EP0980264A2 - Säugetierzelltransduktion zur verwendung in gentherapie - Google Patents

Säugetierzelltransduktion zur verwendung in gentherapie

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Publication number
EP0980264A2
EP0980264A2 EP98930718A EP98930718A EP0980264A2 EP 0980264 A2 EP0980264 A2 EP 0980264A2 EP 98930718 A EP98930718 A EP 98930718A EP 98930718 A EP98930718 A EP 98930718A EP 0980264 A2 EP0980264 A2 EP 0980264A2
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European Patent Office
Prior art keywords
cells
vector
bone marrow
transduction
optionally
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EP98930718A
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English (en)
French (fr)
Inventor
Thierry Vanden Driessche
Marinee Khim Lay Chuah
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Katholieke Universiteit Leuven
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Katholieke Universiteit Leuven
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Priority claimed from EP98200382A external-priority patent/EP0938904A1/de
Application filed by Katholieke Universiteit Leuven filed Critical Katholieke Universiteit Leuven
Priority to EP98930718A priority Critical patent/EP0980264A2/de
Publication of EP0980264A2 publication Critical patent/EP0980264A2/de
Withdrawn legal-status Critical Current

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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
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0669—Bone marrow stromal cells; Whole bone marrow
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to a new method for the ex vivo transduction of mammalian cells.
  • the invention further relates to genetically engineered cells thus obtained and to the use of these cells in gene therapy.
  • the invention in particular relates to the transduction of human bone marrow stromal cells with a vector expressing a blood coagulation factor, in particular factor VIII.
  • Somatic gene therapy involves the genetic engineering of somatic cells and the administration of these cells to a subject in need of therapy. Through genetically engineering the cells, they will acquire one or more desirable properties they did not or did no longer possess, for example the ability to express a particular protein. As an alternative a cell may be genetically engineered to loose an unwanted property.
  • the first important step in genetically engineering cells for use in gene therapy is being capable of efficiently transducing the cells with a vector harboring the gene of interest and to obtain stable expression of that gene in the cells.
  • the present invention has for its general object to provide such a method of efficiently transducing cells ex vivo . It is a more specific object of the present invention to provide such a method for obtaining cells for treating hemophilia A by means of gene therapy.
  • Hemophilia A is a congenital X-chromosome- linked coagulation disorder characterized by uncontrolled crippling hemorrhagic episodes which occurs in approximately 1/10000 males. Hemophilia A is due to a deficiency of coagulation factor VIII (FVIII) , which accelerates the activation of factor X by activated factor IX in the presence of calcium and phospholipids . Ultimately the coagulation cascade leads to the localized generation of thrombin and the conversion of fibrinogen to insoluble fibrin polymers, which in conjunction with platelet aggregation maintains hemostasis .
  • FVIII coagulation factor VIII
  • Hemophilia A is particularly suitable for gene therapy since expression of FVIII does not require precise metabolic regulation and since a slight increase in plasma FVIII levels can potentially convert severe [FVIII: 1-2 ng/ml] to mild hemophilia [FVIII: 2-60 ng/ml] .
  • Gene therapy for hemophilia A should provide constant, sustained synthesis within the patient, thereby obviating the risk of spontaneous bleeding, the need for repeated FVIII infusion and the risk of viral infections associated with plasma-derived FVIII.
  • Retroviral vector- mediated gene transfer offers the potential for long-term gene expression by virtue of its stable chromosomal integration and lack of viral gene expression. Retroviral vectors for the transfer and expression of a B-domain deleted FVIII gene have been described before.
  • Another problem encountered in attempts at achieving long-term human FVIII expression by ex vivo gene therapy approaches using a variety of primary cells is that access of the engineered cells to the bloodstream (such as by intrasplenic or intravenous injection) is a prerequisite to obtain detectable FVIII levels in the circulation.
  • Cells belonging to the lympho-hematopoietic lineage which can be stably transduced with retroviral vectors ex vivo do, however, not secrete FVIII protein.
  • high retroviral transduction efficiency and FVIII expression are needed for gene therapy to be successful.
  • human BM stromal cells can be transduced with an intron-based Moloney murine leukemiavirus (MoMLV) retroviral vector expressing a B-domain deleted human factor VIII cDNA (designated as MFG-FVIII ⁇ B) .
  • MoMLV Moloney murine leukemiavirus
  • Transduction efficiencies were increased 10 to 15-fold by phosphate depletion and centrifugation which obviated the need for selective enrichment of the transduced BM stromal cells. This resulted in high FVIII expression levels in transduced human (180 + 4 ng FVIII/10 6 cells per 24 hr) and mouse (900 ⁇ 130 ng FVIII/10 6 cells per 24 hr) BM stromal cells.
  • Human BM stromal cells transduced with GALV-env pseudotyped PG13-F8 vectors using the optimized transduction method as presented in this invention were subsequently infused into immunodeficient SCID NOD mice.
  • Therapeutic human FVIII expression levels were detected in the plasma of these mice well above the levels needed to convert a severe to a moderate hemophilia A patient and persisted for at least more than a week in vivo. This represents the first protocol showing therapeutic levels of FVIII in vivo using BM stromal cells.
