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WO2002057472A1 - Marqueur susceptible d'etre selectionne pour des cellules et des tissus genetiquement modifies - Google Patents

Marqueur susceptible d'etre selectionne pour des cellules et des tissus genetiquement modifies Download PDF

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WO2002057472A1
WO2002057472A1 PCT/CA2002/000042 CA0200042W WO02057472A1 WO 2002057472 A1 WO2002057472 A1 WO 2002057472A1 CA 0200042 W CA0200042 W CA 0200042W WO 02057472 A1 WO02057472 A1 WO 02057472A1
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cells
gene
ires
expression vector
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Richard L. Momparler
Jacques Galipeau
Nicoletta Eliopoulos
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CENTRE FOR TRANSLATIONAL RESEARCH IN CANCER
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Priority to EP02711670A priority patent/EP1352078A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
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    • 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
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/027—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a retrovirus

Definitions

  • the expression vector of the invention may further be flanked by retroviral long terminal repeat (LTR) sequence at 5' and/or 3' ends of the vector.
  • LTR retroviral long terminal repeat
  • the expression vector may be composed of DNA or RNA selected from the group consisting of eukaryotic, viral, adenoviral, adeno- associated, Simliclei, and Herpes simplex expression vectors.
  • analogs as used herein is intended to mean any functional modified form of the cytidine deaminase. The modification may become from a mutated or modified form of the nucleotidic sequence encoding CD.
  • Fig. 4 illustrates the effect of ARA-C on the proliferation of genetically engineered A549 cells
  • Fig. 5 illustrates the effect of dFdC on the proliferation of genetically engineered A549 cells
  • Fig. 6 illustrates the resistance of CD gene-modified primary human lymphocytes to growth inhibition by ARA-C
  • CD catalyses the deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively and in addition, can deaminate cytosine nucleoside analogues causing their pharmacological inactivation.
  • the CD drug resistance gene has recently shown promise for protecting normal hematopoietic cells from the dose-limiting myelotoxicity of anti-cancer drugs to thereby permit dose escalation for enhanced chemotherapeutic effectiveness.
  • This chemoprotection approach using the human CD gene is the antithesis of a suicide gene therapy strategy utilising the prokaryotic cytosine deaminase.
  • ARA-C an effective antileukemic drug
  • dFdC a promising antitumor agent
  • 5-AZA- CdR which has shown antileukemic and interesting antitumor activity
  • the human CD gene may serve as an ex vivo positive selectable marker with cytosine nucleoside analogues in genetically engineered primary autologous cells.
  • target primary cells for selectable marker gene transfer may include bone marrow stromal cells and lymphocytes but also hematopoietic cells, myoblasts and/or fibroblasts.
  • Gene transfer of human CD into hematopoietic stem cells and/or lymphocytes may be valuable in the treatment of disorders that afflict the hematopoietic system, such as adenosine deaminase deficiency and chronic granulomatous disease, as well as storage disorders such as Gaucher disease and Hunter syndrome.
  • CD for ex vivo selection may be used to augment the proportion of hematopoietic cells transduced with anti-HIV-l genes, such as RevMIO, and consequently give rise to an enriched population of mature T-lymphocytes and monocytic cells with high level antiviral gene expression.
  • the human CD gene may serve as a dominant selectable marker in cancer gene therapy applications employing a non- selectable therapeutic transgene such as a tumor-suppressor gene to inhibit tumor growth or a cytokine gene to strengthen the immune response against neoplastic cells.
  • DMEM Dulbecco's modified essential medium
  • FBS heat-inactivated foetal bovine serum
  • G418 Mediatech, Herndon, VA
  • 2 ⁇ g/ml puromycin Sigma, Oakville,
  • A549 human lung carcinoma cells obtained from American Type Culture Collection (ATCC), were maintained in RPMI 1640 medium
  • NIH 3T3 mouse fibroblast cells from ATCC, were cultured in
  • the murine stem cell virus (MSCV) derived retroviral vector plRES-EGFP a bicistronic construct comprising a multiple cloning site linked by an internal ribosomal entry site (IRES), to the enhanced green fluorescent protein (EGFP) reporter
