced with fresh RPMI medium containing 20% FCS, glutamine and antibiotics on the second day. Cells were harvested after another 24 hours and assayed for expression of the CAT gene.
  • Raji and CEM cells were transfected by DEAE-Dextran using the CellPhectTM kit.
  • Ten ⁇ g vector DNA was combined with DEAE-Dextran according to the manufacturer's protocol and added to 2 x 10 6 cells.
  • the cells were incubated with the DNA-DEAE-Dextran complex for 1 hour.
  • the DNA-DEAE- Dextran mixture was then replaced with fresh RPMI medium containing 20% FCS, glutamine and antibiotics and cells were incubated for two days to allow transient expression of the CAT gene.
  • CAT activity was then assayed as described below.
  • Example 3 Determination of CAT activity in transfected cell lines
  • Cells were harvested by washing off of petri dishes and centrifugation.
  • For CHO and HeLa cells cells were trypsinized prior to collection by centrifugation.
  • Cell pellets were resuspended in 100 ⁇ l 250 mM Tris-HCl, pH 7.4 and lysed by three rounds of freeze-thaw.
  • Cell lysates were incubated at 65°C for 5 min and collected by centrifugation.
  • CAT activity was assayed by incubating cell lysates in a buffer containing 250 mM Tris- HC1, pH 7.4., 4 mM acetyl coenzyme A (Calbiochem, San Diego, CA) and 1 ⁇ l [ 1 C] -chloramphenicol as described (Ausubel et al., (1990) Current Protocols in Molecular Biology) .
  • the acetylated compounds were separated from [ 1 C] -chloramphenicol by thin-layer chromatography (95% chloroform, 5% methanol, v/v) on a silica gel.
  • Acetylated products were visualized by autoradiography using Kodak X-OMAT AR film (Eastman Kodak, Rochester, NY) . Regions of the silica gel containing acetylated and nonacetylated compounds were scraped from the plates and quantitated by scintillation counting.
  • the level of expression of the CAT gene from each of the expression vectors described above is summarized in Table I.
  • pcDL-SRo;-CAT and pcDNAI-CAT produced the highest CAT activity in CHO cells, with pCAT-KX and pCAT-Control vectors producing about 50% and 25% of this activity, respectively.
  • pcDL-SR ⁇ -CAT promoted the highest CAT activity in HeLa cells, although pcDNAI-CAT and pCAT-KX exhibited about 1/3 this activity.
  • K562 cells pcDL-SR ⁇ -CAT exhibited 150% the CAT activity of pCAT-KX.
  • Raji cells the highest CAT activity was obtained using pcDL-SRc.-CAT.
  • pCAT-KX promoted half this level of CAT expression.
  • pCAT-KX promoted a CAT activity similar to pcDL-SRot-CAT in CEM cells and a level 56% higher than pSR ⁇ -CAT in Jurkat cells.
  • [ 14 C] -chloramphenicol were low for pCAT-KX in Jurkat cells compared to other cell lines, independent studies indicate a CAT conversion rate of more than 60% can be obtained in Jurkat cells using pCAT-KX.
  • the negative control lacking a promoter region pSVOKS-CAT
  • pMSG-CAT showed no CAT activity due to the absence of induction by dexamethasone which is required for promoter activation.
  • pCAT-KX containing the human LS promoter when tested against commonly-used expression vectors containing viral promoters, was demonstrated to be the most efficient expression vector in T-cell lines, this vector also induced strong expression in other human cell lines as well as in CHO cells. Most importantly, the pKX expression system can be used for T- cell-specific gene delivery and gene therapy for genetic defects in T-cells. Both T-cell lines, Jurkat and CEM, are LS-producing cells. The predominant expression of the LS promoter-driven expression vector in these two cell lines is attributable to various transcriptional and translational regulatory factors specific to T-cells.
  • the basal expression level of LS is regulated by the eukaryotic transcriptional activator protein Spl through binding to a GGGTGG motif present in the LS promoter region. This indicates that Spl is a probable transcription factor, although other factors are most likely necessary for efficient LS transcription.
  • the presence of Spl in a wide range of cell types may explain the ability of the LS promoter to drive gene expression in cell types other than T-cells.
  • the high levels of LS-driven gene expression in T-cells may be due to the presence of these other T-cell regulatory factors which are absent from other cell types.
  • Example 4 Use of PKX containing ADA gene for gene therapy
  • ADA adenosine deaminase
  • T-cells are purified from the blood of a patient with SCID by separating peripheral blood mononuclear cells (MNCs) from red cells and neutrophils by Ficoll-Hypaque (Pharmacia) density gradient centrifugation. The cells are then treated with monoclonal antibody OKT-3 (Ortho Pharmaceutical Corp., Raritan, NJ) and recombinant human interleukin-2 (Roche, Nutley, NJ) to stimulate T-cell proliferation.
  • OKT-3 Ortho Pharmaceutical Corp., Raritan, NJ
  • human interleukin-2 Roche, Nutley, NJ
  • MOLECULE TYPE geno ic DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • CTTCCTTTCC CCTTGGGGCC
  • CTGTCCTTCC CAGTCTTGCC CCAGCCTCGG GAGGTGGTGG 120
  • MOLECULE TYPE genomic DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE genomic DNA
  • HYPOTHETICAL NO
  • ANTI-SENSE NO