  • the invention in a specific embodiment thus relates to a method for the ex vivo transduction of mammalian cells, in particular bone marrow cells, more in particular bone marrow stromal cells, which method comprises the steps of: a) providing an intron-based retroviral vector comprising a B-domain deleted human factor VIII cDNA; b) pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; c) transducing bone marrow stromal cells with the said pseudotyped vector by optionally pre- incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector solution, optionally supplemented with transduction additives, to the cells, followed by centrifugation of the mixture thus obtained; and d) optionally repeating step c) .
  • the vector is the MFG-FVIII ⁇ B vector as described in Dwarki et al . (1995). Information on the MFG-vector is attainable from Riviere et al . (Proc. Natl . Acad. Sci. USA 92:6733-6737, 1995). Details on the nucleotide sequence of the full-length FVIII gene are described in Truett et al . , DNA 4:333-349, 1985) .
  • the MFG-FVIII ⁇ B vector can be pseudotyped with GALV-env by successively transducing PG13 cells (ATCC CRL 10686) therewith.
  • pseudotyping with GALV- env has been described before for primary human T-cells (Bunnell et al . , Proc. Natl. Acad. Sci. USA 92:7739-7743 (1995) ) .
  • Bunnell et al . did not use the FVIII- containing vector of the invention. Details of the pseudotyping according to the invention are given in example 1. It has been found that in an optimal embodiment of the invention, the transduction protocol comprises both a phosphate starvation and a centrifugation step.
  • the invention is described in detail referring to the transduction of bone marrow stromal cells for expression of factor VIII.
  • the specific steps of ex vivo transduction method can also be used in any combination for the transduction of the same or other cells with the same or other genes. It is for example possible to use other genes of interest, for example other genes encoding factors secreted into the circulation, in particular factor IX, erythropoietin (EPO) , etc..
  • genes may be used that restore stromal function, e.g. for repair of irradiated bone marrow stromal cells after cancer therapy.
  • Alternative cells to be transduced by the method of the invention are for example BM stromal precursor or BM stromal stem cells which are an integral part of the BM stroma.
  • precursor cells for example BM stromal precursor or BM stromal stem cells which are an integral part of the BM stroma.
  • the advantage of using precursor cells is that the progeny of each transduced stromal precursor cell would also contain the transgene in analogy with transduction of BM hematopoietic stem/progenitor cells. This increases the total number of transduced cells in comparison to the number of transduced cells that arises when terminally differentiated cells are transduced.
  • transduction of self-renewing precursor stromal cells may lead to prolonged persistence of engineered cells in vivo as compared to transduction of terminally differentiated cells. In principle any cell type can be used, whereas primary cells are preferred.
  • a vector should contain an expression cassette consisting of either a promoter and an intron or a strong promoter, either one together with the gene of interest.
  • the intron may be located upstream or downstream from the gene.
  • Suitable promoters are viral promoters, e.g. retroviral long terminal repeat (LTR) , cytomegalovirus promoter (CMV) , simian virus 40 promoter (SV40) , adenovirus Major Late Promoter (MLP) , etc. or cellular promoters of either housekeeping genes, e.g. mouse or human small nuclear RNA promoter, elongation factor lc- promoter, etc. or tissue-specific promoters that are highly expressed in target tissue.
  • LTR retroviral long terminal repeat
  • CMV cytomegalovirus promoter
  • SV40 simian virus 40 promoter
  • MLP adenovirus Major Late Promoter
  • cellular promoters of either housekeeping genes, e.g. mouse or human small nuclear
  • Suitable introns for use with these promoters are introns from viral genes, e.g. Moloney murine leukemia virus intron etc., from cellular genes, e.g. ⁇ - globin intron, Factor VIII or Factor IX intron, ApoAl intron, c-1-antitrypsin intron, etc..
  • the transduction protocol of the invention is in particular useful for transduction with retroviral vectors.
  • Each step in the transduction protocol provides for an additional increase in the transduction efficiency. Incorporation of all steps in the method is thus preferred.
  • the general object of the invention of providing an efficient method for ex vivo transduction of mammalian cells is thus achieved according to the invention by the ex vivo transduction by means of a transduction protocol, which method comprises the following elements: a) the mammalian cell is transduced with a gene of interest; b) the transduction is effected by means of a retroviral vector; c) the vector comprises an intron; d) the transduction protocol comprises pseudotyping of the vector; e) the transduction protocol comprises a centrif gation step; f) the transduction protocol comprises phosphate starvation of the cells to be transduced, wherein steps c) to f) are optional and can occur in any possible combination
  • the above method is suitable for transducing genes encoding proteins which can be of importance for gene therapy, i.e. all genes associated with hereditary disorders or acquired and complex disorders (such as cancer, cardiovascular disease, diabetes etc.) wherein therapeutic effects can be achieved by introducing an intact version of the gene into somatic cells, in particular genes encoding proteins that can be secreted into the circulation (hormones, growth factors, lymphokines and cytokines, interferons, antibodies, complement factors, coagulation factors, enzymes etc.) or genes whose products are not secreted and which may have a direct therapeutic effect on the cell in which they are expressed (enzymes, growth factors or growth factor receptors, signal transducing proteins, cytoskeletal components and other structural proteins, etc.) .
  • these genes also include genes encoding proteins that are involved in wound healing, bone formation and repair (such as collagen type I which is defective in osteogenesis imperfecta patients) or proteins that influence osteoporosis, arthritis, osteogenesis imperfecta, chondrodysplasia, and hematopoiesis .