  • pCD-IRES-EGFP Fig. 1, plRES-EGFP comprises a multiple cloning site (mcs) and the EGFP reporter gene parted by an internal ribosomal entry site (IRES).
  • pNeo R -IRES-EGFP is a derivative of plRES- EGFP where the Neo R gene is inserted in the mcs.
  • pCD-IRES- EGFP contains the human CD cDNA in the mcs upstream of the IRES) was synthesised by retrieving the human cytidine deaminase (CD) cDNA sequence by Ncol/Klenow and BamHI digest of the pMFG-CD construct, and ligating it with a Xhol/Klenow and BamHI digest of plRES-EGFP.
  • the retrovector pNeo R -IRES-EGFP encompassing the neomycin phosphotransferase II drug resistance gene, was similarly constructed to serve as an additional control plasmid.
  • the pCD-IRES-EGFP vector (10 ⁇ g) was introduced into GP+E86 packaging cells by calcium phosphate transfection utilising the Cell PhectTM kit (Pharmacia) and cells were subsequently selected for 3 weeks in complete media supplemented with 2.5 ⁇ M cytosine arabinoside (ARA-C) (Upjohn, Don Mills, Ontario).
  • ARA-C cytosine arabinoside
  • the ensuing stable polyclonal producer cell population was used to supply virus for the transinfection (or transduction) of GP+envAM12 amphotropic packaging cells.
  • retrovirus-containing supernatant was harvested from subconfluent ecotropic producer cells, filtered with a 0.45 ⁇ m syringe mounted filter (Gelman Sciences, Ann Arbour, Ml) and applied with 8 ⁇ g/ml PolybreneTM (Sigma Chemical, St. Louis, MO) over target GP+envAM12 cells.
  • these amphotropic producers commenced a 2-week drug selection by culturing in media including 2.5 ⁇ M ARA-C, thence generating the polyclonal population GP+envAM12-CD-IRES-EGFP.
  • the GP+AM12-Neo R -IRES-EGFP polyclonal producer was created in an almost identical manner to that described above, the exception being that drug selection was performed with 400 ⁇ g/ml G418- containing media.
  • GP+E86-Lac Z cells were generated by transinfection of the GP+E86 cell line with filtered retroviral supernatant from the 293GPG-LacZ producer (kind gift from R.C. Mulligan, Children's Hospital, MA) twice a day for 3 consecutive days.
  • pantropic 293GPG packaging cell line is co-transfected with 5 ⁇ g pCD-IRES-EGFP and 70ng pJ6 ⁇ BIeo rationally given by R.C. Mulligan (Children's Hospital, MA). These cells then underwent 4-week selection in 293GPG media supplemented with 100 ⁇ g/ml ZeocinTM (Invitrogen, San Diego, CA) consequently generating the stable polyclonal producer 293GPG-CD-IRES- EGFP. By this same approach, the control 293GPG-IRES-EGFP producer was also conceived.
  • Retroparticles from virus producers were noted to be free from replication competent retrovirus (RCR) by GFP marker rescue assay employing supernatant from transduced target cells. Titration of retrovirus-producers
  • NIH 3T3 cells were utilised, whereas A549 cells were used for titering 293GPG producers.
  • These target cells were plated at a density of 2 to 4 x 10 4 cells per well in 6-well tissue culture dishes and the following day, cells from one test well were trypsinized and counted to ascertain the baseline cell number at time of virus addition.
  • serial dilutions of retroviral supernatants (0.01 to 100 ⁇ l in a final volume of 1ml complete media, supplemented with 8 ⁇ g/ml polybrene, were placed over the adherent target cells.
  • Flow cytometry analysis was realised 72 hours post-transduction to disclose the percentage of GFP-expressing cells.
  • the titer was calculated utilising the equation below by considering the virus dilution that led to 10- 40% GFP positive cells.
  • Retroviral supernatant from 293GPG-CD-IRES-EGFP cells grown to confluence in 293GPG media devoid of tetracycline for over 72 hours to allow VSVG-pseudotyped retroparticle production were placed in a 25cm 2 flask of subconfluent A549 cells with 8 ⁇ g/ml PolybreneTM. This transduction procedure was executed once a day for 3 consecutive days yielding A549- CD-IRES-EGFP cells. As a control, A549 cells were likewise transduced with viral particles from 293GPG-IRES-EGFP producers hence giving rise to A549-IRES-EGFP cells.
  • the gel was immersed in denaturing solution (0.5M NaOH; 1.5M NaCl) and then in neutralising buffer (0.5M Tris-HCI pH7; 1.5M NaCl, pH7), each for 45 minutes.
  • denaturing solution 0.5M NaOH; 1.5M NaCl
  • neutralising buffer 0.5M Tris-HCI pH7; 1.5M NaCl, pH7
  • the DNA in the gel was transferred onto a Hybond-NTM nylon membrane (Amersham, Oakville, Ontario) using 10X Standard Saline Citrate (SSC) for an approximately 48 hour downward transfer with the Turbo BlotterTM device (Schleicher & Shuell, Keene, NH).