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Abstract

Vecteur d'expression contenant un promoteur viral qui favorise l'expression génique à efficacité élevée dans des cellules mammaliennes. Ce vecteur d'expression contient le promoteur de leucosialine humain et présente des niveaux d'expression sensiblement plus élevés dans les lymphocytes T humains que les vecteurs d'expression classiques contenant des promoteurs viraux.
PCT/US1995/004583 1994-04-15 1995-04-13 Vecteur d'expression contenant un promoteur non viral WO1995028490A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2812305A1 (fr) * 2000-07-28 2002-02-01 Commissariat Energie Atomique Element d'adn promoteur de l'activite de transcription, actif dans les cellules humaines et methode generale pour en ajuster l'activite
AU2003219959B2 (en) * 2002-03-01 2008-07-31 Sagres Discovery, Inc. Novel compositions and methods for cancer

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
C.S. SHELLEY ET AL.: "Structure of the human sialophorin (CD43) gene", BIOCHEMICAL JOURNAL, vol. 270, pages 569 - 576 *
G. FERRARI ET AL.: "Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions", BLOOD, vol. 80, no. 5, pages 1120 - 1124 *
Journal of Cell Biology, 1991, 115 (3 Part 2), page 308a, abstract 1784 *
K. MITANI ET AL.: "Long-term expression of retroviral-transduced adenosine deaminase in human primitive hematopoietic progenitors", HUMAN GENE THERAPY, vol. 4, pages 9 - 16 *
S. KUDO ET AL.: "A short, novel promoter sequence confers the expression of human leukosialin, a major sialoglycoprotein on leukocytes", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 266, no. 3, pages 8483 - 8489 *
V.W. VAN BEUSECHEM ET AL.: "Long-term expression of human ADA in rhesus monkeys transplanted with retrovirus-infected bone-marrow cells", PNAS, vol. 89, pages 7640 - 7644 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2812305A1 (fr) * 2000-07-28 2002-02-01 Commissariat Energie Atomique Element d'adn promoteur de l'activite de transcription, actif dans les cellules humaines et methode generale pour en ajuster l'activite
WO2002010334A2 (fr) * 2000-07-28 2002-02-07 Genome Express Element d'adn promoteur de l'activite de transcription, actif dans les cellules humaines et methode generale pour en ajuster l'activite
WO2002010334A3 (fr) * 2000-07-28 2002-04-18 Commissariat Energie Atomique Element d'adn promoteur de l'activite de transcription, actif dans les cellules humaines et methode generale pour en ajuster l'activite
US7892730B2 (en) 2000-12-22 2011-02-22 Sagres Discovery, Inc. Compositions and methods for cancer
AU2003219959B2 (en) * 2002-03-01 2008-07-31 Sagres Discovery, Inc. Novel compositions and methods for cancer

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