  • Other genes also include genes involved in the protection of the BM stroma form the detrimental side-effects on BM stromal function of (i) cancer therapy (such as by chemo- or radiotherapy) or (ii) viral infections (e.g. HIV) .
  • the invention relates in particular to a method in which the gene of interest encodes a gene encoding a factor secreted into the circulation, more in particular a gene encoding a factor involved in blood coagulation.
  • the mammalian cell is transduced with a gene encoding factor VIII, factor IX, factor X, factor V, factor VII, factor XII, factor XIII, Von illebrand factor (v F) , tissue factor (TF) and all other proteins directly or indirectly influencing blood coagulation.
  • the mammalian cell is preferably a primary cell, in particular a primary bone marrow cell.
  • the mammalian cell can also be a bone marrow cell selected from the group consisting of bone marrow stromal stem cells, bone marrow stromal stem cells, bone marrow stromal precursor cells, bone marrow mesenchymal cells, bone marrow hematopoietic stem cells, bone marrow hematopoietic progenitor cells, cells belonging to the lymphohematopoietic lineage, fibroblasts, endothelial cells, chondroblasts, chondrocytes, myoblasts, myocytes, osteoblasts, epithelial cells.
  • mesenthelial cells Other cells that can be transduced are mesenthelial cells, keratinocytes, hepatocytes .
  • Suitable retroviral vectors are Moloney murine leukemia virus, Gibbon ape leukemia virus, Rous sarcoma virus, myeloproliferative sarcoma virus, lentivirus, human foamy virus, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV) , bovine leukemia virus (BLV) .
  • the vector can be pseudotyped with other envelopes that utilize the GLVR-1 receptor for viral entry, in particular the 10A1 envelope.
  • the method comprises: a) providing an intron-based retroviral vector comprising any gene, in particular a gene that is secretable in the blood stream, more in particular a blood coagulation gene; b) pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; c) transducing bone marrow stromal cells with the said pseudotyped vector by optionally pre-incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector solution, optionally supplemented with transduction additives to the cells, followed by centrifugation of the mixture thus obtained; and d) optionally repeating step c) .
  • GLV Gibbon ape leukemia virus
  • the invention in another embodiment relates to a method for the ex vivo transduction of cells, comprising: a) providing an intron-based retroviral vector comprising any gene, in particular a gene that is secretable in the blood stream, more in particular a blood coagulation gene; b) pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; c) transducing cells to be transduced, in particular bone marrow stromal precursor cells, with the said pseudotyped vector by optionally pre-incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector solution, optionally supplemented with transduction additives to the cells, followed by centrifugation of the mixture thus obtained; and d) optionally repeating step c) .
  • GLV Gibbon ape leukemia virus
  • a further embodiment of the invention relates to a method for the ex vivo transduction of bone marrow stromal cells, comprising the steps of: a) providing an intron-based retroviral vector comprising a B-domain deleted human factor VIII cDNA
  • MFG-FVIII ⁇ B (designated as MFG-FVIII ⁇ B) ; b) optionally pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; and/or c) optionally transducing bone marrow stromal cells with the said pseudotyped vector by pre-incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector-containing solution, optionally supplemented with transduction additives to the cell; and/or d) optionally centrifuging the mixture thus obtained; and/or e) optionally repeating step c) and d) .
  • GLV Gibbon ape leukemia virus
  • Still a further embodiment of the invention relates to a method for the ex vivo transduction of bone marrow stromal cells, comprising the steps of: a) providing a retroviral vector comprising a B-domain deleted human factor VIII cDNA; b) optionally pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; and/or c) optionally transducing bone marrow stromal cells with the said pseudotyped vector by pre-incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector-containing solution, optionally supplemented with transduction additives to the cell; and/or d) optionally centrifuging the mixture thus obtained; and/or e) optionally repeating step c) and d) .
  • a retroviral vector comprising a B-domain deleted human factor VIII cDNA
  • b) optionally pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope
  • An alternative embodiment of the invention relates to a method for the ex vivo transduction of bone marrow stromal cells, comprising the steps of: a) providing an intron-based retroviral vector comprising a gene of interest; b) optionally pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; and/or c) optionally transducing bone marrow stromal cells with the said pseudotyped vector by pre-incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector-containing solution, optionally supplemented with transduction additives to the cell; and/or d) optionally centrifuging the mixture thus obtained; and/or e) optionally repeating step c) and d) .
  • GLV Gibbon ape leukemia virus
  • the invention also provides a method for the ex vivo transduction of bone marrow stromal cells, comprising the steps of: a) providing a retroviral vector comprising a gene of interest; b) optionally pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope; and/or c) optionally transducing bone marrow stromal cells with the said pseudotyped vector by pre-incubating the cells for a suitable period of time in cell culture medium without phosphate and subsequently adding a vector-containing solution, optionally supplemented with transduction additives to the cell; and/or d) optionally centrifuging the mixture thus obtained; and/or e) optionally repeating step c) and d) .
  • a retroviral vector comprising a gene of interest
  • b) optionally pseudotyping the said vector with the Gibbon ape leukemia virus (GALV) envelope
  • GLV Gibbon ape leukemia virus
  • the present invention further relates to the transduced cells thus obtained.
  • the invention in particular relates to bone marrow stromal cells being transduced with an intron-based retroviral vector comprising a B-domain deleted human factor VIII cDNA, which vector has been pseudotyped with the Gibbon ape leukemia virus (GALV) envelope.