  • the membrane was then irradiated in a BioslinkTM UV linker with 0.3 J/cm 2 and hybridised in Express HybTM solution (Clontech) containing PCR-amplified 32 P-labeled cDNA for human CD.
  • the membrane was washed and exposed using a Phosphor ImagerTM.
  • MTT Growth inhibition assay
  • Stably transduced test and control A549 cells were assayed for CD enzyme activity. Concisely, these adherent cells (2-5 x 10 7 cells) were trypsinized, washed with phosphate-buffered saline (PBS) and resuspended in 5mM Tris-HCI (pH 7.4) and 5mM dithiothreitol. The cell suspension was freeze-thawed rapidly three times, centrifuged at high speed (12 000 rpm) for 15 minutes and the supernatant consisting of the cytosolic extract was collected.
  • PBS phosphate-buffered saline
  • 5mM Tris-HCI pH 7.4
  • dithiothreitol 5mM dithiothreitol
  • One unit of enzyme activity was defined as the amount of enzyme that catalyses the deamination of one nmole of 3 H-cytidine per minute at 37°C.
  • Total protein concentration was determined using the Bio-RadTM protein assay (Bio-Rad Laboratories, Mississauga, Ontario) with bovine serum albumin as the standard. Transduction of primary human lymphocytes and growth suppression assay
  • lymphocytes were activated with phytohemagglutinin (PHA) for 72hrs and subsequently co-cultivated over subconfluent 293GPG-CD-IRES-EGFP or control 293GPG-IRES-EGFP virus producers tetracycline withdrawn three days earlier for optimal high titer virion generation.
  • PHA phytohemagglutinin
  • LipofectamineTM was added for a final concentration of 6 ⁇ g/ml, and co-cultivation permitted to proceed for 48 hours. Lymphocytes were then carefully collected and three days later plated at a density of 600 000 cells per well of48-well tissue culture dishes in the absence or presence of various concentrations of ARA-C in a final volume of 800 ⁇ l complete media.
  • % cell survival (number of cells in test well/number of cells grown in media only) x 100. (Flow cytometry analysis for CD3 expression was conducted and all cells confirmed to be lymphocytes.)
  • CD-IRES-EGFP transduced stromal cells were mixed in various proportions with untransduced stromal cells and plated at a density of 25 000 cells/well of 6-well plates without ARA-C and with ARA-C at a final concentration of 2.5 ⁇ M. Drug exposure was ceased 7 days later, and cells expanded for an additional week in DMEMTM with 10% FBS and 50U/ml Pen/Strep and analysed by flow cytometry analysis. Moreover, the cell combination where CD-IRES-EGFP modified stroma constituted 10% of total cell number was plated at 25 000 cells/well in 6-well dishes with a range of ARA-C concentrations. Pursuant to a 1-week drug exposure (preventing total confluence by cell passaging) and subsequent week of cell expansion, percent GFP positive cells and mean GFP fluorescence were evaluated by flow cytometry analysis.
  • IRES-EGFP, GP+envAM12-Neo R -IRES-EGFP, 293GPG-CD-IRES-EGFP, and 293GPG-IRES-EGFP were revealed to be 99.2, 100, 50.4, and 65.2, respectively, as compared to values of less than 3% for parental unaltered cells.
  • their retroviral supernatant was utilised in a titration assay and the extrapolated viral titers were 1.5 x 10 6 , 1.3 x 10 6 , 1 x 10 6 , and 0.5 x 10 6 infectious particles per ml.
  • CD-IRES-EGFP did not sustain any rearrangements or deletions before integration as proviral DNA into the genome of transduced cells
  • Southern blot analysis was carried out on gene-modified A549 cells. This procedure revealed a DNA band corresponding to the 3683bp fragment expected from Nhel digest of integrated unrearranged CD-IRES-EGFP proviral DNA (Fig. 3). Genomic DNA from the indicated cell lines was digested with Nhel, which cuts once in each flanking LTR, and fractionated on 1 % agarose gel. Hybridisation of the blot with a 32 P-labeled CD cDNA probe permits detection of integrated, unrearranged proviral DNA of the predicted 3683bp size. Molecular weights are indicated).
  • A549-CD-IRES-EGFP cells selected with 2.5 ⁇ M ARA-C the DNA band detected was of higher intensity than that of cells exposed to 1 ⁇ M ARA-C, and notably more so than that of CD- transduced cells which were not drug selected (Fig. 3).
  • Expression of CD enzyme activity in gene-modified A549 cells was performed on the cytosolic extract of A549 cells transduced with CD-IRES-EGFP retroviral particles and on that of control cells, i.e. untransduced A549 cells, as well as A549 cells modified with IRES-EGFP virions.
  • Fig. 8 The sensitivity of genetically engineered A549 cells to the toxicity of cytosine nucleoside analogues ARA-C and dFdC was evaluated by MTT assay. As depicted in Fig. 8 (Parental A549 cells, A549 cells transduced with control IRES-EGFP retroparticles, and A549 cells transduced with CD-IRES- EGFP virions and expanded without ARA-C, with 1 ⁇ M ARA-C, or 2.5 ⁇ M ARA-C for 12 days, were subsequently exposed to ARA-C for 4 days and cell survival quantified by MTT assay. Percent survival is plotted against drug concentration (log scale).
  • ARA-C at a concentration of 1 ⁇ M caused a substantial decrease in the % cell survival of untransduced A549 and A549-IRES-EGFP cells to values of 15.3 + 0.6 and 10.6 ⁇ 0.4, respectively.
  • survival by A549-CD-IRES-EGFP cells was over 3-fold greater at 47.4 ⁇ 1.9%.
  • the sensitivity of the A549-CD-IRES- EGFP cells previously treated with 1 ⁇ M, as well as 2.5 ⁇ M ARA-C was also assessed.
  • the % survival of the 2.5 ⁇ M ARA-C-selected CD-modified cells was 70.7 ⁇ 1.3, thus significantly higher than the above-mentioned 47.4 ⁇ 1.9% cell survival of A549-CD-IRES-EGFP cells which had not undergone post-transduction drug selection (Fig.4).
  • Percent survival is plotted against drug concentration (log scale). Average + SEM, n > 8). In contrast however, A549-CD-IRES-EGFP cells demonstrated considerably less drug sensitivity, with 86.4 ⁇ 2.4% survival at the same concentration of dFdC. Likewise, the 2.5 ⁇ M ARA-C selected CD-IRES-EGFP modified cells revealed at 0.1 ⁇ M dFdC, 92.1 ⁇ 1.7% cell survival. Furthermore, treatment with the highest concentration of 1 ⁇ M dFdC considerably reduced cell survival of unselected A549-CD-IRES-EGFP cells to 17.2 ⁇ 0.7% whereas the 2.5 ⁇ M ARA-C selected cells showed 53.3 ⁇ 1.8% survival (Fig. 5). Effect of ARA-C on growth of genetically-engineered primary human lymphocytes
  • IRES-EGFP retrovirions 5.5 ⁇ 1.7% survival
  • CD-IRES-EGFP viral particles did not significantly affect that of lymphocytes transduced with CD-IRES-EGFP viral particles
  • a cell mixture consisting of 10% CD- IRES-EGFP vector-bearing stromal cells within a population of untransduced marrow stroma was exposed to increasing concentrations of ARA-C for one week and subsequently cultured for another week.
  • Flow cytometry analysis revealed that the degree of selective expansion of the GFP positive cells was dependent on the dose of ARA-C (Fig. 6).
  • the 11.7% GFP + cells without ARA-C escalated to 51.5% and 99.6% when selected in 0.25 and 2.5 ⁇ M ARA-C, respectively (Fig. 8).
  • CD engineered stroma does not alter in vivo engraftment capacity
  • 10% CD-IRES-EGFP positive mouse stroma enriched to >99% by ARA-C exposure was thereafter gene- modified to also express ⁇ -galactosidase and implanted by intramuscular injection in immunocompetent syngeneic mice.
  • sections of muscle harvested 2 weeks post-implantation contain engrafted ex vivo drug selected, transgene expressing cells.
  • Retroviral gene transfer permits stable and efficient integration of a foreign gene in the chromosomal DNA of the host cell and subsequently in all progeny cells.
  • high level and long term expression of a beneficial exogenous gene is difficult to achieve in gene therapy studies.
  • gene therapy of many hereditary and acquired affliction commands that the desired therapeutic gene be efficiently translated in the majority of target cells. Accordingly, it is anticipated that if the genetically engineered cells represent only a small portion of the target cells, their outnumbering by the unaltered cells will ultimately ensue.
  • One possible means to overcome this obstacle may be to expand the proportion of gene- modified cells in vitro through dominant selection relying on the co- expression of a drug resistance gene.
  • a bicistronic retroviral vector enclosing the human CD cDNA and the GFP reporter gene is generated. It is noted that its expression lead to functional levels of CD in genetically engineered human cell line A549, primary human lymphocytes, and primary murine bone marrow stromal cells.
  • the convenience of this CD-IRES-EGFP retroviral construct (Fig. 1), where GFP represents the non-selectable therapeutic gene, was the accorded ability to track the gene-modified cells via the green fluorescence (emitted at 507nm following blue light excitation at 488nm) which was visualised by fluorescence microscopy and quantified by flow cytometry analysis. Therefore, GFP expression here reflected CD expression.
  • the CD-IRES-EGFP retrovector is integrated as an intact proviral DNA in the genome of transduced cells, since no rearrangements, nor deletions are revealed by Southern blot analysis of gene-modified cells (Fig. 3).