  • GALV Gibbon ape leukemia virus
  • the invention according to a further aspect thereof relates to these cells for use in the therapeutical treatment of coagulation disorders, in particular hemophilia A.
  • the therapy of hemophilia A consists for example of returning bone marrow stromal cells, that have been genetically engineered with the gene for factor VIII by the method of the invention to a subject in need of therapy. This type of therapy is called gene therapy.
  • gene therapy For other types of treatment other types of genes can be transduced to the genetically engineered cells.
  • the invention also relates to the use of the cells for the preparation of a therapeutic composition for the treatment by means of gene therapy of disorders of the blood, in particular coagulation disorders, more in particular hemophilia A.
  • BM stroma Clinical application for hemophilia A using BM stroma requires injection or infusion of about 10 8 autologous BM stromal cells prepared with the method of the invention to convert a severe to a mild hemophiliac based on an in vitro production of 400 ng/10 6 cells per 24 hr, assuming that all the engineered cells engraft j-n vivo and continue to produce these high levels of FVIII. To completely correct the bleeding phenotype, an infusion of at least 7xl0 8 BM stromal cells will be required. At least 10 10 BM stromal cells can be enriched from 1 liter of total BM, which can routinely be obtained from a single BM isolation without any adverse side effects to the donor.
  • FIG. 1 Development of an adherent stromal layer after long-term BM culture. Photomicrographs were taken after 5 days (A) , 10 days (B) and 14 days (C) under phase-contrast . A confluent monolayer of subcultured cells at the time of transduction is also shown (D) . Adherent stromal cells, suspension cells and erythrocytes are indicated as A, S and E, respectively.
  • FIG. 2 Immunohistochemical staining of human BM stromal cells. Human BM stromal cells were stained for human prolyl 4-hydroxylase, as a fibroblast-specific marker .
  • FIG. 3 Determination of GLVR-1 versus GLVR-2 mRNA expression by quantitative RT-PCR.
  • Purified total RNA (1, 2.5 and 5 ⁇ g) from adherent human BM stromal cells was reverse transcribed.
  • Two ⁇ l of the reaction mixture containing the cDNA obtained form either 1 ⁇ g RNA (1) (lanes 1-8) , 2.5 ⁇ g RNA (11) (lanes 9-16) or 5 ⁇ g RNA (111) (lanes 18-22) was serially diluted and subjected to PCR with GLVR-1 and GLVR-2 specific primers as described in Materials and Methods.
  • Quantification was performed using a Phosphorimager after background subtraction (B) using experimental samples that fell within the linear range of the assay corresponding to (lanes 13-16) (A) for GLVR-1 (cDNA obtained from 2.5 ⁇ g RNA was diluted 16-128-fold) and (lanes 9-14) (A) for GLVR-2 (cDNA obtained from 2.5 ⁇ g RNA was diluted 132 -fold) .
  • FIG. 4 FVIII production (A), Southern blot analysis (B) and titration (C) of FVIII-retroviral vectors pseudotyped with GALV-env (PG13-F8) .
  • FVIII production in the PG13-F8 clone #5 compared to the MFG- FVIII ⁇ B clone #XF2 was quantified with a functional chromogenic FVIII assay (A) .
  • Genomic DNA of the MFG- FVIII ⁇ B producer clone #XF2 (lanes 1,2), the PG 13-F8 producer clone #5 (lanes 3,4) and the control plasmid pMFG-FVIIl ⁇ B (lanes 5,6) were digested with Sma I (lanes 1, 3, 5) or Nhe I (lanes 2, 4, 6) and subjected to Southern blot analysis with a FVIII-specific probe (B) . Bands corresponding to proviruses containing only non- deleted FVIII sequences were indicated by arrows; molecular weight markers (in kb) were indicated on the right.
  • Relative viral titer of the MFG-FVIII ⁇ B and PG13- F8 producer cell clones was determined in function of cell number by RNA dot blot analysis of PEG-precipitated viral vector particles using a FVIII-specific probe as described in the Materials and Methods (C) . Quantification was performed using a Phosphorimager after background subtraction.
  • FIG. 6 Analysis of transduction efficiency by quantitative PCR/Southern blot analysis. Genomic DNA of the transduced human BM stromal cells was subjected to PCR/Southern blot analysis using primers specific for the FVIII-retroviral vector and ⁇ -actin specific primers for normalization. Bands corresponding to the amplified FVIII or ⁇ -actin-specific fragments were indicated by arrows (A) . The intensities of the PCR-amplified fragments relative to the maximum transduction efficiency (i.e.
  • mice were injected as indicated intrasplenically (i.s.) with l-3xl0 6 human BM stromal cells transduced with PG13/F8 under optimized conditions. FVIII expression was measured over time with a human FVIII-specific ELISA. The experiment was repeated with BM stroma from unrelated donors and the results of one representative experiment were given.
  • the present example demonstrates the expression of factor VIII in human BM stromal cells and compares the transduction efficiency and expression levels when using various vectors and different transduction protocols.
  • BM stromal cells were obtained from the iliac crest and/or sternum of healthy BM donors since large amounts of enriched bone marrow could be isolated relatively easily by needle aspiration from these sites. BM donors provided their informed consent to participate in the procedure. BM was collected in an equal volume of MyeloCult H5100 medium (StemCell Technologies, Vancouver, Canada) supplemented with 250 U/ml heparin. Erythrocytes were removed by sedimentation for 30 min at room temperature using Plasmasteril
  • mice After expanding the BM stromal cells for an additional 7 days, the non-adherent suspension cells were decanted while the adherent stromal layer was trypsinized for transduction or further characterization.