  • the more intense cDNA signal detected with CD gene-modified A549 cells selected with 2.5 ⁇ M ARA-C, versus that with 1 ⁇ M ARA-C treated cells, or the weakest band seen with unselected cells, indicates an augmented copy number of the proviral sequences. This higher copy number suggests selection and enrichment of the CD positive cells with multiple integrants and/or amplification of the CD gene by ARA-C exposure of genetically-engineered cells. It was discovered that it is possible to increase CD expression via amplification of the proviral CD gene with ARA-C exposure of CD-transduced fibroblast cells.
  • CD-IRES-EGFP transduced A549 cells as compared to corresponding control IRES-EGFP- modified cells, disclosed an over 1000-fold increment (Table 1).
  • CD activity is markedly augmented in cells following stable transduction with CD- encoding retroparticles.
  • lymphocytes may be a valuable tool in numerous cell and gene therapy studies.
  • the transfer of therapeutic genes into lymphocytes may be used in the treatment of cancer, graft versus host disease (GVHD), AIDS, and autoimmune diseases.
  • GVHD graft versus host disease
  • Enhancing with the CD gene the population of transduced lymphocytes expressing a second non-selectable therapeutic gene may be beneficial, particularly in situations where poor transfer efficiency into lymphocytes is the limiting factor for successful gene therapy applications.
  • ex vivo enrichment of gene-modified lymphocytes may be useful for the therapy of patients with adenosine deaminase deficiency and for the modulation of GVHD following hematopoietic cell transplantation for leukemia and lymphoma.
  • the ex vivo enrichment using CD of donor T lymphocytes co-expressing the HSV-TK suicide gene may allow in vivo elimination with gancyclovir of all alloreactive donor cells following allogeneic bone marrow transplantation.
  • ARA-C dose escalation for in vitro selection of gene-modified stromal cells brought about expression augmentation, as evidenced by GFP fluorescence increments.
  • CD-IRES-EGFP positive cells were attained with the use of moderate drug concentrations, the more powerful doses of ARA-C selected the cells with the superior CD expression, thus strongest drug resistance phenotype.
  • Amplification of the CD proviral DNA has also occurred with intensive ARA-C treatment of CD-IRES-EGFP transduced marrow stromal cells.
  • Characteristics of an optimal drug resistance gene as a potential positive selectable marker comprise its effectiveness at imparting significant drug resistance, its small cDNA not imposing size constraints on the therapeutic gene included in the bicistronic vector, and very importantly its inability to raise an immune response.
  • hematopoietic cells can be dominantly selected not only through the concomitant expression of a drug resistance gene but also of a cell-surface reporter.
  • Human and mouse hematopoietic cells have been gene-modified to express the cell surface protein human CD24 and were subsequently selected by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • Retroviral gene transfer of the murine cell surface marker Heat Stable Antigen (HSA) or a truncated variant of human CD34 into human cells has also allowed the enrichment of gene-modified cells through FACS or immunoaffinity columns, respectively. In clinical applications it is very likely that the expression of a non-human selectable marker by transduced human cells will raise an immune reaction.
  • HSA Heat Stable Antigen
  • Another selectable protein is the human low-affinity nerve growth factor receptor for which cell sorting is utilised to enrich genetically engineered cells based on cell-surface marking.
  • cell surface reporters the possibility exists that untransduced cells may be co-selected with gene-modified cells due to intercellular exchange of proteins on the surface of cells. This obstacle may be overcome with the use of cytoplasmic reporter proteins such as the green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • Many investigators have utilised the GFP reporter for the in vitro selection through FACS of transfected or transduced cells expressing high degree of fluorescence. Nevertheless, FACS may impose significant physical stress on gene-modified cells that may be detrimental particularly to sorted primary cells.
  • a further advantage of the human CD gene as a positive selectable marker is that the drugs that it confers resistance to, cytosine nucleoside analogues, and which are required for ex vivo selection, are antimetabolites that will not cause DNA damage in CD-enriched cells.

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Abstract

L'invention concerne un gène marqueur susceptible d'être sélectionné, un procédé de production de cellules génétiquement modifiées permettant l'expression d'un vecteur bicistronique et des applications en thérapie génique ex vivo. L'invention concerne également la sélection de cellules génétiquement modifiées provenant de population mixte, avant leur implantation in vivo. L'invention concerne en outre le gène humain de la cytidine déaminase (CD) tenant lieu de marqueur dominant susceptible d'être sélectionné ex vivo dans des cellules primaires à gène modifié avec des analogues de la cytosine nucléoside. Un rétrovecteur bicistronique comprenant la séquence codant la CD humaine et le gène rapporteur de la protéine fluorescente verte (GFP) améliorée est utilisé dans la transduction de cellules. L'invention concerne finalement l'introduction du gène CD dans les applications en thérapie cellulaire et génique pour la sélection dominante ex vivo de cellules génétiquement modifiées.
PCT/CA2002/000042 2001-01-16 2002-01-16 Marqueur susceptible d'etre selectionne pour des cellules et des tissus genetiquement modifies Ceased WO2002057472A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA002434387A CA2434387A1 (fr) 2001-01-16 2002-01-16 Marqueur susceptible d'etre selectionne pour des cellules et des tissus genetiquement modifies
US10/466,705 US20040115800A1 (en) 2002-01-16 2002-01-16 Selectable marker for genetically engineered cells and tissues
EP02711670A EP1352078A1 (fr) 2001-01-16 2002-01-16 Marqueur susceptible d'etre selectionne pour des cellules et des tissus genetiquement modifies