  • the mouse BM stroma was isolated by sacrificing bnx mice (Harlan, Zeist, the Netherlands) and flushing the femur and tibia with MyeloCult M5300 medium (StemCell Technologies) .
  • BM cells harvested either from one femur or two tibiae were cultured in a 10 cm petri dish containing 10 ml of MyeloCult M5300 long-term culture medium supplemented with freshly prepared hydrocor isone hemisuccinate (10 "6 M, Sigma) , 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin and 250 ng/ml amphotericin B (Life Technologies) (designated as MBM medium) .
  • Adherent mouse BM stromal cells were obtained by growing the cells for 3-7 days. After expanding the BM stromal cells for 7 days, the non-adherent suspension cells were decanted while the adherent stromal layer was trypsinized for transduction.
  • the MFG-FVIII ⁇ B and the GCsamENF ⁇ splicing vectors were described previously (Dwarki et al . , 1995, supra ; Chuah et al . , 1995, supra) and can be reconstructed quite easily from these descriptions.
  • the B-domain deleted FVIII gene was driven from the 5 ' MoMLV LTR and was cloned downstream of the MoMLV intron used to generate subgenomic env mRNA.
  • a Kozak consensus sequence for translational initiation was introduced in the MFG-FVIII ⁇ B vector and the 3' untranslated region (UTR) of the FVIII gene was deleted.
  • the MFG-FVIII ⁇ B vector lacked a neo R selectable marker, whereas the GCsamF ⁇ EN vector expressed FVIII and the neomycin phosphotransferase II (NPTII) proteins from a single polycistronic transcript that was driven from the 5 ' MoMLV LTR by virtue of the internal ribosome entry site (IRES) .
  • NPTII neomycin phosphotransferase II
  • Viral supernatant was first collected over 24 hr from a confluent plate of MFG-FVIII ⁇ B producer cells (clone #XF2) and filtered through a 0.45 ⁇ m filter to remove residual producer cells.
  • 8xl0 6 PG13 cells were subjected to successive daily transductions with 5 ml of MFG-FVIII ⁇ B viral vector-containing conditioned medium in the presence of polybrene (8 ⁇ g/ml, Sigma) .
  • the resulting producer cells were designated as PG13-F8 and individual clones were obtained by limiting dilution.
  • FVIII production by each of the individual PG13-F8 clones was quantified using a functional chromogenic assay as described below. Clones that expressed the highest levels of FVIII were further screened for viral production by RNA dot blot analysis (see below) and subsequently subjected to Southern blot analysis as described previously (Sambrook et al . , Molecular Cloning 16-32 (1989), Chuah et al . , 1995, supra) to exclude the presence of rearranged proviral sequences. Briefly, genomic DNA was extracted with the high pure PCR template preparation kit (Boehringer, Mannheim, Germany) and 23 ⁇ g of DNA was restricted with Sma I or Nhe I.
  • Hybridizations were performed by probing the Southern blot membrane with a FVIII-specific probe corresponding to a random primed 1095 bp Bgl II - Spe I restriction fragment of plasmid pMT2LA.
  • the membranes were washed stringently at 65°C in 2xSSC and 0.1% SDS for 30 min, followed by 0.5xSSC and 0.2% SDS for an additional 30 min.
  • FVIII activity in the transduced stromal cells and the viral producer cell clones was quantified by measuring the FVIII-dependent generation of factor Xa from factor X using a chromogenic assay (Coatest FVIII, Chromogenix, Molndal, Sweden) as described previously (Chuah et al . , 1995, supra) . Briefly, 24 hr-conditioned culture medium was harvested in phenol-red free media to avoid calorimetric interference in the FVIII chromogenic assay. Human plasma purified FVIII (Octapharma,
  • Supernatant containing retroviral vector particles was obtained by seeding 4xl0 6 producer MFG- FVIII ⁇ B and 30xl0 6 producer PG13-F8 in 10 ml of D10 per 75 cm 2 flask, unless indicated otherwise. These cells were grown at 32 °C and the supernatant was harvested after 24 hr. Supernatants were aliquoted and immediately frozen on dry ice prior to storing at -80°C until use. Vector titer in the culture medium was determined by RNA dot blot analysis as described previously (Yang et al, Hum. Gene Ther. 6:1203-1213 (1995)).
  • Hybridizations were performed by probing the membrane with a FVIII-specific probe corresponding to a random primed 1095 bp Bgl II - Spe I restriction fragment of pMT2LA.
  • the membranes were washed stringently at 65°C in 2xSSC for 30 min followed by 0.5xSSC for an additional 30 min. After background subtraction, signal intensities were quantified using a Phosphorimager (Molecular Dynamics, Sunnyvale, CA) .
  • Additional controls consisted of serially diluted viral vector supernatants with known functional titer based on vectors containing a neo R gene (GCsamF8EN) .
  • Functional titers expressed as G418 R cfu/ml were determined by transduction of NIH-3T3 cells as described previously (Chuah et al . , 1994 and Chuah et al . , 1995, supra) .