Applications Claiming Priority (2)

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US26119001P 2001-01-16 2001-01-16
US60/261,190 2001-01-16

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WO2002057472A1 true WO2002057472A1 (fr) 2002-07-25

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EP (1) EP1352078A1 (fr)
CA (1) CA2434387A1 (fr)
WO (1) WO2002057472A1 (fr)

Citations (4)

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WO1997020935A2 (fr) * 1995-12-06 1997-06-12 Cambridge University Technical Services Limited Vecteurs viraux
US5770428A (en) * 1993-02-17 1998-06-23 Wisconsin Alumni Research Foundation Chimeric retrovial expression vectors and particles containing a simple retroviral long terminal repeat, BLV or HIV coding regions and cis-acting regulatory sequences, and an RNA translational enhancer with internal ribsome entry site
WO1999053046A2 (fr) * 1998-04-14 1999-10-21 Chiron Corporation Technique sans clonage d'expression d'un gene interessant
WO2001068667A1 (fr) * 2000-03-14 2001-09-20 The Johns Hopkins University School Of Medicine Arret de la proliferation de tumeurs fortement glycolitiques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770428A (en) * 1993-02-17 1998-06-23 Wisconsin Alumni Research Foundation Chimeric retrovial expression vectors and particles containing a simple retroviral long terminal repeat, BLV or HIV coding regions and cis-acting regulatory sequences, and an RNA translational enhancer with internal ribsome entry site
WO1997020935A2 (fr) * 1995-12-06 1997-06-12 Cambridge University Technical Services Limited Vecteurs viraux
WO1999053046A2 (fr) * 1998-04-14 1999-10-21 Chiron Corporation Technique sans clonage d'expression d'un gene interessant
WO2001068667A1 (fr) * 2000-03-14 2001-09-20 The Johns Hopkins University School Of Medicine Arret de la proliferation de tumeurs fortement glycolitiques

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Title
CONSALVO M ET AL: "5-FLUOROCYTOSINE-INDUCED ERADICATION OF MURINE ADENOCARCINOMAS ENGINEERING TO EXPRESS THE CYTOSINE DEAMINASE SUICIDE GENE REQUIRES HOST IMMUNE COMPETENCE AND LEAVES AN EFFICIENT MEMORY", JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 154, no. 10, 15 May 1995 (1995-05-15), pages 5303 - 5312, XP000654904, ISSN: 0022-1767 *
CRYSTAL R G: "IN VIVO AND EX VIVO GENE THERAPY STRATEGIES TO TREAT TUMORS USING ADENOVIRUS GENE TRANSFER VECTORS", CANCER CHEMOTHERAPY AND PHARMACOLOGY, SPRINGER VERLAG, BERLIN, DE, vol. 43, 1999, pages S90 - S99, XP000891470, ISSN: 0344-5704 *
ELIOPOULOS N ET AL: "Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.", GENE THERAPY, vol. 9, no. 7, April 2002 (2002-04-01), April, 2002, pages 452 - 462, XP002201262, ISSN: 0969-7128 *
MULLEN C A ET AL: "TREATMENT OF MICROSCOPIC PULMONARY METASTASES WITH RECOMBINANT AUTOLOGOUS TUMOR VACCINE EXPRESSING INTERLEUKIN 6 AND ESCHERICHIA COLI CYTOSINE DEAMINASE SUICIDE GENES", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD, US, vol. 56, 15 March 1996 (1996-03-15), pages 1361 - 1366, XP002937745, ISSN: 0008-5472 *
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EP1352078A1 (fr) 2003-10-15
CA2434387A1 (fr) 2002-07-25

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