  • BM stromal cells were seeded at a concentration of 10 4 cells per well in 0.4 ml Iscove's modified Dulbecco ' s medium (IMDM) supplemented with freshly prepared hydrocortisone (10 "6 M, Sigma) , 10% heat- inactivated FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 ⁇ g/ml streptomycin and 250 ng/ml amphotericin B (designated as 110 medium) in 8-chamber slides (Life Technologies) and incubated at 37 °C. Medium was removed from chamber slides containing confluent cells followed by fixation with acetone at -20°C for 5 min.
  • IMDM Iscove's modified Dulbecco ' s medium
  • TSA Tris saline buffer
  • Immunostaining was performed by first incubating the cells for 30 min in 100 ⁇ l 5-fold diluted pre-immune rabbit serum (Dako, Glostrup, Denmark) for the fibroblast-specific immunostaining or pre-immune swine serum for the endothelial cell-specific immunostaining (Dako), after which supernatant was removed.
  • the fibroblast-specific immunostaining the cells were incubated overnight with a monoclonal mouse antibody that reacted with human prolyl 4-hydroxylase (clone 5B5, 155 ⁇ g/ml Dako) diluted 1/50-1/100 in TSB.
  • RNA expression levels in the human BM stromal cells were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) .
  • Total RNA was first purified using the chaotropic Trizol method followed by phenol-chloroform extraction and isopropanol precipitation (Chomczynski , P., Biotechniques 15:532-534, 536-537 (1993)). The precipitated RNA was then redissolved in H 2 0 and spectrophotometrically quantified. The first strand cDNA was synthesized starting from 1, 2.5 and 5 ⁇ g purified total RNA using a Superscript II reverse transcriptase kit (Life Technologies) .
  • the cDNA was subsequently amplified directly by PCR using 2 ⁇ l of the reaction mixture, that was subjected to serial 2-fold dilutions.
  • PCR was performed with a Techne/Progene thermocycler using Taq polymerase and oligonucleotide pairs that discriminated specifically between GLVR-1 ( 5 ' -GCAGTTTTCTGTGCCCTTATCGTC-3 ' and 5 ' -GGAGTTTATTTGGTTGCTGACGG-3 ' ) and GLVR-2 ( 5 ' -TTCAGGAAGCAGAGTCCCCAGT-3 ' and 5' -TGTCGATGTGGATTTTGCAG-3 ' ) .
  • PCR was performed by denaturation for 5 min at 94 °C, followed by 26 cycles of 45 sec at 94°C, 1 min at 60°C, 2 min at 72°C and a final extension for 7 min at 72°C.
  • the RT-PCR reaction products obtained from 1 ⁇ g, 2.5 ⁇ g and 5 ⁇ g RNA were subjected to serial 2-fold dilution (ranging from 2- to 128-fold dilution) to ensure that detection of the PCR amplified fragments fell within the linear range of the quantitative PCR.
  • BM stromal cells Human and murine BM stromal cells were seeded at a density of 10 5 cells/ml per well in a 6-well plate containing 1 ml 110 medium supplemented with freshly prepared hydrocortisone (10 ⁇ 6 M, Sigma) for human stroma or hydrocortisone-hemisuccinate (10 ⁇ 6 M, Sigma) for mouse stroma. The next day, the 110 medium was aspirated and BM stromal cells were washed once with 5 ml of PBS followed by transduction with the amphotropic MFG-FVIII ⁇ B vector (for murine and human stroma) or the GALV-env pseudotyped PG13-F8 vector (for human stroma) under standard or optimized conditions.
  • Standard conditions involved overnight incubation of the stromal cells with vector- containing supernatant at 32 °C by virtue of the increased stability of retroviral vectors at 32°C versus 37°C.
  • Vector-containing supernatants were supplemented with 4 ⁇ g/ml protamine sulphate and hydrocortisone (or hydrocortisone-hemisuccinate) .
  • vector-containing supernatant with protamine sulphate and hydrocortisone (or hydrocortisone- hemisuccinate) was added to the cells followed by a centrifugation step at 32°C for 1 hr at 1400 g and an overnight incubation at 32°. Cells were returned to 37°C the next day.
  • a phosphate starvation step was included that involved a 9-10 hr incubation of the stromal cells in 110 medium containing IMEM without phosphate. A total of 4 rounds of transductions were performed successively under the same conditions over the next 4 days followed by two washings with PBS.
  • High molecular weight genomic DNA was isolated from the transduced BM stromal cells using the high pure PCR template preparation kit (Boehringer, Mannheim, Germany) . To determine transduction efficiency, PCR was performed with a Techne/Progene thermocycler using Taq polymerase and oligonucleotide pairs specific for the FVIII cDNA in the MFG-FVIII ⁇ B or GCsamF ⁇ EN retroviral vectors (5 ' -GAGCTCTCCACCTGCTTCTTTCTG-3 ' and 5 'CCCTTCTCTACATACTAGTAGGGC-3 ' ) yielding a specific 594 bp PCR product.
  • ⁇ -actin-specific primers 5 « -CATTGTGATGGACTCCGGAGACGG-3 ' and 5' -CATCTCCTGCTCGAAGTCTAGAGC-3 ' ) were added to the PCR reaction mixture yielding a 232 bp ⁇ -actin specific PCR product .
  • a producer clone transduced with GCsamF8EN and containing 6 integrated proviral copies was used as a control .
  • PCR was performed by denaturation for 8 min at 95°C, followed by 28 cycles of 1 min at 95°C, 1 min at 59°C, 2 min at 72°C and a final extension for 5 min at 72 °C.
  • Fig. 1A The kinetics of the development of an adherent BM stromal cell layer of different BM donors did not vary significantly and a representative example is given is Fig. 1.
  • Fig. 1A During the first 5 days (Fig. 1A) , most BM cells remained in suspension except for the presence of adherent macrophage-like cells. Less than 5% of the suspension cells were erythrocytes . Cells with a spindle- like morphology started to adhere between day 6-10 (Fig. IB) whereas suspension cells gradually disappeared from the cultures. The adherent cells continued to proliferate and formed cell aggregates that extended to generate patchy areas of an adherent layer (Fig. IC) . The hemopoietic foci and residual suspension cells were removed from the cultures (Figs.
  • IC, ID IC, ID
  • adherent cells were trypsinized and expanded to generate an adherent stromal layer (Fig. ID) . More than 95% of the adherent stromal cells exhibited the distinctive spindle- like morphology that was retained even after long-term culture (at least 4 weeks) .
  • BM contains endothelial and fibroblast- like cells
  • the identity of the spindle-like stromal cell layers was confirmed by immunohistochemical analysis. Immunostaining for fibroblasts was performed using a murine monoclonal antibody against the ⁇ -subunit of human prolyl 4-hydroxylase which catalyses the hydroxylation of proline residues in collagens to hydroxyproline .
  • the majority of the stromal cells (84 ⁇ 8.0 %) stained for the prolyl 4-hydroxylase antigen indicating that they are fibroblastic (Fig. 2A) whereas no staining was observed when the primary or secondary antibody was omitted and when using the NIH-3T3 mouse cell fine as a negative control, as expected (data not shown).
  • Detection of GLVR-1 and GLVR-2 specific PCR- amplified fragments from the BM stromal cells fell within the linear range of the assay for the RT-PCR reaction products obtained from 1 ⁇ g RNA (Fig. 3A, lanes 1-8) and 2.5 ⁇ g RNA (Fig. 3A, lanes 9-16) (r 2 0.97-0.99). No PCR amplified product could be detected in the negative controls that did not contain RT (Fig.
  • GALV-env pseudotyped FVIII- retroviral vectors were generated (designated as PG13-F8) in an attempt to obtain high transduction efficiencies.
  • PG13-F8 clones Six out of the 23 PG13-F8 clones that were screened, expressed functional FVIII ranging from 5 to 200 ng
  • RNA dot blot analysis yielded an equivalent average functional titer for MFG-FVIII ⁇ B of 6 ⁇ 3xl0 4 G418 R cfu/ml based on 23 independent batches of viral supernatant.
  • the titer of the PG 13-F8 clone #5 was equivalent to (1.8 ⁇ 0.6)xl0 5 cfu/ml based on 17 independent batches of viral supernatant.
  • the optimized transduction protocol with the MFG-FVIII ⁇ B vector also yielded significantly higher FVIII expression levels (p ⁇ 0.001) (900 ⁇ 130 ng FVIII/10 6 cells per 24 hr) as compared to the standard protocol (140 ⁇ 14 ng FVIII/10 6 cells per 24 hr) (Fig. 5B) .
  • the FVIII expression levels in mouse BM stromal cells transduced with the MFG-FVIII ⁇ B vector were consistently higher than in human BM stroma transduced with the same vector possibly due to the higher proliferative capacity of the mouse BM stroma at the inception of transduction, resulting in higher transduction efficiencies and FVIII expression levels.
  • FVIII expression was significantly higher (p ⁇ 0.001) with centrifugation (320 ⁇ 37 ng FVIII/10 6 cells per 24 hr) than without (110 ⁇ 13 ng FVIII/10 6 cells per 24 hr) (Fig.5A).
  • centrifugation step alone strongly increased FVIII expression levels in transduced human BM stromal cells.
  • FVIII expression without phosphate starvation corresponded to 320 ⁇ 37 ng FVIII/10 6 cells per 24 hr whereas significantly higher (p ⁇ 0.05) levels were obtained after phosphate starvation (390 ⁇ 12 ng FVIII/10 6 cells per 24 hr) (Fig. 5A) .
  • phosphate starvation had contributed to a significant but only moderate increase in FVIII expression levels in transduced human BM stromal cells.
  • human or mouse BM stromal cells can be exploited as an alternative BM-derived target cell for hemophilia A gene therapy since they could express relatively high levels of human FVIII (400-900 ng/10 6 cells per 24 h) when transduced with FVIII retroviral vectors.
  • the high levels of FVIII could be attributed to the use of an intron- based vector, the development of an optimized transduction protocol and the generation of a GALV-env pseudotyped FVIII retroviral vector.
  • Human BM stromal cells were first transduced with FVIII retroviral vectors in vitro using the optimized transduction method with GALV-env pseudotyped MFG-FVIII ⁇ B vectors as presented in this invention and were subsequently infused into immunodeficient SCID NOD mice.
  • Therapeutic human FVIII expression levels were detected in the plasma of these mice well above the levels needed to convert a severe to a moderate hemophilia A patient and persisted for at least 1 week in vivo . This represents the first in vivo study showing therapeutic levels of FVIII using BM stromal cells.
  • engraftment of the FVIII-engineered cells could be achieved even in the absence of myelo-ablation and without having to seed the cells into artificial fibers or neo-organs.
  • One advantage of the present method is that no myeloablation is required. Because of this, the gene therapeutic method described herein is clinically acceptable for hemophilia patients. Furthermore, the in vivo expression levels and kinetics are comparable to what has been reported previously using neo-organs (Dwarki et al . , 1995, supra) . 2. Materials and methods
  • Human BM stromal cells were seeded at a density of 10 5 cells/ml per well in a 6-well plate containing 1 ml 5 110 medium supplemented with freshly prepared hydrocortisone (10 ⁇ 6 M, Sigma) for human stroma. The next day, the 110 medium was aspirated and BM stromal cells were washed once with 5 ml of PBS followed by transduction with the GALV-env pseudotyped PG13-F6 vector 0 under optimized conditions. Vector-containing supernatant was collected at 32 °C.
  • Vector containing supernatants were supplemented with 4 ⁇ g/ml protamine sulphate and hydrocortisone and added to the cells followed by a centrifugation step at 32°C for 1 hr at 1400 g and an 5 overnight incubation at 32°C. Cells were returned to 37°C the next day.
  • a phosphate starvation step was included that involved a 9-10 hr incubation of the stromal cells in 110 medium containing IMEM without phosphate.
  • a total of ⁇ rounds of transductions were performed successively 0 under the same conditions over the next 2 weeks followed by two washings with PBS.
  • BM stromal cells that were stably transduced 5 with the PG13-F6 retroviral vector were trypsinized, washed with PBS and resuspended at a cell density of 7.5 to 15xl0 6 per ml of PBS.
  • Four to 5 weeks old male NOD-SCID recipient mice were injected intra-splenically through a small incision using a 27G needle with 1.5 to 3xl0 6 BM 0 stromal cells per 200 ⁇ l . Control mice received an intrasplenic injection of 200 ⁇ l PBS.
  • FVIII in vitro activity in the transduced 5 stromal cells and the viral producer cell clones was quantified by measuring the FVIII -dependent generation of factor Xa from factor X using a chromogenic assay (Coatest FVIII, Chromogenix, Molndal, Sweden) as described previously (Chuah et al . , 1995, supra) . Briefly, 24 hr-conditioned culture medium was harvested in phenol-red free media to avoid colorimetric interference in the FVIII chromogenic assay. Human plasma purified FVIII (Octopharma, Langenfeld, Germany) of known activity was used as a FVIII standard and 1 U was defined as 200 ng FVIII/ml.
  • FVIII in vivo expression was determined by collecting mouse plasma at regular intervals and performing a human FVIII-specific ELISA. A 96-well microtite plate was coated with 2 monoclonal antibodies to human FVIII which do not cross-react with mouse FVIII: N77110M (Biodesign, Kennebunkport, ME) and ESH2 (American Diagnostica, Greenwich, CT) at a concentration of 25 ⁇ g/ml. After incubation overnight at 4°C, the plate was washed three times with PBS.
  • Blocking agent PBS, 10% horse serum, 1 mM CaCl 2
  • PBS 10% horse serum
  • 1 mM CaCl 2 a blocking agent
  • Plasma samples were diluted 1:5 in TNTC buffer (50 mM Tris pH 7.2, 5 mM CaCl 2 , 0.1% Tween 20, 0.5 M NaCl) and 100 ⁇ l was added into each well.
  • Mouse plasma spiked with known concentrations of serially diluted purified human FVIII was used to generate a standard curve.
  • the plate was incubated for 1 hr at 37°C, the wells were washed 3 times with PBS, 0.05% Tween 20 and 100 ⁇ l of the second antibody was added.
  • the second antibody was serum from a hemophiliac with a high inhibitor titer, diluted 1:1000 in blocking agent PBS + 10% horse serum + 1 mM CaCl 2 .
  • the wells were washed and 100 ⁇ l of TMB was added to the well.
  • the reaction was stopped by adding 100 ⁇ l H 2 S0 4 1.5 M.
  • the absorbance at 450 nm was determined using a microplate reader. Normal mouse plasma did not interfere with the assay and the limit of sensitivity was 1 ng/ml for purified plasma-derived human FVIII added to normal mouse plasma .
  • GALV-env pseudotyped PG13-F6 retroviral vector yielded an in vitro production of 490 ⁇ 40 ng FVIII ng/10 6 cells per 24 hr.
  • One to 3xl0 6 transduced BM stromal cells were injected intrasplenically into NOD-SCID mice and FVIII expression was measured with a human FVIII-specific ELISA (Fig. 7C) .
  • Therapeutic levels of FVIII could be detected that were at least 10 -fold higher than the therapeutic levels needed to convert severe to moderate hemophilia A (2 ng/ml) .
  • Therapeutic levels persisted for at least one to two weeks after which animals were sacrificed for PCR analysis to determine the homing pattern of the injected cells. Control animals that received PBS only did not produce human FVIII, as expected.
  • Therapeutic human FVIII expression levels were detected in vivo in the plasma of these mice well above the levels needed to convert a severe to a moderate hemophilia A patient. This is the first demonstration that transduced BM stromal cells can be used to achieve therapeutic levels of a protein in vivo . More in particular, this represents the first protocol showing therapeutic levels of FVIII in vivo using BM stromal cells. Engraftment of the FVIII-engineered cells was relatively efficient since only a single injection of 1- 3xl0 6 cells was needed to obtain FVIII expression.

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